CN101948861B - Methods for cultivating male sterile line and restorer thereof in plant - Google Patents

Abstract

The invention discloses a method for cultivating plant male sterile lines and restorer lines thereof. In this method, a recombinant expression vector containing an expression cassette is introduced into the target plant to obtain the male sterile plant; the expression cassette includes in turn from upstream to downstream: loxp site, anther or stamen organ primordia-specific promoter , the reverse complement gene of the gene encoding the ethylene receptor ETR1 protein, the terminator and the loxp site. The present invention also constructs the restorer line of the above-mentioned sterile line, which is to introduce the Cre gene into the above-mentioned target plant to obtain the restorer line. Experiments have proved that after crossing the restorer line and the sterile line, the fertility of the F1 generation can be restored, thereby creating a new, convenient and feasible male sterility and heterosis utilization with independent intellectual property rights (sterile line creation method) technical system. This invention converts the discovery of basic research into the application field, and provides a new perspective and experience for exploring the application potential of heterosis and future precision breeding.

Description

Breeding method of plant male sterile line and restorer line thereof

technical field

The invention relates to a method for cultivating plant male sterile lines and restorer lines thereof.

Background technique

Since the 1970s, the utilization of heterosis has made a significant contribution to rice production in my country. Facing the new and important demand of "high yield, high quality, high efficiency, safety, and ecology" put forward by my country's economic development for agricultural production, whether to further explore the application potential of heterosis has become a severe challenge for contemporary scientists.

The formation of heterosis is based on the combination of two different parents. To further explore its potential on the basis of existing applications, in addition to strengthening the research on the formation mechanism of heterosis, it is also urgent to establish effective methods to create male sterility traits, so as to effectively expand the screening of hybrid combinations and the production of excellent combinations. on the application.

At present, most of the male sterility traits widely used in breeding work and production come from natural mutations and their trait-transferred lines. The source of male sterility traits is very limited, which is a serious constraint factor for screening and especially the application of expanded hybrid combinations. Although American scientists have proved in the early 1990s that artificially controlled male sterility traits can be created through biotechnology methods, according to current international rules, all innovations with potential applications are protected by intellectual property rights. Therefore, finding new ideas and methods for creating artificially controlled male sterility traits with independent intellectual property rights has become one of the unavoidable and urgently needed key problems faced by countries and regions that want to take the initiative in exploring the application potential of heterosis. one.

For a long time, people have been using conventional breeding methods to select sterile lines, or transfer sterility from one variety to another. These methods have a long period of time and slow results, and cannot meet the urgent needs of production development. At present, many genes that control male sterility of plants have been cloned, but because plant sterility is affected by nuclear genes, mitochondrial genes, chloroplast genes and environmental factors, the mechanism of action is relatively complicated, and people's understanding of it is still insufficient, so It is difficult to obtain obvious results in a short period of time. Compared with male sterile breeding by conventional methods, genetic engineering male sterile has some characteristics: the breeding cycle is shortened, the fertility is relatively stable, less affected by the environment, less dependent on genotype, Environmental pollution is less. Mariani et al. in 1990 pioneered a new approach to create male sterility in plants. They used the anther tapetum-specific promoter (TA29) and ribonuclease gene (Barnase) from tobacco to transform tobacco and rape, and the anther tapetum of transgenic tobacco and rape was destroyed due to the action of ribonuclease , forming pollen abortion, while other organs of the transgenic tobacco developed normally. In addition to the Barnase gene, many other functional genes have been applied to the research of genetic engineering male sterility.

Contents of the invention

The object of the present invention is to provide a method for cultivating male sterile plants.

The method for cultivating male sterile plants provided by the present invention is to introduce the recombinant expression vector containing the following expression cassettes into the target plant to obtain the male sterile plants: from upstream to downstream, including: loxp site, anther or stamen Organ primordia-specific promoter, reverse complement of gene encoding ethylene receptor ETR1 protein, terminator and loxp site.

