CN118324884A - Tomato SlBBX5 gene and its application in plant drought resistance and plant architecture development - Google Patents

Abstract

The invention belongs to the field of plant molecular biology, and particularly relates to tomato SlBBX and a coding protein and application thereof. The full length of cDNA sequence of the gene is shown as SEQ ID No.1, and the amino acid sequence of the coded protein is shown as SEQ ID No. 2. The invention clones the full-length sequence of SlBBX from tomato cDNA, constructs an over-expression vector, and successfully transfers the over-expression vector into wild tomato AC by an agrobacterium-mediated genetic transformation method. Through phenotype identification and physiological and biochemical analysis of the transgenic plants, slBBX genes are found to positively regulate and control drought resistance of tomatoes. After drought treatment, slBBX over-expressed transgenic plants can improve drought resistance of tomatoes, and SlBBX5 over-expressed transgenic plants are dwarfed and have more internodes.

Description

Tomato SlBBX5 gene and its application in plant drought resistance and plant architecture development

Technical Field

The invention belongs to the field of plant molecular biology, and specifically relates to tomato SlBBX5 and its encoding protein and application.

Background technique

Under the conditions of a changing climate, plants are subject to a variety of abiotic or biotic stresses, among which drought is the main threat. Due to the limited supply of water resources in the world, the demand for food from the rapidly growing population in the future may further aggravate the impact of drought (Somerville and Briscoe. 2001). The yield loss in the field under drought is usually between 30% and 90%; the yield loss varies among different crop species (Hussaines All. 2019). Therefore, understanding the molecular mechanism of plant drought stress response and cultivating drought-resistant varieties are of great significance for increasing crop yields and solving food security issues.

B-box/BBX proteins constitute a family of plant-specific transcription factors that contain one or two ‘B-box’ domains in their N-terminal regions (Gangappa S N and Botto J F). BBX proteins play important roles in seed germination, seedling photomorphogenesis, shade avoidance, flowering photoperiod regulation, and responses to biotic and abiotic stresses (Gangappa S N and Botto J F).

It is reported that BBX proteins have an important signal transduction pathway, which is induced by abiotic or biotic stress. Taking Arabidopsis as an example, after 2 hours of heat treatment at 42℃, it was found that BBX18 overexpressing plants had a significant inhibitory effect on heat resistance. In addition, BBX18 can also reduce the germination rate and survival rate of Arabidopsis after treatment by regulating the expression of heat-responsive genes (DGD1, HSP70, HSP101, APX2) (Wang et al. 2013). In Arabidopsis, BBX24 overexpression enhances its salt tolerance (Nagaoka et al. 2003). Compared with wild-type plants, BBX24 transgenic plants have significantly increased root length in 50mM and 100mM NaCl culture media (Nagaoka et al. 2003). However, the BBX24 gene was not induced under salt stress, indicating that BBX24 tolerance to salt stress may not be directly regulated. Interestingly, BBX24 directly interacts with H-protein promoter binding factor 1 (HPPBF-1) (Nagaoka et al. 2003). The zinc finger protein BBX19 interacts with ABF3 to negatively regulate drought tolerance in chrysanthemum (Xu Y et al. 2020). BBX regulatory factors may also be involved in the regulation of plant defense responses. The rice gene OsCOL9 encodes a BBX protein belonging to the Col protein family II class, and the expression of this gene at the mRNA level is enhanced after infection with the rice blast fungus. In addition, OsCOL9 gene knockout rice plants showed higher pathogen sensitivity (Liu et al. 2016).

Tomato is one of the most consumed vegetables in the world, and China is the country with the largest tomato planting area and the highest output in the world, accounting for about 6% of the total vegetable production, and plays an important role in the vegetable industry (data source: Food and Agriculture Organization of the United Nations (latest)). At the same time, due to the small size of the tomato genome, the availability of a large number of mutant resources and molecular biology tools, it is also regarded as a classic model plant and has very important scientific research value. Therefore, a systematic and complete study of tomato drought-resistant genes and the exploration of its drought-resistant mechanism can lay a theoretical foundation for the breeding of drought-resistant tomato varieties.

Summary of the invention

In order to solve the above problems, the present invention provides tomato SlBBX5 and its encoded protein and application.

First, the present invention provides a tomato S1BBX5 protein, which is:

1) a protein consisting of the amino acids shown in SEQ ID No. 2; or

2) A protein derived from 1) with equivalent activity, wherein one or more amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID No. 2.

