CN107987141B - Application of a maize nuclear factor gene ZmNF-YA1 in plant stress resistance transformation - Google Patents

Abstract

The invention discloses the application of a maize nuclear factor gene ZmNF-YA1 in the transformation of plant stress resistance. In the expression vector, the ZmNF-YA1 gene is introduced into the plant by using transgenic technology; by detecting the expression of the transgene and measuring the stress resistance of the plant, the transgenic plants and their offspring with increased or decreased resistance to stress are screened out, creating a plant with a high level of resistance in plant breeding. New germplasm of applied value. The invention utilizes the ZmNF-YA1 gene to participate in the regulation of plant stress resistance through gene expression regulation, and is of great significance for cultivating high-yield transgenic crops.

Description

Application of a maize nuclear factor gene ZmNF-YA1 in plant stress resistance transformation

technical field

The invention belongs to the field of bioengineering breeding of crops, and in particular relates to the application of a maize nuclear factor gene ZmNF-YA1 in the transformation of plant stress resistance; that is, a scheme for changing maize stress resistance by constructing a transgenic overexpression structure and a transgenic approach and use.

Background technique

NF-Y (nuclear factor-y) is a ubiquitous transcription factor complex in eukaryotes, consisting of three different subunits (NF-YA/CBF-B, NF-YB/CBF-A and NF- YC/CBF-C), and regulate the expression of target genes by acting on the promoters of other regulatory factors. The complete NF-Y transcription complex binds to the CCAAT motif in the target gene promoter to regulate the transcription of the target gene. NF-YA is a DNA sequence-specific subunit that binds to the core pentameric nucleotide CCAAT motif (motif) in the gene promoter region. The NF-Y complex can function as a transcriptional activator or repressor, and its binding to DNA and transcriptional regulatory activity are also regulated by other transcription factors, which act by interacting with the NF-Y subunit.

In yeast and mammals, each NF-Y subunit is encoded by a single gene that has multiple splices and multiple post-translational modifications. In mammals, the physiological functions of the NF-Y complex and its regulatory pathways include a variety of molecular and biochemical processes, such as endoplasmic reticulum stress, DNA damage and repair, and cell cycle regulation. But in plants, each NF-Y subunit is encoded by a gene family, so the NF-Y complex has a diversity of different subunits. In Arabidopsis, the NF-YA subunit is encoded by 10 genes, the NF-YB subunit is encoded by 13 genes, and the NF-YC subunit is encoded by 13 genes. Rice has at least 11 NF-YA genes, 12 NF-YB genes, and 8 NF-YC genes. In maize, 14 NF-YA genes, 18 NF-YB genes and 18 NF-YC genes have been found. GUS expression analysis of 36 NF-Y subunit genes in Arabidopsis thaliana revealed that the members of each subfamily have complex and diverse expression patterns, suggesting that the functions of different NF-Y complexes composed of these family members are diverse and can be regulated. expression of multiple genes.

Existing work shows that all three NF-Y subunit family members are involved in the regulation of plant growth and development. In Arabidopsis LEAFY COTYLEDON1 induces the development of vegetative cells into embryos, and two NF-Y subunit genes (AtLEC1/AtNF-YB9) regulate embryogenesis and seed maturation. AtNF-YB9 and LEC1-LIKE (L1L/NF-YB6) regulate Arabidopsis embryonic development by inducing the expression of genes related to embryogenesis and cell differentiation. Several NF-YA subunit genes in Arabidopsis are expressed in embryos, such as NF-YA1, YA2, A3, A4, A6, A7, A8, and A ^] . Lines overexpressing NF-YA1, A5, A6, or A9 were hypersensitive to ABA during seed germination, and male gametogenesis, embryogenesis, seed morphology, and germination were affected. These NF-Y overexpressing materials can form somatic embryos directly from vegetative organs. NF-YA3 and -A8 had the highest expression levels from globular embryo to torpedo embryo stage. The nf-ya3nf-ya8 double mutant was embryonic lethal, but the nf-ya3 and nf-ya8 single mutants did not show obvious phenotypic changes, suggesting that these two genes are functionally redundant during early embryogenesis in Arabidopsis.

In addition to affecting plant embryogenesis and seed maturation, NF-Y transcription factors also regulate vegetative or/and reproductive growth of plants. Overexpression of AtNF-YB2 promotes primary root elongation by stimulating cell division and cell elongation. In the legume model plant Medicago truncatula, MtHAP2-1 is expressed in the nodule meristem and plays an important role in the differentiation of nodule cells. The study of overexpressing PvNF-YC1 in common bean and interfering with this gene expression by RNAi revealed that NF-Y plays an important role in the process of root nodule organ formation. In green cuttings, Yu et al. found that a NF-YC protein regulates the growth direction of pollen tubes by interacting with PwFKBP12.

It has been reported that NF-Y regulates plant drought tolerance. AtNF-YB1 and its maize homolog ZmNF-YB2 were overexpressed in Arabidopsis and maize, respectively, and the growth and survival rate of transgenic plants were better than those of non-transgenic plants under drought conditions. ChIP assay results showed that changes in AtNF-YB1 expression did not affect dehydration-responsive element binding proteins or ABA-dependent drought resistance pathways, suggesting that AtNF-YB1 may function through an ABA signaling-independent drought resistance pathway . Multiple works have shown that NF-Y interacts with bZIP, and the bZIP protein is involved in the ABA signaling pathway.

AtNF-YA5 gene was up-regulated by drought, osmotic stress and salt stress. The transcriptional regulation of AtNF-YA5 is regulated by an ABA-dependent mechanism, and miR169 is involved in its post-transcriptional modification. Under drought conditions, miR169 was down-regulated, and the process was ABA-dependent. 35S::miR169 and nf-ya5 mutant plants were more sensitive to drought stress. AtNF-YA5 regulates the stomatal size of guard cells, and AtNF-YA5 also improves the drought tolerance of plants by activating the expression of stress-responsive genes in other cells, such as oxidative stress response-related genes. In addition to AtNF-YA5, overexpression of other AtNF-YA genes such as YA2, YA3, YA7, and YA10 can also improve plant tolerance to drought ^[148] . The expression levels of 11 wheat NF-Y genes under drought conditions were significantly different from those under normal conditions, of which 8 were down-regulated and 3 were up-regulated. A rice NF-YA gene (OsHAP2E), a target of miR169, is induced by salt stress and may be regulated by an ABA-dependent pathway. OsNF-YA7 was induced by drought stress, but its expression was not responsive to ABA. The drought tolerance of rice plants overexpressing OsNF-YA7 was improved. Analysis of the OsNF-YA7 promoter revealed three DRE/CRT elements, which were regulated by the ABA-independent drought-regulated transcription factor OsDREB2. Therefore, it is speculated that OsNF-YA7 regulates the drought tolerance of plants mainly through an ABA-independent pathway. Drought stress reduced the transcriptional expression level of OsNF-YA2 and increased drought tolerance in osnf-ya2 indel mutants, suggesting that this gene may be a negative regulator of drought in rice. Overexpression of the wheat NF-YA gene TaNF-YA10-1 in Arabidopsis can improve the drought tolerance of plants and promote root and plant growth, but TaNF-YA10 overexpressed plants are more sensitive to salt stress, indicating that this gene is in response to Different regulatory functions in drought stress and salt stress.