Further, in the above expression cassette, the nucleotide sequence of the loxp site is shown in the 7th-40th position of sequence 3 in the sequence listing, and the nucleotide sequence of the anther or stamen organ primordia-specific promoter is shown in sequence 1 in the sequence listing As shown, the nucleotide sequence of the gene encoding the ethylene receptor ETR1 protein is shown in Sequence 2 in the sequence listing.

Further, the construction method of the above-mentioned recombinant expression vector is:

The reverse complementary gene of the gene encoding the promoter and the ethylene receptor ETR1 protein is inserted between the multiple cloning sites of an intermediate vector (specifically: the promoter is inserted between the EcoRI and SacI sites; the ethylene receptor ETR1 protein The reverse complementary gene of the coding gene is inserted between the BglII and SpeI sites) to obtain the recombinant expression vector;

The intermediate vector is to insert the fragment shown in sequence 3 in the sequence listing between the multiple cloning sites of the pCAMB2300 plasmid (such as between the pvsI sta and the SmaI restriction site), to obtain the intermediate vector.

The above-mentioned target plants are dicotyledonous plants or monocotyledonous plants, preferably Arabidopsis thaliana.

Another object of the present invention is to provide a method for cultivating a restorer line of the sterile line obtained by the above method.

The method for cultivating the above-mentioned restorer line provided by the present invention is to introduce the Cre gene into the above-mentioned target plant to obtain the restorer line. The nucleotide sequence of the Cre gene is shown in sequence 4 in the sequence listing.

Specifically, the above-mentioned Cre gene is introduced into plants through an expression vector: the expression vector inserts the Cre gene fragment shown in sequence 4 in the sequence listing between the multiple cloning sites of pCAMBIA2300 (such as XbaI and BamHI restriction sites between dots) to obtain the expression vector.

The present invention uses the CRE/loxP site-specific recombination system to connect the antisense ETR1 mediated by the organ-specific promoter AP3 at both ends of the loxP site, and successfully constructs a transgenic Arabidopsis male sterile line; the constructed male sterile line with The Arabidopsis restorer line of the CRE gene mediated by the 35S promoter was crossed with the above-mentioned transgenic male sterile line to delete the sterility gene in the Arabidopsis F1 generation and restore the fertility of the Arabidopsis F1 generation (Table 4).

The present invention observes the morphology of the floral organs of the transgenic Arabidopsis thaliana male sterile line, examines the pollen with Alexander staining microscope, and the pollen germination experiment finds that the filaments of the genetically modified Arabidopsis male sterile male sterile line are obviously shortened, the anthers are shrunk, the fertile pollen is greatly reduced, and the pollen cannot be produced. Normal germination (the germination rate is only 2.67%), the seed setting rate is significantly lower than that of the control plants, and the fertility is shown as semi-sterility or total sterility. The F1 generation showed fertility recovery.

In this way, using the CRE/loxP system, we can artificially construct male sterile lines and restorer lines, forming a method of using male sterility to create new germplasm resources.

The present invention converts the discovery of basic research (down-regulation of organ-specific ETR1 expression can lead to male sterility) into a new idea and new method with source innovation in the field of application, and accumulates new ideas for exploring the application potential of heterosis and future precision breeding. experience of.

Description of drawings

Figure 1 is the electropherogram of the AP3 promoter; M is Marker 15000; 1: the AP3 promoter fragment amplified using the total genomic DNA of wild-type Arabidopsis thaliana as a template.

Figure 2 shows the acquisition of aETR1 gene (abbreviated as ETR1), M: Marker 2000plus; 1: ETR1 fragment amplified using cDNA after reverse transcription of wild-type Arabidopsis total RNA as a template.

Figure 3 is the verification of the intermediate vector loxP-MCS-loxP (pCAMBIA2300), M: Marker 15000; 1: loxP-MCS-loxP (pCAMBIA2300) plasmid is the loxP-MCS-loxP fragment (156bp) amplified by the template.