The present invention also provides a gene encoding the tomato S1BBX5 protein.

Preferably, the sequence of the gene is shown as SEQ ID No.1.

The invention also provides an overexpression vector, a host cell and an engineering bacterium containing the gene.

The invention also provides application of the gene in improving the drought resistance of plants.

In a specific embodiment of the present invention, the gene is constructed into an overexpression vector and transferred into the plant genome, so that the gene is overexpressed in the plant to improve the drought resistance of the plant.

The present invention also provides the use of the gene in regulating plant type development.

In a specific embodiment of the present invention, the gene is constructed into an overexpression vector and transferred into the plant genome, so that after the gene is overexpressed in the plant, the plant becomes dwarfed.

SlBBX5 is a member of the tomato BBX gene family. Its N-terminal region contains two 'B-box' domains and encodes 358 amino acids. Subcellular localization shows that it is located in the nucleus. Tissue expression analysis showed that the gene was expressed in all tissues (roots, stems, leaves, flowers, and fruits) of the common cultivated species AC, with the highest expression level in flowers, indicating that the SlBBX5 gene may play an important role in plant growth and development. The expression pattern showed that the SlBBX5 gene not only responded to drought stress, but also may be induced by a variety of hormone regulatory factors such as ABA and GA. The full-length sequence of SlBBX5 was cloned from tomato cDNA, and an overexpression vector was constructed. It was successfully transferred into wild-type tomato AC by Agrobacterium-mediated genetic transformation. Through phenotypic identification and physiological and biochemical analysis of transgenic plants, it was found that the SlBBX5 gene positively regulates the drought resistance of tomatoes. After drought treatment, SlBBX5 overexpression transgenic plants can improve the drought resistance of tomatoes, and SlBBX5 overexpression transgenic plants are dwarfed and have more internodes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the analysis of cis-acting elements in the promoter region of the SlBBX5 gene.

FIG2 shows the tissue expression pattern analysis of the SlBBX5 gene (R: root; S: stem; L: leaf; FL: flower; FR: fruit).

Figure 3 shows the expression profile of SlBBX5 after various stress and hormone treatments (time: h). Note: 0 h is taken as 1 for each treatment.

FIG. 4 shows the PCR electrophoresis diagram of the bacterial solution of the SlBBX5 overexpression vector.

Figure 5 is a schematic diagram of genetic transformation of SlBBX5 tomato. Note: A: pre-culture; B: callus differentiation into resistant seedlings; C: resistant seedlings rooting; D: positive seedlings transplanted into pots.

Figure 6 shows the PCR identification of some SlBBX5 transgenic tomato positive plants. Note: M: DL 2000 Marker; 1-22: transgenic plants; CK ^- : negative control with water as template.

Figure 7 shows the expression analysis of SlBBX5 transgenic tomato. Note: WT: wild type AC; OE13, OE14, OE5, OE6, OE7: transgenic lines overexpressing SlBBX5.

Figure 8 shows the plant type observation diagram of each strain of SlBBX5 gene. Note: WT: wild type AC; OE14, OE5, OE13: SlBBX5 overexpression strains, the significance analysis was performed using the variance analysis-multiple comparison method, and the ones with significant differences (P < 0.05) from the wild type (WT) were marked with "*".

Figure 9 shows the morphological determination of SlBBX5 overexpression transgenic plants. Note: WT: wild type AC; OE5, OE14, OE13: SlBBX5 overexpression lines, the significance analysis was performed using the variance analysis-multiple comparison method, and the ones with significant differences (P < 0.05) from the wild type (WT) were marked with "*" and the ones with extremely significant differences (P < 0.01) from the wild type (WT) were marked with "***".

Figure 10 shows the phenotypic analysis of SlBBX5 transgenic tomatoes and wild-type tomatoes after drought treatment. Note: WT: wild-type AC; OE14, OE5, OE13: SlBBX5 overexpression lines.

Figure 11 shows the phenotypic differences between SlBBX5 transgenic lines and wild type WT under simulated drought treatment on MS medium. Note: Three biological replicates for each line. WT: wild type AC; OE5, OE6: overexpressed SlBBX5 transgenic lines, significance analysis was performed using ANOVA-multiple comparison method, and those with significant differences (P < 0.05) from the wild type (WT) were marked with "*"; Control: normal growth control group on MS medium; Drought: mannitol treatment group.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular cloning laboratory manual (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001), or the conditions recommended by the manufacturer's instructions.