Several works have reported that NF-Y transcription factors regulate plant responses to heat and cold stress. Overexpression of AtNF-YA2 or AtNF-YC1 in Arabidopsis can improve cold stress resistance of plants. Transcriptome analysis showed that some abiotic stress-responsive genes were down-regulated in AtNF-YA2-overexpressing plants, but the mechanism by which down-regulation of these genes would increase the cold resistance of AtNF-YA2-overexpressing plants was unclear. AtNF-YC1 may regulate freezing stress through a C-REPEAT BINDING FACTORs (CBFs)-dependent pathway.

The increasing impact of heat stress on crop yield is a current research topic in plant science. Sato et al. found that DREB2A can interact with DNA POLYMERASE II SUBUNIT B3-1 (DPB3-1/NF-YC10), especially under heat stress conditions. DPB3-1/NF-YC10 can form trimers with NF-YA2 and NF-YB3 to regulate the expression of genes related to heat stress mediated by DREB2A. Overexpression of DPB3-1/NF-YC10 increased the expression level of the heat stress-responsive gene HEAT SHOCK TRANSCRIPTIONFACTOR A2 (HSFA2), while the expression level of HSFA2 was down-regulated in the nf-yc10 mutant ^[158] . Overexpression of NF-YC10 did not affect the growth of Arabidopsis plants. Overexpression of Arabidopsis AtNF-YC10 in rice could improve the heat tolerance of rice plants without affecting plant growth and development. NF-YC10 belongs to a monophyletic group and is highly conserved in terrestrial plants, thus serving as a candidate gene for the creation of heat stress-resistant crops.

NF-Y transcription factors are also involved in the endoplasmic reticulum (ER) stress response. In Arabidopsis thaliana, NF-YC2, NF-YA4, and NF-YB3 form a transcriptional complex with bZIP28, which upregulates the expression of ER stress-related genes and prevents the accumulation of misfolded and unfolded proteins in the ER. Alleviates the decline in protein synthesis. Although the existing bioinformatics analysis results and microarray data show that the expression of members of the NF-Y gene family is regulated by a variety of stresses, there are few studies on the specific stress resistance of plants and their mechanisms.

In many reports, the expression patterns of plant NF-YA gene family are spatiotemporally specific. Expression analysis of Arabidopsis NF-YA family members in different tissues found that some members share common organ expression patterns, such as AtNF-YA4 and AtNF-YA7 have similar expression patterns in leaves and flowers, suggesting that their Functional redundancy. Zhang et al. systematically identified 50 ZmNF-Y genes, and analyzed their expression patterns using microarray data, and concluded that some ZmNF-Y family members are specifically expressed in vegetative organs and reproductive organs, and different members have different effects on biological There are also differences in responses to stress and abiotic stress. The response of ZmNF-Y to biotic stress was analyzed by inoculating the pathogenic bacteria Fusarium moniliformes, Rhizoctonia solani and Anthracis turfica respectively. It was found that ZmNF-YA3, ZmNF-YA8 and ZmNF-YA12 were up-regulated, while ZmNF-YA1 was down-regulated. Luan Mingda et al. conducted subcellular localization analysis of seven ZmNF-YAs regulated by mature miR169, and found that these ZmNF-YAs were localized in the nucleus, but lacked the ability to activate transcription. The expression patterns of ZmNF-YA and miR169 in response to stress were analyzed, and it was found that in maize leaves, the expression of ZmNF-YA and miR169 responded to PEG, ABA and salt stress, but there was no expected negative correlation between them. In maize roots, short-term stress (0-48 h) caused down-regulation of miR169 expression, while long-term stress (15 days) up-regulated the expression of miR169, and most ZmNF-YAs showed opposite expression changes to miR169 under short-term stress. In response to adversity stress, the expression level of miR169 was positively correlated with maize root elongation, while ZmNF-YA14 was negatively correlated with root elongation, that is, the miR169/ZmNF-YA14 regulatory module may be involved in maize root response to adversity stress control process. In addition, overexpression of ZmNF-YA14 up-regulated the expression of peroxidase-related genes, actively scavenged the accumulation of intracellular reactive oxygen species, and enhanced the resistance of maize to salt stress.

The maize NF-Y family has a large number of genes. The maize genome sequence was searched, and it was found that there were 36 possible members of the maize NF-YA family, 28 members of the NF-YB family, and 25 members of the NF-YC family. However, the functional research of these NF-Y family genes is still in its infancy, and the search only finds research reports that over-expression of the ZmNF-YB2 gene in maize improves the drought tolerance of plants and patents filed by Monsanto. There are few reports on the specific application of maize nuclear factor gene ZmNF-YA1 in plant stress resistance transformation.

SUMMARY OF THE INVENTION

In view of the current research status, the present invention provides the application of a maize nuclear factor gene ZmNF-YA1 in the transformation of plant stress resistance. The use of ZmNF-YA1 gene to participate in the regulation of plant stress resistance through gene expression regulation is of great significance for the cultivation of high-yield transgenic crops.

The application of the maize nuclear factor gene ZmNF-YA1 in the transformation of plant stress resistance is to identify and clone the ZmNF-YA1 sequence from maize, construct a fusion gene with the full-length or partial sequence, and the promoter of the latter is connected to The fusion gene is inserted into the plant expression vector before the target gene coding frame (in positive sense form) or RNAi structure, and the recombinant gene is introduced into plant cells by transgenic technology to obtain transgenic plants; by detecting transgene expression and character identification of plants , screen out the transgenic plants and their progeny with significantly changed target traits, and create new germplasm and new varieties with application prospects in plant breeding. The specific plans are as follows:

The application of a maize nuclear factor gene ZmNF-YA1 in the transformation of plant stress resistance, characterized in that: the ZmNF-YA1 gene is cloned from maize, and the gene is recombined into a plant in a sense or antisense form or an RNAi structure form for expression In the vector, a fusion gene is formed; the recombinant gene is introduced into the plant by transgenic technology; by detecting the expression of the transgene and measuring the stress resistance of the plant, the transgenic plants and their offspring with increased or decreased resistance to stress are screened out, creating a new technology in plant breeding. A new germplasm with application value; wherein, the nucleotide sequence of the ZmNF-YA1 gene cDNA is shown in SEQ ID No.1, and the encoded amino acid sequence is shown in SEQ ID No.2.