Fig. 4 is a map of the recombinant expression vector loxP-PAP3: antiETR1-loxP (pCAMBIA2300).

Figure 5 is the vector verification of loxP-PAP3::antiETR1-loxP (pCAMBIA2300) M: Marker 2000plus; 1: AP3 promoter fragment (727bp) amplified with the loxP-PAP3::antiETR1-loxP plasmid as a template 2: loxP-PAP3 ::antiETR1-loxP plasmid is the AtETR1 fragment (2217bp) amplified as the template 3: loxP-PAP3::antiETR1-loxP plasmid is the target, and the AP3 promoter digestion product is obtained by double digestion; 4: loxP-PAP3::antiETR1- Electrophoresis pattern of loxP plasmid; 5: PAP3: antiETR1 fragment was obtained by double digestion with loxP-PAP3::antiETR1-loxP plasmid; 6: ETR1 gene was obtained by double digestion with loxP-PAP3::antiETR1-loxP plasmid fragment.

Fig. 6 is a picture of hygromycin resistance screening of Arabidopsis seedlings positively transfected with loxP-PAP3::antiETR1-loxP gene.

Figure 7 is the genome identification of positive transgenic Arabidopsis thaliana with loxP-PAP3::antiETR1-loxP gene, M: Marker2000plus; 1-9: the genome of positive transgenic Arabidopsis thaliana with sterile phenotype is used as template PCR amplification; 10-13 : Genome PCR amplification of transgene-positive Arabidopsis thaliana without sterility phenotype; 14: PCR amplification using wild-type Arabidopsis genome as template (control).

Figure 8 is the observation of the phenotype of Arabidopsis positively transfected with loxP-PAP3::antiETR1-loxP, A, E: flowers on the inflorescence of Arabidopsis; (left: wild type. Right: transgenic Arabidopsis) B, F: inflorescence The first flower; (left: wild type. Right: transgenic Arabidopsis) C, G: the third flower in the inflorescence; (left: wild type. Right: transgenic Arabidopsis) D, H: wild type Siliques and transgenic plants; I: Comparison of whole plant levels of wild-type Arabidopsis and transgenic loxP-PAP3::antiETR1-loxP Arabidopsis.

Figure 9 is a comparison of pollen viability and pollen germination between positively transfected loxP-PAP3::antiETR1-loxP plants and wild-type plants, A: wild-type Arabidopsis pollen Alexander staining, wild-type pollen is active, purple-red, and round in shape , the granules are full, and the whole anther is full; B: Alexander staining of the pollen of Arabidopsis thaliana positively transgenic for PAP3: antiETR1 gene, the pollen of the transgenic Arabidopsis thaliana is inactive and green, and the shape of the pollen is irregular, and the anthers are found to be shrunken.

Figure 10 is a comparison of pollen germination between positively transfected loxP-PAP3::antiAtETR1-loxP plants and wild-type plants, A is the wild type; B is the transgenic type.

Figure 11 is the test of transgenic male sterile Arabidopsis thaliana carpel and stamen fertility, A: positive transgenic loxP-PAP3::antiETR1-loxP gene Arabidopsis pistil confers wild-type pollen; B: partial enlarged view of Figure A, in the figure It shows that transgenic Arabidopsis thaliana obtained normal siliques after pollination; C: Carpels of wild-type plants were pollinated by transgenic male sterile lines (arrows point to the siliques developed after pollination); D: Partial enlarged view of Figure C ; Wild-type Arabidopsis thaliana did not get normal siliques after infecting sterile Arabidopsis thaliana pollen; E: Positively transfected loxP-PAP3::antiETR1-loxP sterile plants pollinated themselves; F: Partial enlarged view of Figure E; Figure F shows Normal siliques were not obtained from sterile plants pollinated by themselves.

Figure 12 shows the acquisition of the Cre gene, M: Marker 15000; 1: the Cre fragment amplified using the pMM23 plasmid as a template.