This experiment used the common cultivar AC (S. lycopersicum cv. Ailsa Craig) as the recipient material for gene cloning and genetic transformation, hormone treatment and expression pattern analysis. All tomato test materials and transgenic lines used for subsequent experiments were planted in the network room base on the campus of Southwest University.

Example 1 Cloning and bioinformatics analysis of S1BBX5 gene

Using the cDNA of tomato variety AC as a template, the SlBBX5 gene was cloned by PCR, with a total length of 1077 bp (shown in SEQ ID No. 1) and encoding 358 amino acids (shown in SEQ ID No. 2). The primers are as follows:

OESlBBX5-F:ACTAGTCGAAATAATGGGAACGGAGA;

OESlBBX5-R:CTCGAGACAAACGATGGAACGACACCG.

During the development and differentiation process, organisms need to integrate signals from different tissues, development and environment to regulate gene expression. Cis-acting elements play an important role in gene response to stress and response. Based on the tomato genome sequence, the cis-acting elements in the -3000bp promoter region upstream of the SlBBX5 gene were analyzed. As shown in Figure 1, cis-acting elements related to hormone and stress response were found in the promoter region of the SlBBX5 gene: abscisic acid response element (ABRE), salicylic acid response element (TCA-element), cis-regulatory elements involved in MeJA response, and cis-acting elements involved in light response (Box4, G-Box).

Example 2 Analysis of tissue expression pattern of S1BBX5 gene

The specific expression of the gene in tissues indicates that it plays an important role in plant growth and development. Real-time PCR was used to analyze the tissue expression pattern of the SlBBX5 gene using cDNA from the roots, stems, leaves, flowers and fruits of tomato AC as templates. Tomato β-Actin (Solyc11g005330.1.1) was used as an internal reference gene for correction. The primer sequences are as follows:

QSlBBX5-F: TACCAGTACAGAACAACAACGA;

QSlBBX5-R:TGATGATGAAACACTTTGGCTG;

TomACTIN(Q)Fw GTCCTCTTCCAGCCATCCAT

TomACTIN(Q)RvACCACTGAGCACAATGTTACCG

qRT-PCR reaction system and procedure:

The results showed that as shown in Figure 2B (with root expression normalized to 1), the SlBBX5 gene was expressed in roots, stems, leaves, flowers and fruits, with the highest expression in flowers and the second highest expression in leaves, which was consistent with the website prediction (as shown in Figure 2A). Therefore, the drought response gene SlBBX5 may have an important function in the process of plant growth and development.

Example 3 Analysis of adversity and hormone expression patterns of S1BBX5 gene

BBX proteins play an important role in plant growth and development, photomorphogenesis, hormone signal transduction and stress response. In order to explore whether the SlBBX5 gene is also involved in the stress response, the common cultivated tomato variety AC was treated with stress and hormones to observe whether the expression of SlBBX5 is affected by these factors.

Hormone expression pattern: Use five-leaf, one-heart wild-type tomato plant AC to spray 100 μmol/L ABA, GA, JA, and Br on the leaves of the plant, and spray the control group with distilled water until the leaves drip water. In order to overcome the effect of photoperiod on gene expression, we took functional leaves from the same part at different time points, normalized their expression levels according to the untreated CK expression level, compared the treated results with the untreated results, and calculated the corresponding expression levels. After the samples were collected, they were frozen with liquid nitrogen and stored in an ultra-low temperature refrigerator at -80°C;

Analysis of adversity expression pattern: The wild-type tomato plant AC with five leaves and one heart was subjected to the following adversity treatments, and the soil was irrigated with PEG (100mmol/L) and NaCl (100mmol/L), and the leaves of the plant were sprayed with MV (methyl viologen, 100μmol/L) until no water droplets formed on the leaf surface. Each treatment was irrigated or sprayed with equal volumes of treatment solution and control solution (water). After the samples were collected, they were frozen with liquid nitrogen and stored in an ultra-low temperature refrigerator at -80℃.

The expression patterns after drought treatment, salt (NaCl) treatment, and ABA, GA, JA, Br, and MV treatment were analyzed by qRT-PCR. The primers and conditions used for qRT-PCR were the same as those in Example 2.