In the application of the above-mentioned maize nuclear factor gene ZmNF-YA1 in the transformation of plant stress resistance: the ZmNF-YA1 gene has a cDNA form or a genomic gene form, and its coding sequence is inserted into a plant expression in a sense form or an antisense form or an RNAi structural form. The vector is constructed into a fusion gene; the promoter is a stress-inducible promoter or a constitutive promoter.

Wherein: the plant stress resistance includes drought tolerance, salt tolerance or heat tolerance; the plant is corn, wheat or bluegrass.

Expression analysis of ZmNF-YA1 gene

In the research on the mechanism of maize drought tolerance, the drought-tolerant inbred line Q319 and the drought-sensitive inbred line 65232 were used as materials, and the chip hybridization technology was used to compare the drought treatment at the seedling stage (3-leaf maize seedlings in sand culture were irrigated with 18% PEG solution, 12h, 24h, 48h after treatment and 24h after resumption of watering) on the transcriptome of different genotypes, a batch of transcription factors with different trends in different genotypes were selected. Then, the expression intensity of these genes was verified by quantitative RT-PCR technology. ZmNF-YA1/GRMZM2G000686 gene was selected for research. Under suitable growth conditions, the expression abundance of ZmNF-YA1 in roots was significantly higher than that in leaves. In roots, the expression level of drought-tolerant inbred line Q319 was 2.5 times that of drought-sensitive inbred line 65232; in leaves, the expression levels of Q319 and 65232 were similar, about 1/4 of that in roots of 65232. When osmotic stress was treated for 24h, in the roots of drought-tolerant inbred line Q319, the expression abundance decreased, about 70% of that before treatment, while the expression in the roots of 65232 increased significantly and was higher than that of Q319; in leaves , The changes of Q319 and 65232 showed an opposite trend, that is, Q319 increased significantly, and 65232 decreased to a level lower than 1/10 in the root before treatment. After osmotic stress treatment for 48h, the expression abundance of ZmNF-YA1 in roots increased slightly in Q319 compared with 24h, but decreased significantly in 65232, slightly higher than the level before treatment; in leaves, ZmNF-YA4 in Q319 Compared with 24h, the expression abundance of 65232 increased significantly, which was about 4 times of that before treatment, while 65232 only showed a slight increase. After rehydration for 24 h, the transcript abundance of ZmNF-YA1 in roots of 65232 and Q319 was almost equal, slightly higher than the expression level of root 65232 before untreated; The expression abundance of 65232 increased slightly, and the abundance was higher than that of Q319. That is, the expression of ZmNF-YA1 was inhibited by osmotic stress in Q319 roots and induced by osmotic stress in Q319 leaves; whereas it was induced by osmotic stress in 65232 roots and inhibited by osmotic stress in 65232 leaves. That is, their responses to osmotic stress were significantly different, which may be related to their drought resistance.

Production of genetically modified corn

The ZmNF-YA1 gene coding frame was fused with the promoter Prd29A induced by dehydration stress, and recombined into the plant expression vector to construct the vector pCambia1300-Prd29A::ZmNF-YA4-PCaMV35S::bar, using Agrobacterium-mediated method or gene The T-DNA region of the plasmid was transferred into the maize backbone inbred line by gun bombardment, and the transformed seedlings were transplanted and survived, bagged and selfed, and the seeds were harvested. Through herbicide screening and molecular biology detection methods (PCR, Southern blotting, RT-PCR), the offspring of the transformed plants were detected, and the transgenic plants were obtained. At the same time, according to the specific sequence of ZmNF-YA1 gene, RNAi structure was constructed and recombined into plant expression vector to carry out maize genetic transformation to obtain transgenic maize plants. By selfing and molecular characterization for 3 consecutive generations, transgenic homozygous lines were generated. Under normal cultivation conditions, transgenic maize plants had normal morphology, growth and development. The expression intensity of ZmNF-YA1 in the overexpressed lines was significantly higher than that in the non-transgenic plants, while the expression intensity of ZmNF-YA1 in the transgenic lines was significantly lower than that of the non-transgenic plants. .

If using the method of biolistic bombardment, take the immature embryos (1.0-1.5mm in size) of inbred plants of maize (Zea mays L.) 9-15 days after self-pollination, inoculate them on the induction medium, and culture for 4-6 weeks A crisp, pale yellow type II callus was obtained, which was then subcultured every 10-15 days on the subculture medium. The obtained type II callus was used as the recipient material for genetic transformation.

Gene gun pellets were prepared by conventional methods. That is, take 1.0 μm gold powder, wash it with 70% ethanol, let it stand for 15 minutes, and remove the supernatant by centrifugation; then wash it thoroughly with sterile water for 3 times, then 50% sterilized glycerol (the final concentration is 60 mg/ml ammunition) for storage. When in use, vortex for 5 minutes to break the agglutination of gold powder, add 5 μl of plasmid T-DNA (1 μg/μl), 50 μl of 12.5M CaCl ₂ , and 20 μl of 0.1 M spermidine in sequence, and vortex while adding samples. Then, continue vortexing for 2-3 minutes and let stand for 1 minute. After the supernatant was discarded by centrifugation, 70% ethanol was added to stand. The supernatant was then discarded by centrifugation, resuspended in absolute ethanol, and sampled and added to the microprojectile carrier. The dosage of micro bomb is 0.5mg per bomb. A 0.4 cm thick medium was poured into a petri dish with a diameter of 9 cm, and then the callus was placed into the petri dish at a high density and bombarded once per dish. The bombardment parameters are taken as follows: the distance between the splittable disc and the carrier is 2.5cm, the distance between the carrier and the blocking net is 0.8cm, and the flight distance of the microprojectile is 6-9cm. Other parameters are set according to the instruction manual. After bombardment, the material was cultured in the dark for 3 days, and then the material was transferred to a new medium with unchanged composition for 3 weeks, so that the transferred target gene was fully expressed. The material is transferred to medium supplemented with a selection agent (eg, 0.1% of the herbicide glufosinate) for selection. Three consecutive generations were screened for 15 days each. Tissue pieces that became sticky and dead were eliminated during passage. The screened resistant callus was transferred to a medium without screening agent, and after 16 hours of light/day, the culture was resumed for one generation, and then transferred to a differentiation medium to differentiate seedlings. The seedlings produced by the callus are rooted in the rooting medium, and after the seedlings are strong, they are transferred into flowerpots, and when they grow to a height of about 10 cm, they are planted in the field and self-cultivated.