Figure 13 shows 35S: CRE vector identification M: Marker 2000plus; 1: Using pMM23 plasmid as a template, XbaI/BamHI double enzyme digestion of 35S: CRE to obtain CRE digestion product; 2: Cre fragment amplified using pMM23 plasmid as a template.

Figure 14 shows the detection of insert fragments in the offspring of the transgenic male sterile line × wild type hybrid. M: Marker 2000plus1-30: Genomic PCR was performed using the genome of the F1 generation of the transgenic male sterile line × wild type hybrid as a template. The detection target was loxP-PAP3: antiETR1-loxP fragment (3500bp), as can be seen from the figure, this fragment still exists in the F1 generation.

Figure 15 shows the genome detection of positive transgenic loxP-PAP3::antiETR1-loxP gene male sterile Arabidopsis and positive transgenic 35S:Cre gene restorer line. A: Positive loxP-PAP3::antiETR1-loxP transgenic male sterile Phenotype Arabidopsis thaliana and the positive 35S:Cre gene restorer line hybridization offspring genome as template PCR amplification (detection of Cre gene), the figure shows that the Cre fragment still exists in the F1 generation genome (pointed by the arrow); B : Positive transformation of loxP-PAP3::antiETR1-loxP showing male sterile phenotype of Arabidopsis and positive transformation of 35S:Cre gene restorer line crossing the genome as a template for PCR amplification (detection of AP3ETR1 sequence detection of inserts between loxP) The figure shows that the loxP-PAP3::antiETR1-loxP fragment has been deleted in the F1 generation genome (pointed by the arrow).

Figure 16 shows the growth of the F1 generation after the male sterile plants positively transfected with loxP-PAP3::antiETR1-loxP were inoculated with 35S:CRE pollen and wild-type Arabidopsis pollen respectively. The plant in Figure A-1 in Figure A is loxP- PAP3::antiETR1-loxP×35S: CRE F1 generation figure shows that the fertility of the hybrid offspring of the transgenic sterile plant and the restorer line is the same as expected, that is, the offspring of the transgenic sterile line×restorer hybrid can grow normally and obtain the next generation; Figure A-2 is the loxP-PAP3::antiETR1-loxP×WT F1 generation. In the figure, it can be seen that the fertility of the offspring of the cross between the transgenic sterile plant and the wild type is the same as the expected result, that is, the offspring of the transgenic sterile line×restored cross are sterile. Breeding plants; Figure B is an enlarged view of Figure A.

Detailed ways

The present invention will be further described below in conjunction with specific examples, but the present invention is not limited to the following examples.

In the following examples, unless otherwise specified, all are conventional methods.

In the present invention, loxP-MCS-loxP (pCAMBIA2300) has the same meaning as pCAMBIA2300-loxP-MCS-loxP; loxP-PAP3: antiETR1-loxP (pCAMBIA2300) has the same meaning as pCAMBIA2300-loxP-PAP3: antiETR1-loxP.

Embodiment 1, the acquisition of sterile line and its verification

1. Obtaining CMS

(1) Construction of recombinant vector

1. AP3 promoter cloning

Using the Arabidopsis thaliana AP3 promoter sequence on NCBI, design a pair of primers as follows:

AP3F: 5'-AACCATGGAACTTGTGATTCCTTCTTAAAC-3';

AP3R: 5'-AA GGATCC ATTTCTTCTCTCTTTGTTTAATC-3'.

Using the genomic DNA of Arabidopsis thaliana (Arabidopsis ecotype Col-0; purchased from Arabidopsis Biological Resource Center, USA) as a template, using the above primer pair, the partial sequence with AP3 promoter function was obtained by PCR method, and the electrophoresis of the PCR product (717bp) See Figure 1 for the picture. Confirmed by sequencing. Except for the introduced restriction site, the nucleotide sequence of the PCR product is shown in Sequence 1 of the Sequence Listing, which is named AP3.