Character Detection and Utilization of Transgenic Maize Plants

For the transgenic homozygous plants obtained by continuous bagging and selfing, it was first determined that the transgenic plants grew and developed normally under normal cultivation conditions, and then the resistance differences between the transgenic plants and the control plants under stress conditions were analyzed.

Heat tolerance test: Transgenic homozygous maize plants grown at 28°C (light, 13h/d)/22°C (dark, 11h/d) were moved to 36°C (light) for 2h, and then at 39°C. Continuous heat treatment for 4 days (light 13h/d, dark 11h/d), and then resume growth at 28°C. After heat treatment, the non-transgenic control (wt) almost completely died. Compared with the non-transgenic plants, the heat resistance of bluegrass pla of.

Cold tolerance test: Transgenic homozygous maize plants grown at 28°C (light, 13h/d)/22°C (dark, 11h/d) were placed at 16°C (light for 13h/d) and 10°C (light for 13h). /d) for 1 day, followed by continuous cold treatment at 4°C for 3 days (illumination for 13 h/d), and then resumed growth at 28°C. In the cold treatment, the non-transgenic control (wt) grew slowly, which was significantly smaller than that of the transgenic line after treatment; at 4°C for 1 day, a few transgenic overexpression plants and non-transgenic control had damaged leaves, and the leaf tips tended to wilt, while the transgenic RNAi The structural maize plants were sensitive to chilling injury and were severely damaged; after 3 days of treatment at 4°C, growth was resumed for 1 day, and the damage degree of the transgenic overexpression lines was significantly lower than that of the control. The plants were severely damaged, and almost all the seedlings died.

Drought tolerance experiment: Mature seeds of homozygous lines transfected with ZmNF-YA1 gene and its RNAi structure or antisense form were taken after being fully dried and sown in soil trays and grown under suitable conditions. Watering was stopped at the 4-leaf stage of the plants, treated with drought for one week, and then watered again, and the growth status and survival rate of the plants were observed to determine the drought resistance of the plants. The results showed that the reduction of ZmNF-YA1 gene expression at seedling or jointing stage reduced the drought resistance of plants, while the overexpression of ZmNF-YA1 gene significantly improved the drought resistance of plants.

The seeds of the transgenic ZmNF-YA1 gene and its RNAi structure or antisense form of maize and non-transgenic control inbred lines were sown in flowerpots and fields. Drought stress treatment was carried out during the pollination and grain filling stages, that is, by controlling watering and avoiding rain, the relative soil moisture content was maintained at about 55-60% for 15 days, and the traits of transgenic plants and the detection of physiological indicators were carried out. Pollinate, harvest the ears and test the seeds. It was found that the drought resistance of the transgenic ZmNF-YA1 overexpression lines was significantly higher than that of the non-transgenic control and the transgenic RNAi structure or antisense form. Not only did the plant suffer light symptoms, and the growth recovered quickly after the stress was relieved, but also the economic traits such as grain yield per plant were significant. better than the non-transgenic control and trans-RNAi structure; and the plant drought resistance and grain yield of the trans-RNAi structure or antisense form were significantly worse than those of the non-transgenic control. See Example 3 and Example 4 for the resistance detection and plant screening of transgenic bluegrass plants.

Through the resistance detection test, the drought tolerance, heat tolerance and cold tolerance of the transgenic overexpression plants promoted by the stress-inducible promoter were significantly improved compared with the non-transgenic control. On this basis, based on various test results, excellent transgenic plants were selected to be homozygous for bagging and self-crossing, and the combining ability was determined. In maize single-cross breeding.

Detailed ways

Example 1: Transgenic ZmNF-YA1 gene to create a drought-tolerant and salt-tolerant inbred line of maize

1) The establishment of the receptor system is based on the backbone inbred lines used in agricultural production in my country, such as Zheng 58, Chang 7-2, DH4866 and so on. The seeds are soaked in 70% ethanol for 8 minutes, then soaked in 0.1% mercuric chloride for 8-12 minutes, and then washed with sterile water 3-5 times. Shake the seeds continuously during sterilization to ensure complete surface sterilization. After sterilization, the seeds were germinated in a sterile triangular flask, and a small amount of sterile water was placed in the flask for 1-2 days under dark conditions (25-28°C). After the seeds germinated (whitened), they were germinated on modified MS medium in the dark. When the embryo elongates only 3-4 cm, peel off the coleoptile and 2-3 young leaves, exposing the growth cone at the top of the shoot tip.

2) Corn shoot tip transformation and plant regeneration to construct a fusion gene (sense form) carrying the full sequence of the maize nuclear factor gene ZmNF-YA1, transgenic ZmNF-YA1 using the stress-inducible promoter RD29A/B or CaMV35RNA promoter or maize Ubiquitin promoter start up. The fusion gene was then recombined into the Mini-Ti plasmid of Agrobacterium tumefaciens with the plant herbicide resistance gene bar in the T-DNA region to obtain a genetic transformation vector.

Agrobacterium tumefaciens (such as LBA4404, etc.) with binary vector (Mini--Ti plasmid with selection agent resistance gene and ZmNF-YA1 gene) was added to antibiotic-supplemented LB medium (each liter of medium contained: trypsinization 10 g of peptone, 5 g of yeast extract, 10 g of NaCl, pH 7.0, autoclaving) were shaken and cultured at 28°C, and the shaking rate was 110 rpm (revolution/min), so that the bacteria were in the logarithmic growth phase. It was then centrifuged at 3000 rpm for 10 minutes and the supernatant was discarded. The bacteria were washed with 1/2 concentration of liquid seed germination medium (that is, the composition of seed germination medium was halved, and the agar powder was removed), and then collected by centrifugation. The cells were then suspended in a 1/2 concentration liquid modified MS medium supplemented with 100 μmol/l of acetosyringone (As), and diluted 5--20 times for transformation. During transformation, pour the bacterial solution into a petri dish with a diameter of 4.5 cm, and tilt the petri dish to soak the sterile seedlings with exposed shoot apex growth cones in the bacterial solution. The soaked bud tips were then blotted dry with sterile filter paper, and the germinated seeds were cultured on the modified MS medium for 2-3 days in the dark at a temperature of 22-24°C. The sterile shoots were then incubated under scattered light for 2 days. The sterile seedlings after light culture were transplanted into flower pots covered with upper layer of vermiculite and lower layer of loam, and the tops of the plants were covered with vermiculite. The plants were then grown under natural light, with a day temperature of 22-28°C and a night temperature of 15-21°C, and watered with 1/2 of the modified MS medium inorganic salts the next day.