2. Acquisition of the ethylene receptor atETR1 gene

Using the ETR1 gene (At1g66340) of Arabidopsis retrieved from NCBI, the specific primers were designed as follows:

atETR1F: 5'-CCTTAATTAAATGGAAGTCTGCAATTGTATT-3';

atETR1R: 5'-ACCATGGTTACATGCCCTCGTACAG-3'.

Arabidopsis thaliana Col-0 total RNA was reverse-transcribed into cDNA, and the above primers were used to perform PCR amplification on the cDNA. The electrophoresis of the PCR product (2217bp) is shown in FIG. 2 . Confirmed by sequencing. Except for the introduced enzyme cutting site, the nucleotide sequence of the PCR product is shown in Sequence 2 of the Sequence Listing, and it is named atETR1.

3. Construction of the intermediate vector loxP-MCS-loxP (pCAMBIA2300)

First design the multiple cloning site base sequence loxP-MCS-loxP (shown in sequence 3 in the sequence listing, the nucleotide sequence of the loxp site is the 7th-40th of sequence 3 in the sequence listing with loxP sites at both ends and positions 111-143; the MCS multiple cloning site is positions 41-142):

The sequence was cloned into pGEM-TEasy vector, and the sequence was correct.

The loxP-MCS-loxP sequence is excised from the cloning vector, and inserted between the pvsI sta and SmaI restriction sites of the pCAMBIA2300 plasmid ( http://www.cambia.org.au ) (loxP-MCS-loxP can replace 35s in pCAMB2300: GFP sequence), the constructed intermediate vector was named loxP-MCS-loxP (pCAMBIA2300).

The intermediate vector loxP-MCS-loxP (pCAMBIA2300) was verified by PCR and had a correct structure (Figure 3).

4. Construction of recombinant expression vector loxP-PAP3: antiETR1-loxP (pCAMBIA2300)

The AP3 promoter obtained in step 1 and the atETR1 obtained in step 2 are respectively inserted between the EcoRI and SacI sites of the intermediate vector in step 3 and between the BglII and SpeI sites (that is, the AP3 promoter obtained in step 1 and the step 2 obtained atETR1 was inserted between the two loxp sites) to obtain a recombinant expression vector (Figure 4), named loxP-PAP3: antiETR1-loxP (pCAMBIA2300). The vector was verified by PCR and enzyme digestion, and the structure was correct (Fig. 5).

(2) Acquisition of CMS

The constructed transgenic plasmid (loxP-PAP3: antiETR1-loxP(pCAMBIA2300)) was transformed into Arabidopsis thaliana (Col) by Agrobacterium-mediated Floral Tip method. The developing floral tissue was directly immersed in overnight cultured Agrobacterium solution containing 5% sucrose and 200 μl/l surfactant Silwet L-77. 33 hygromycin-resistant regenerated plants were obtained through plate selection with hygromycin Hyg+ (50 mg/L) ( FIG. 6 ), and the regenerated transformed plants were transferred to the greenhouse for growth. Nine transgenic Arabidopsis plants grown after a period of time showed a sterile phenotype.

In order to identify whether the transgenic Arabidopsis thaliana with hygromycin resistance was indeed transferred to the loxP-PAP3::antiETR1-loxP fragment, we performed genomic PCR detection on the transgenic positive Arabidopsis seedlings. Because the PCR primers for identifying the loxP-PAP3::antiETR1-loxP fragment in the positive seedling genome are designed according to the DNA sequence characteristics of the loxP-PAP3::antiETR1-loxP fragment (the forward primer is designed on AP3, specifically AACCATGGAACTTGTGATTCCTTCTTAAAC; the reverse primer On atETR1, specifically ACCATGGTTACATGCCCTCGTACAG), so in the PCR amplification reaction using the Arabidopsis genome as a template, only the genomic DNA integrated with the loxP-PAP3::antiETR1-loxP fragment will have a corresponding specific size band band appears. The test results showed that the 9 transgenic resistant plants with male sterility phenotype all had the expected specific band amplification, and the 4 transgenic resistant plants without male sterility phenotype also had the expected specific band amplification Amplification; while the wild-type plant control has no amplification of the corresponding fragment (Fig. 7). The experiment preliminarily showed that the 9 male sterile phenotype resistant plants were positive transgenic seedlings.