3) Resistance detection and selection of transgenic plants

After the transformed plants grow 3 leaves, spray 0.12% herbicide glufosinate aqueous solution, and it is advisable to drop droplets from the plants. Untransformed control plants stopped growing 4 days after spraying and began to die 9 days later. After the transformed plants were sprayed, some individual changes were similar to those of the control plants, while others continued to grow without obvious changes. When the surviving plants grew to 5 leaves, they were colonized in the field, and they were bagged and self-crossed to produce seeds. The leaves of the transplanted surviving plants were taken for molecular biological detection to determine the transgenic plants. The transgenic plants (T0) were then bagged and selfed. T1 seeds from different TO plants were sown in greenhouses or fields with protective facilities, sprayed with 0.18% aqueous solution of herbicide glufosinate, and plant resistance was observed. The herbicide-resistant plants screened in the T1 generation continued to be bagged and selfed, and their progeny continued to be identified by molecular biology and tested for resistance. Through several generations of selfing homozygosity and resistance detection and selection, the transgenic maize homozygous line was finally obtained. In the identification and selection of drought resistance, transgenic lines were planted in flowerpots, greenhouses and fields, respectively, and subjected to drought stress treatment at the 3-leaf stage, jointing stage and pre-flowering stage, tasseling and powder-scattering stage, and grain-filling stage, and the changes in physiological parameters were determined. Grain yield, strains with strong drought resistance, significantly increased grain yield compared with non-transgenic control materials, and slightly increased grain yield of inbred lines with the recipient under suitable cultivation conditions were screened out. This line can be used to formulate corn drought-resistant herbicide-resistant hybrids.

Seeds of homozygous transgenic lines were sown in pots filled with sand, watered with 0.5% or 0.7% NaCl aqueous solution, and 1/3 MS medium inorganic salt solution supplemented with 0.5% or 0.7% NaCl was watered after emergence. The emergence rate, leaf necrosis degree, and plant survival rate at the 5-leaf stage were counted, and transgenic lines with excellent salt tolerance were selected for inbred line selection and new variety cultivation.

Example 2: Transgenic ZmNF-YA1 gene to create heat-resistant inbred line of maize

1. Acquisition of maize transgenic plants

(Hoechst Schering AgrEvo GmbH,含有除草剂glufosinateammonium)水溶液，浓度为9.6ml—10.8ml

以植株掉液滴为宜。未转化对照植株在喷洒后4天后停止生长，9天后开始死亡。转化植株在喷洒后，一些个体变化同对照植株相似，另一些个体持续生长，变化不明显。待存活植株长到5叶时，将其定植到田间，套袋自交结籽。Seed germination, plasmid construction and genetic transformation of an elite inbred line of maize are the same as those in Example 1. After the transformed plants grow 3 leaves, spray the herbicide

(Hoechst Schering AgrEvo GmbH, containing the herbicide glufosinateammonium) aqueous solution, the concentration is 9.6ml-10.8ml

It is advisable to drop the droplets from the plant. Untransformed control plants stopped growing 4 days after spraying and began to die 9 days later. After the transformed plants were sprayed, some individual changes were similar to those of the control plants, while others continued to grow without obvious changes. When the surviving plants grew to 5 leaves, they were colonized in the field, and they were bagged and self-crossed to produce seeds.

2. Homozygous and progeny analysis of transgenic plants

水溶液处理，观察统计抗性和敏感性个体比例；采用PCR技术检测外源基因，并统计外源基因在子代植株中的分离比例。存活植株移栽到大田，套袋自交。T2代植株除套袋自交结籽外，采用PCR技术检测外源基因进行Southernblotting验证，并采用RT-PCR技术检查转基因表达强度。对于入选的转基因株系测定植株在不同温度(28～39℃)和光强下净光合速率的变化，测定单株产量和生物量，并以未转基因植株为对照。选出优良的转基因株系后在大田栽培条件下进行玉米产量性状的观测和比较，选择高产高光效株系进入耐热性鉴定试验和玉米育种实验。T1 generation plants grow to 3-leaf stage with 10.8ml

Treated with aqueous solution, the proportion of resistant and sensitive individuals was observed and counted; PCR technology was used to detect exogenous genes, and the proportion of exogenous genes segregated in progeny plants was counted. The surviving plants were transplanted to the field, and selfed by bagging. In addition to bagging and self-crossing of T2 generation plants, PCR technology was used to detect foreign genes for Southern blotting verification, and RT-PCR technology was used to check the intensity of transgene expression. For the selected transgenic lines, the changes of the net photosynthetic rate of the plants at different temperatures (28-39 °C) and light intensities were determined, and the yield and biomass per plant were determined, and the non-transgenic plants were used as the control. After selecting excellent transgenic lines, observation and comparison of maize yield traits were carried out under field cultivation conditions, and high-yield and high-light-efficiency lines were selected for heat tolerance identification experiments and maize breeding experiments.

3. Heat resistance identification test of transgenic lines

Seedling stage heat tolerance test: Transgenic homozygous maize plants grown at 28°C (light, 13h/d)/22°C (dark, 11h/d) were moved to 36°C (light) for 2h growth, and then incubated at 39°C. Continuous heat treatment at ℃ for 4 days (light 13h/d, dark 11h/d), and then resume growth at 28℃. After heat treatment, almost all of the non-transgenic control (wt) died, and only a few of the transgenic lines had little difference with the non-transgenic control, while the heat tolerance of most lines was significantly better than that of the control, and some of them were transgenic Lines with no obvious damage are the preliminarily selected transgenic heat-resistant lines.

Heat-resistance test at the initial stage of pollination and grain-filling: The seeds of the preliminarily selected transgenic heat-resistance lines were sown in the greenhouse and grown under suitable growth conditions until the pollination stage, and then the greenhouse temperature was adjusted to 36°C (light, 13h/d)/30°C ℃ (dark, 11h/d) for 7 days, then adjust the temperature to 38℃ (light, 13h/d)/30℃ (dark, 11h/d) for 7 days, and count the number of green leaves and ears after 20 days . After harvesting, parameters such as ear development, seed setting rate, and 100-grain weight were observed, and transgenic lines with excellent heat tolerance were screened.