2. Phenotype observation of sterile lines

1. Morphological observation of flower organs

Utilize conventional paraffin section technique, compare above-mentioned 9 strains of male sterile plants positively transfected with loxP-PAP3::antiETR1-loxP gene with wild-type plant flower organ morphology, the results are shown in Figure 8 (because the 9 strains have the same phenotype, Figure 8 is only The result of 1 plant is shown), the positive loxP-PAP3::antiETR1-loxP gene plants have normal flower shape and structure, calyx and petals are complete, and there is no obvious difference between the early development of pistil and normal wild-type plants, but with As the plants grow further, the filaments of the transgenic plants are significantly shorter than those of the wild type, and anther atrophy begins to appear; at the whole plant level, it can be seen that the transgenic plants have a severe sterility phenotype, and the seed setting rate is significantly lower than that of the control plants. Fertility is manifested as semi-sterility or total sterility.

2. Alexander staining microscopic examination of pollen morphology

The pollen morphology of transgenic plants was observed more finely by Alexander staining microscope. It was found that the anthers of the 9 transgenic plants with male sterility phenotype were shrunken and irregular in shape, and the fertile pollen content in the anthers was very small, and most of the pollen was dyed green in the pollen staining experiment, indicating that this part of the pollen had no life. (Fig. 9); while the wild-type plants showed full anther structure, full pollen grains and smooth surface structure, and the pollen was dyed purple in the pollen staining experiment, indicating that this part of the pollen was energetic.

3. Pollen germination experiment

Experimental procedure: take isolated Arabidopsis pollen and suspend in germination solution (10% sucrose, 100ml plus 1-2 drops of 0.1% boric acid, 1mmol/L CaCl ₂ , 1mmol/L Ca(NO) ₃ , 1mmol/L MgSO ₄ ), adjusted to pH 6.5 with KOH. Shake lightly at 30 rpm for 4 hours in a light incubator at 25C. Observe under the microscope and count the germination rate.

Under in vitro conditions, by observing the pollen cultured on the medium for 24 hours, it was found that the pollen tubes of wild-type plant pollen can be elongated, and the germination rate can reach 41%; while the pollen of transgenic plants with male sterile phenotype The germination rate was only 2.67% (Table 1), and only a few pollen germinated with short pollen tubes (Fig. 10).

Table 1. Statistics of pollen germination

3. Fertility detection of carpels and stamens

Because the AP3 promoter used in this experiment is a B-type gene-specific promoter, in the second and third rounds of expression in the floral organ of the ABC model, the antisense AtETR1 gene transferred using it as a promoter should theoretically not affect Carpel function. In order to detect whether the carpel function of the transgenic plants is changed and whether the stamen fertility is affected by the antisense atETR1 gene, we tested the fertility of the stamens and pistils of the transgenic plants. Methods as below:

(1) Infect the wild-type Arabidopsis pollen to the pistil of Arabidopsis positive transgenic loxP-PAP3::antiETR1-loxP gene to detect the fertility of transgenic Arabidopsis carpels, and found that the positive transgenic loxP-PAP3::antiETR1 The pistils of the -loxP gene plants were fertile, and the obtained siliques grew normally and had plump seeds ( FIG. 11 ).

(2) Infect the pollen of the positively transferred loxP-PAP3::antiETR1-loxP gene Arabidopsis thaliana on the pistil of the wild-type Arabidopsis to detect the fertility of the positive transloxP-PAP3::antiETR1-loxP gene Arabidopsis stamens, As a result, it was found that wild-type Arabidopsis pistils pollinated by positively transfected loxP-PAP3::antiETR1-loxP gene did not obtain normal growth of siliques ( FIG. 11 ).

(3) Infect the pollen of Arabidopsis positive transgenic loxP-PAP3::antiETR1-loxP gene to its own pistil to detect the fertility of transgenic Arabidopsis anthers, and found that the positive transgenic loxP-PAP3::antiETR1-loxP gene Normal growth of siliques was not obtained from the pistils pollinated by A. thaliana ( FIG. 11 ).

According to statistics of the number of siliques of transgenic plants crossed with different lines (Table 2), combined with the phenotypic results obtained above, it is preliminarily shown that the antisense ETR1 we transferred into Arabidopsis only changed the Arabidopsis silique through the ethylene signaling system. The developmental fate of stamens in mustard mustard, but did not change the function of pistils.

Table 2. Statistics on the number of siliques of offspring of transgenic plants crossed with different lines

Example 2, the acquisition of recovery lines and their verification

1. Obtaining the Restoration System

1. Cre gene cloning

According to the published Cre gene sequence in phage P1, the following specific primers were designed:

CRE F: 5'-CATCTAGAATGTCCAATTTACTGACCG-3';

CRE R: 5'-TTAAGCTTCTAATCGCCATCTTCCAG-3'.

The gene (1032 bp) was obtained by PCR method using pMM23 plasmid (promega) as a template (Fig. 12), and it was confirmed to be correct by sequencing (the nucleotide sequence is shown in sequence 4 in the sequence listing).

2. Construction of expression vector

The CRE gene was connected between the XbaI and BamHI restriction sites of pCAMBIA2300 to form a recombinant vector named 35S:CRE. The vector was verified by PCR and enzyme digestion, and the structure was correct ( FIG. 13 ).

3. Acquisition of Restoration Department

The 35S:CRE obtained in step 2 was transformed into Arabidopsis thaliana (col) by using the Agrobacterium-mediated Floral Tip method to obtain the trans 35S:CRE Arabidopsis thaliana.

After PCR detection (primers were CREF: 5'-CATCTAGAATGTCCAATTTACTGACCG-3'; CRE R: 5'-TTAAGCTTCTAATCGCCATCTTCCAG-3'), 33 positive strains were obtained.

2. Verification of the recovery system

Observation was carried out by crossing the sterile line with the wild type and crossing the sterile line with the above-mentioned restorer line.

1. Hybrid F1 seed resistance

The progeny seeds obtained after crossing the positive loxP-PAP3::antiETR1-loxP gene male sterile line in Example 1 and the wild type (WT, col) were sown after adding Hyg ⁺ (50mg·L ^-1 ) monoclonal resistance On MS plate medium; sow the seeds obtained after crossing the positive loxP-PAP3::antiETR1-loxP gene male sterile line and the positive 35S:CRE gene restorer line after adding Kan ⁺ 50mg·L ^-1 /Hyg+50mg ·Resistance analysis was carried out on the L ^-1 double-resistance MS lithographic medium, and the results showed that resistance segregation appeared in all hybrid offspring seeds. According to the results of the chi-square test, the resistance chi-square value of the two hybrid offspring is less than 3.841 (critical value) (Table 3), so the difference between the theoretical value and the actual value can be considered the same at the 5% significance level, This result is in line with Mendel's segregation law.