Example 3: Transgenic ZmNF-YA1 gene to create drought-tolerant and salt-tolerant bluegrass

Using different varieties of Poa pratensis L, such as prizes, new song Laid, Rugby-A, Midnight, etc., the shoot tips of seedlings were used as test materials to induce callus expansion at the base, and the expanded tissues were cut and placed in the subculture. Induced clump shoots on subculture and clump media, the latter continued to proliferate on subculture and clump media, providing transgenic recipient material. The exogenous gene was introduced into recipient cells by Agrobacterium-mediated method, and transformed cells and plants were obtained by selection. In the genetic transformation mediated by Agrobacterium, the use of surfactant Silwet L-77 treatment and reduced pressure treatment effectively increased the transformation frequency, and established an efficient genotype-constrained small bluegrass transgenic technology system. The specific operations are as follows.

Prepare various media

Basic medium: modified MS medium (MS medium inorganic salts, 10.0 mg L ^-1 of thiamine hydrochloride, 1.0 mg L ^-1 of pyridoxine hydrochloride, 1.0 mg L ^-1 of niacin, 2.0 mg L ^-1 of glycine , inositol 100.0mg L ^-1 , biotin 0.05mg L ^-1 , casein hydrolyzate 500mg L ^-1 ), sucrose 30g L ^-1 , agar powder 6.5g L ^-1 , pH 5.8--6.0. for seed germination. For liquid medium, remove the agar powder.

Induction medium: minimal medium supplemented with a combination of different plant growth regulating substances (hormones). For most bluegrass genotypes, 2,4-D concentrations were 0.01-1.0 mg.L ^-1 , 6-benzylpurine (6-BA) or kinetin or zeatin concentrations were 0-5 mg.L ^-1 . Use solid medium. The optimum hormone concentration varies within this range depending on the genotype of the culture material.

Subculture and clumping medium: solid medium, the basic medium is added with different combinations of plant growth regulators, the concentration of 2,4-D is generally 0-0.2mg L ^-1 , 6-BA or kinetin or zeatin The concentration is 0-3 mg L ^-1 , which varies according to the bluegrass genotype.

Rooting medium: basic medium plus 0.002-2.0mg L ^- 1 naphthalene acetic acid (NAA) or indole butyric acid (IBA) or indole acetic acid (IAA), using solid medium for rooting or strong seedlings of rootless seedlings nourish. The optimum hormone concentration varies within this range depending on the genotype of the culture material.

Seed sterilization and germination and induction of clumps

The seeds are sterilized with 70% alcohol for 4-5 minutes, 0.2% mercuric chloride for 10-15 minutes, and rinsed 3-5 times with sterile water. Shake the seeds continuously during sterilization to ensure complete surface sterilization. After sterilization, the seeds were sown on moist sterile filter paper, and the Petri dishes were sealed and placed in the dark or low light for germination at 20-28°C. The shoot tips were excised between 10 and 15 days and cultured on induction medium (adding 2,4-D 0.2 mg L ^-1 , 6-BA 2 mg L ^-1 ) to induce basal swelling, and the swelling rate was 100%. After culturing on the induction medium for 20 days, the swollen tissue at the base of the shoot tip (1-3mm in diameter) was excised and transferred to the subculture and clumping medium (2,4-D 0.1 mg L ^-1 , 6-BA 2 mg L ^{- 1} ) Induce the formation of cluster buds. After 15-20 days, most of the swollen bases degenerate into cluster buds, the clustering rate is about 75%, and the number of buds per cluster is mostly 15-20. Divide the clump buds and transfer them to the subculture and clump-forming medium, and the clump bud cells proliferate rapidly, generally subculture once every 15 days. To obtain larger seedlings, remove 2,4-D from the subculture and clump medium, and keep 2 mg of L-16-BA. Complete plants can be regenerated by culturing isolated shoot apex and shoot basal swellings and clump buds at 24±2°C under 500-1000Lx light (14h/d).

Genetic transformation of bluegrass

ZmNF-YA1 gene cloning, fusion gene construction and transformation plasmid recombination are the same as Example 1. Agrobacterium tumefaciens (such as AGL0 and LBA4404) with binary vector (Mini-Ti plasmid with plant selection marker gene) was added to LB with antibiotics Culture medium (each liter of medium contains: tryptone 10g, yeast extract 5g, NaCl 10g, pH 7.0, autoclave sterilization) under shaking at 28 ° C, the shaking rate is 170-180rpm (revolution/min), so that Bacteria were in logarithmic growth phase (OD ₆₀₀ =0.4-0.6). It was then centrifuged at 3000 rpm for 10 minutes and the supernatant was discarded. Bacteria were washed with 1/2 concentration of liquid minimal medium, and then collected by centrifugation. Then suspend the cells with 1/2 concentration liquid basic medium, dilute 5-20 times, add acetosyringone (As) 100 μmol L ^-1 and surfactant Silwet L-77 (concentration is 0.1-0.3%) ) is used for conversion.

The bluegrass bushy buds were cut to expose the growth point of the bud tip, put into the above bacterial solution for infiltration and treated with 0.95-0.10 atmospheric pressure (0.95×10 ⁵ Pa--1×10 ⁴ Pa) for 4-8 minutes. Then use sterile filter paper to absorb the bacterial liquid, transfer the clump buds to subculture and clump medium for 2-3 days, and then transfer to the antibiotic Cefotaxime 250mgL ^-1 or carbenicillin (Carb) 500 mgL ^-1 medium was cultured in the dark to inhibit bacterial growth. On the antibacterial medium, the clumps of buds gradually recovered, and after 10-15 days, they were transferred to the screening medium containing the selection agent for screening. Screening was performed for 3 consecutive generations, 15-20 days per generation. Selection agent resistance gene bar or antibiotic resistance gene hpt (hygrin phosphotransferase gene). The resistant tissue pieces were then transferred to subculture and clump medium to differentiate seedlings. The latter grow roots on rooting medium. Rooted seedlings were moved into pots and watered every 5 days with an inorganic salt solution of 1/2 of the basic medium and every other day. The leaves of the transplanted surviving seedlings were taken for molecular biology detection. Through PCR and Southern blot analysis, the transformation rate (the number of sprouts producing transgenic plants/the number of sprouts infected with Agrobacterium*100) was about 2-5%. Induced basal enlargement is more sensitive to light. When the concentration of 2,4-D is low (<0.1mg.L-1) and the light intensity is >1000Lx, it is easy to differentiate and root, but not easy to differentiate into clumps. When the light is weak (500Lx), it is easy to expand and take root. At a more suitable 2,4-D concentration, the light intensity is about 1000Lx, the growth is good (the swollen tissue is yellowish green), and it is easier to form clumps than at 500Lx (the swollen tissue tends to be white), and the number of buds per clump is also more. . When the concentration of 2,4-D is high, the effect of light intensity is not great, the swelling is large, and it is not easy to take root, but the clump formation rate is low. At this stage, it should be cultivated under strong light at a suitable concentration of 2,4-D to facilitate the formation of clumps later. In the first 5-8 days of subculture of clump bud blocks, if the light intensity is >1000Lx, the tissue is easy to take root, which is not conducive to the differentiation of buds. Therefore, in the first 5-8 days after subculture, the clump buds are cultivated under low light (about 500Lx), and when several small buds grow, they are cultivated under the light intensity > 1000Lx, and the number of buds per cluster can reach 15 -25.

Determination of Screening Agent Concentration

After the herbicide aqueous solution was sterilized by filtration, it was added to the induction medium from which the casein hydrolyzate was removed (when the temperature of the medium was lower than 50°C). The concentrations of glufosinate were 0.1%, 0.15%, 0.2% and 0.25%, namely four gradient concentrations. The induced clump buds are cut into single buds and then cultured on a medium containing different concentrations of chlorsulfuron, and more than 100 seedlings are inoculated on each selective medium. Subculture was performed once every 15 days, and three generations were continuously screened. According to the survival rate after screening, the appropriate screening doses of herbicides for different varieties of seedlings were determined. For example, the selection concentration of glufosinate for the variety prize is 0.2%, and the seedling survival rate is more than 10% under this concentration of continuous screening for 3 generations. Survival seedlings transgenic PCR test is generally positive.

Transplantation of seedlings and selection of transgenic plants

Plantlets are induced to root on a suitable rooting medium. When the roots are 2-3cm long, put the seedlings under natural light for 1-2 days, then remove the sealing film and harden the seedlings for 1-2 days, and transplant them into flowerpots in the morning or evening (loam under the flowerpot and leeches above). Stone), water 1/2 basic medium inorganic salt solution once every 5 days, and water once every 3 days. The growth temperature of transplanted seedlings is 15-25 ℃. When the seedlings grow to 4-5 tillers, they should be irrigated with enough water, and the drought will last for more than 1 month. After the leaves of the plants are all wilted for a week, the watering will be resumed. The plants that recover faster after rewatering are selected for cloning propagation. Some cloned seedlings are transplanted into flowerpots with loam soil, and when the plants grow to about 5 tillers, over-water 0.3%-0.5% NaCl aqueous solution, and water them continuously for 3 days to make the soil salt content reach the set concentration (not high). In 0.5% NaCl), and then maintained for 1-3 months, the surviving plants were selected and cloned. At the same time, the cloned seedlings of transgenic plants were screened repeatedly by spraying the aqueous solution of the herbicide glufosinate with constant concentration. Space isolation or bagging of the lines with excellent glufosinate tolerance to salt and drought tolerance is carried out, so that they can set seeds under isolation conditions.

The harvested transgenic plant seeds were sown in flowerpots, and the herbicide glufosinate at a concentration of 0.25% was sprayed at the 4-5 leaf stage. The surviving plants were tested for salt tolerance and drought tolerance again, and excellent resistant plants were screened out. Salt and drought tolerance transgenic stable line, which can be used for environmental greening after propagation.

Example 4: Transgenic ZmNF-YA1 RNAi structure or antisense form to create heat-tolerant bluegrass

The ZmNF-YA1 antisense form or RNAi structure was fused with the promoter Prd29A induced by dehydration stress, and then recombined into a plant expression vector to transform Bluegrass, and a transgenic plant was obtained. After the latter is transplanted and survived, it grows to 5-6 tillering stages under natural conditions, and is sprayed with 0.25% glufosinate solution. The surviving plants are propagated by asexual cloning, and the transgenic molecules are detected. The obtained transgenic bluegrass plants were molecularly identified and homozygous for 2 consecutive generations, resulting in transgenic homozygous lines. Using transgenic plants as materials, heat and cold stress treatments were carried out respectively, and it was clear that there were differences in stress resistance between transgenic plants and control plants. The heat treatment method is as follows.

Transgenic bluegrass plants produced a large number of tillers after 2 months of transplanting. Select neat and consistent transgenic bluegrass plants and transplant them into flowerpots and paper cups. After the plants have grown for 2 months, they are cut, and the heat resistance is measured when the new leaves grow to about 10 cm. During processing, the materials were put into an artificial climate box, the photoperiod was controlled to be 12h/d, and the relative humidity was 70%. During the heat stress treatment at 40℃, the temperature is 30℃(2h)→32℃(2h)→34℃(2h)→36℃(2h)→38℃(2h)→40℃(3d)→23℃(3d) Gradient change. Under heat stress at 46℃, press 30℃(2h)→32℃(2h)→34℃(2h)→36℃(2h)→38℃(2h)→40℃(3h)→46℃(5h)→ 23°C (7d) gradient change. During the treatment period, the growth status of the materials was observed, including the degree of wilting, survival rate, leaf color changes, and differences between lines. At the same time, relevant physiological parameters, including chlorophyll content, membrane damage, and chlorophyll fluorescence, were measured before treatment, at 40°C for 2h, 12h, 3d, after recovery at 3d, at 46°C for 5h, and at 10d. Physiological Experiment Guide" (edited by Tang Zhangcheng). Fv/Fm values were measured after half an hour of dark adaptation.

In the case of short-term heat stress treatment at 46°C, different bluegrass plants were treated at 46°C for 5 hours and then placed at 23°C for recovery. The growth of plants in different treatment periods was observed, and relevant physiological indicators were measured, such as chlorophyll content, membrane damage, and chlorophyll fluorescence. changes, etc. After treatment at 46°C, the reduced ZmNF-YA1 gene expression line was better than the control line. Although all plants survived at 23°C for 10 days, the growth vigor was very different. Non-transgenic plants have poor growth vigor, severe heat stress damage, and slow recovery; reduced ZmNF-YA1 gene expression plants recover quickly, with more tillers, strong growth, and long new leaves, showing that they are less damaged in heat stress and have good performance. of heat resistance. During the heat treatment, the leaf Fv/Fm showed a decreasing trend and gradually increased during the recovery period. The content of malondialdehyde in different Poa annua lines increased during the first 2 days of heat treatment and recovery, and the non-transgenic control was significantly higher than that of the plants with reduced ZmNF-YA1 gene expression, indicating that the latter had a lower degree of cell membrane lipid peroxidation. During the heat stress treatment and recovery period, the ion extravasation rate of the non-transgenic plants was significantly higher than that of the line with reduced ZmNF-YA1 gene expression, and the membrane damage showed a maximum value at 2 days of recovery, and the latter had a significant increase in the heat stress treatment and recovery period. The plasma membrane stability was higher than that of the control, indicating that reducing ZmNF-YA1 gene expression significantly improved the heat tolerance of plants.