Table 3. Chi-square test for the resistance of offspring of transgenic sterile lines crossed with wild type and restorer lines respectively

2. PCR analysis of hybrid offspring

1) Genomic PCR analysis was performed on the progeny plants obtained from the F1 generation seeds obtained by crossing the male sterile Arabidopsis thaliana positively transfected with the loxP-PAP3::antiETR1-loxP gene and the wild type through resistance screening, and using the upstream and downstream of the inserted fragment Primers for PCR amplification (primers are AACCATGGAACTTGTGATTCCTTCTTAAAC and ACCATGGTTACATGCCCTCGTACAG). The results showed that all the plants that could grow normally on the Hyg ⁺ 50mg·L ^-1 MS medium had the expected amplified fragments. (Figure 14)

2) The progeny obtained by crossing the male sterile Arabidopsis thaliana positively transfected with loxP-PAP3::antiETR1-loxP gene and the positively transfected 35S:Cre gene restorer line plants, using the upstream and downstream primers of Cre gene (specifically, the primers are CATCTAGAATGTCCAATTTACTGACCG and TTAAGCTTCTAATCGCCATCTTCCAG. ) for PCR amplification. The results are shown in Figure 15, all the plants that can normally take root on the Kan+50mg·L ^-1 /Hyg+50mg·L ^-1 MS double-resistance medium have an expected amplified fragment of about 1.0kb (Cre).

At the same time, using loxP primers and loxP-PAP3::antiETR1-loxP expression cassette fragment primers (primers are specifically ACACGTGGCTAGCATAACTTCGT and CCCCGAATTCATAACTTCGTA), positively transfected loxP-PAP3::ant iETR1-loxP gene male sterile Arabidopsis and positively transfected 35S: Genomic DNA of the progeny obtained from the hybridization of the Cre gene restorer line plants was detected by PCR. As shown in FIG. 15 , no bands appeared. This result indicated that the Cre gene could normally delete the exogenous insertion sequence in the loxP site.

3. Fertility of hybrid seeds

The fertility of the offspring of transgenic sterile plants crossed with wild type and restorer lines was the same as expected, and the offspring of transgenic sterile lines × wild type crossed were all sterile plants (Table 4 and Fig. 16A-2, Fig. 16B-2) , the offspring of the transgenic sterile line × restorer line are fertile plants (Table 4 and Fig. 16A-1, Fig. 16B-1).

Table 4. Fertility detection of transgenic sterile plants crossed with wild type and restorer lines respectively

Fertility Analysis of F1 Generation of Hybrid Combination Detect total tree fertile plants sterile plant Transgenic male sterile line (♀)×wild type (♂) 30 0 30  Transgenic sterile line (♀) × restorer line (♂) 30 29 1

Claims (2)

1. a method of cultivating the male sterile Arabidopis thaliana is the recombinant expression vector that contains following expression cassette to be imported in the Arabidopis thaliana obtain said male sterile Arabidopis thaliana: comprise successively from the upper reaches to downstream: reverse complemental gene, terminator and the loxp site of loxp site, flower pesticide or stamen organ anlage specific promoter, the proteic encoding sox of ethylene receptor ETR1;

In the said expression cassette, the nucleotide sequence in loxp site shown in the 7-40 position of sequence in the sequence table 3, the nucleotide sequence of flower pesticide or stamen organ anlage specific promoter shown in sequence in the sequence table 1, the nucleotide sequence of the proteic encoding sox of ethylene receptor ETR1 is shown in sequence in the sequence table 2;

The construction process of said recombinant expression vector is: the reverse complemental gene of said promotor and the proteic encoding sox of said ethylene receptor ETR1 is inserted between the MCS of an intermediate carrier, obtain said recombinant expression vector;

Said intermediate carrier is that the fragment shown in the sequence in the sequence table 3 is inserted between the MCS of pCAMBIA2300 plasmid, obtains said intermediate carrier.

2. method of cultivating the recovery system of the sterile Arabidopis thaliana that the said method of claim 1 obtains; Be that the Cre gene is imported in the Arabidopis thaliana in the said method of claim 1; Obtaining said recovery is that the nucleotide sequence of said Cre gene is shown in sequence in the sequence table 4;

Said Cre gene imports in the Arabidopis thaliana through an expression vector: said expression vector is that the Cre gene fragment shown in the sequence in the sequence table 4 is inserted between the MCS of pCAMBIA2300, obtains said expression vector.

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