In the long-term heat stress treatment at 40°C, after 1 day of treatment, the plants wilted slightly, and there was little difference between them; after 3 days of treatment, the degree of wilting of different lines showed significant differences, and the non-transgenic plants wilted more heavily, reducing ZmNF. The -YA1 gene expressing line has significantly improved heat tolerance, straight leaves and less water loss. During the recovery treatment, all the non-transgenic controls died, and the lines with reduced ZmNF-YA1 gene expression had good heat resistance, and the survival rate of some lines was higher than 70%. The damage to plants caused by stress treatment at 40°C for 3 days was much greater than that caused by stress treatment at 46°C for 5 hours, that is, prolonged heat stress treatment would cause damage to bluegrass cells that was difficult to repair.

The lines with significantly improved heat resistance compared with the control were cloned and propagated, and 0.2% herbicide glufosinate solution (the dose was 3 times that of field weeding) was sprayed to eliminate plants with weak resistance. The rest of the plants were isolated and pollinated, and seeds were mixed according to the strains for the breeding of new varieties.

Different transgenic lines from the same variety also showed significant differences in the degree of heat tolerance, which may be related to the difference in the integration site and expression intensity of foreign genes in transgenic plants. Judging from the analysis of the recovery rate, survival rate of plants after stress treatment and the measured values of physiological indicators during stress treatment and recovery process, short-term heat stress treatment at 46°C was relatively suitable for the primary selection of materials, with a short period and better screening effect. Under heat stress at 40°C for 3 days or more, transgenic lines with excellent heat tolerance can be effectively screened, and the latter can be directly used for the breeding of new heat-tolerant bluegrass varieties.

sequence listing

<110> Shandong University

<120> Application of a maize nuclear factor gene ZmNF-YA1 in the transformation of plant stress resistance

<141> 2018-1-29

<160> 2

<170> PatentIn Version 3.5

<210> 1

<211> 1270

<212> cDNA

<213> Artificial sequences

<220>

<221> Corn

<222>(1)…(1270)

<223> Nucleotide sequence of ZmNF-YA1 gene cDNA

<400> 1

agagatagga aagggcccaa cagctcaaca gaaaagccaa gcaaaggctg ctgcatactg 60

gaaggccctc tgtctgtgtg cgagcgcaag agaaagggag tcagagagag agagagaggg 120

aggagacctt gcagaggagc gaagcaagca aggtgggaaa gaggcagcag caagggcggc 180

gggctgccgg aaggggaaca tgctccctcc tcatctcaca gagaatggcg cggtgatgat 240

tcagtttggc catcagatgc ctgattacga ctccccggct acccagtcaa ccagtgagac 300

gagccatcaa gaagcgtctg gaatgagcga agggagcctc aacgagcata ataatgacca 360

ttcaggcaac cttgatgggt actcgaagag tgacgaaaac aagatgatgt cagcgttatc 420

cctgggcaat ccggaaacag cttacgcaca taatccgaag cctgaccgta ctcagtcctt 480

cgccatatca tacccatatg ccgatccata ctacggtggc gcggtggcag cagcttatgg 540

cccgcatgct atcatgcacc ctcagctggt tggcatggtt ccgtcctctc gagtgccact 600

gccgatcgag ccagccgctg aagagcccat ctatgtcaac gcgaagcagt accacgctat 660

tctccggagg agacagctcc gtgcaaagct agaggcggaa aacaagctcg tgaaaagccg 720

caagccgtac ctccacgagt ctcggcacct gcacgcgatg aagagagctc ggggaacagg 780

cgggcggttc ctgaacacga agcagcagcc ggagtccccc ggcagcggcg gctcctcgga 840

cgcgcaacgc gtgcccgcga ccgcgagcgg cggcctgttc acgaagcatg agcacagcct 900

gccgcccggc ggtcgccacc actatcacgc gagagggggc ggtgagtagg gagccccgac 960

actggcaact catccttggc ttatcagcga ttcgactcgg ctctccctcg tctgaaactg 1020

aactctctgc aactactgta actgtaacta aactgggtgt gcccggattg gcggtcgttc 1080

tgttctacta ctagtacctg ctacgcgtcg ttgggttggg tctggactag agagcgtgct 1140

ggttctttga tgaacttggc tggacttgag ggtgttgact agcgcgaagc tgagttccat 1200

gtaaaacttt tgcttcaaga ccgatgactg gcggcataat aagtagcagt aataaccatt 1260

cttctgtgtc 1270

<210> 2

<211> 249

<212> PRT

<213> Artificial sequences

<220>

<221> Corn

<222>(1)…(249)

<223> Amino acid sequence encoded by ZmNF-YA1 gene

<400> 2

MLPPHLTENG AVMIQFGHQM PDYDSPATQS TSETSHQEAS GMSEGSLNEH NNDHSGNLDG 60

YSKSDENKMM SALSLGNPET AYAHNPKPDR TQSFAISYPY ADPYYGGAVA AAYGPHAIMH 120

PQLVGMVPSS RVPLPIEPAA EEPIYVNAKQ YHAILRRRQL RAKLEAENKL VKSRKPYLHE 180

SRHLHAMKRA RGTGGRFLNT KQQPESPGSG GSSDAQRVPA TASGGLFTKH EHSLPPGRH 240

HYHARGGGE 249

Claims (2)

1. The application of the over-expression corn nuclear factor gene ZmNF-YA1 in cultivating drought-tolerant corn or bluegrass; wherein, the nucleotide sequence of the ZmNF-YA1 gene cDNA is shown as SEQ ID No.1, and the coded amino acid sequence thereof is shown as SEQ ID No. 2.

2. The application of reducing the expression of a corn nuclear factor gene ZmNF-YA1 in cultivating heat-resistant meadow bluegrass; wherein, the nucleotide sequence of the ZmNF-YA1 gene cDNA is shown as SEQ ID No.1, and the coded amino acid sequence thereof is shown as SEQ ID No. 2.