As can be seen from Figure 3, under high salt treatment, compared with untreated plants, the expression level of SlBBX5 gene was up-regulated within 0.5h of the initial treatment, and then rapidly down-regulated within 1h, and then maintained a low expression level. At 24h of treatment, the expression level reached the highest. Under PEG simulated drought treatment, compared with untreated plants, the expression level of SlBBX5 gene decreased rapidly within 0.25h of the initial drought, and rapidly up-regulated and reached the highest level within 1h. Under MV treatment, the expression was rapidly upregulated at the beginning, but the expression began to decrease 1h after treatment, and then maintained a low expression level; in hormone treatment, under GA treatment, SlBBX5 gene was upregulated, reaching the highest level 0.25h after treatment; but after ABA treatment, the gene expression level decreased significantly at the beginning, and rapidly decreased 0.25h after treatment, and then upregulated in the later stage, reaching the highest level 1h after treatment; after Br treatment, the gene expression level increased rapidly, reaching the highest level in 1h; under JA treatment, the gene expression level was upregulated within 1h at the beginning of treatment, and rapidly downregulated in 6h. The results show that SlBBX5 gene not only responds to drought stress, but also may be induced by multiple hormone regulatory factors such as ABA and GA, indicating that SlBBX5 may be a positive regulatory factor in response to adversity.

Example 4 Vector Construction and Genetic Transformation

1. Vector Construction

① Primer design

Primer 3Plus software was used to design primers and the corresponding adapter sequences were added.

The primers are as follows:

OESlBBX5-F:ACTAGTCGAAATAATGGGAACGGAGA

OESlBBX5-R:CTCGAGACAAACGATGGAACGACACCG

② Reorganization

First, the target gene was amplified with specific primers and recovered. Then, the pHG plasmid was extracted by shaking the bacteria, and the vector was double-digested with XbaI and XhoI restriction endonucleases, and the digested products were recovered. Then, the digested vector was connected to the target gene fragment by T4 ligase, and transformed into Escherichia coli. PCR detection was performed with primer pair 35S+OES1BBX5-R, and the amplified fragment size was 1323 bp (Figure 4). The positive clone was shaken to extract the plasmid, and after successful sequencing, it was transformed into Agrobacterium.

35S:TTCGCAAGACCCTTCCTCTA

OESlBBX5-R:CTCGAGACAAACGATGGAACGACACCG

2. Agrobacterium-mediated transformation of tomato

Agrobacterium-mediated transformation of tomato by leaf disc method. Reference: Dr. Jinhua Li’s dissertation, 2013.

(1) Seed disinfection: Select tomato seeds (AC) with full grains and no impurities and place them in tissue culture bottles. Disinfect them with 75% alcohol on a clean bench for 1 min, pour out the alcohol, add a certain amount of 20% sodium hypochlorite and disinfect for 20 min, rinse with sterilized water 9 to 10 times, replace the sterilized tissue culture bottles appropriately during this period, seal and culture at 220 rpm and 25°C for 2 to 3 days; after the seeds turn white, transfer them to the prepared MS solid culture medium on a clean bench and culture them under suitable growth conditions until two cotyledons grow.

(2) Cutting seedlings:

① Cut off two cotyledons in the seedling cutting dish (the angles of the two cotyledons are between 45° and 180°), cut off the two ends of the cotyledons, keep the middle part of the cotyledons (farther away from the growth point), and transfer them to the dish containing seedling cutting solution.

②Then place the cut leaves with the front side facing up on the pre-culture medium and culture in a dark place for 24 to 36 hours.

(3) Shaking bacteria:

A single clone of the Agrobacterium plaque containing the overexpression vector that has been tested for plaques and has the correct band was picked up and placed in 3-5 mL YEB liquid medium (500 μg/mL Str, 500 μg/mL Rif, 50 μg/mL Kan) and cultured at 28°C for 1 day.

(4) Activation:

The above bacterial solution was added to 30-50 ml of liquid culture medium (YEB+500 μg/mL Str+500 μg/mL Rif+50 μg/mL Kan), and cultured at 28°C with shaking for 8-12 h until the OD600 value was around 1.0.

(5) Infection:

① Divide the shaken bacterial solution into two 50 mL sterile centrifuge tubes and centrifuge at 4°C, 4000 rpm for 10 min; discard the supernatant, resuspend the bacteria in YEB liquid medium without antibiotics, and centrifuge at 4°C, 4000 rpm for 10 min;

② Pour off the supernatant and resuspend the bacteria with the resuspension solution. Transfer the leaves that have been kept in the pre-culture medium away from light to a sterile empty dish, pour the resuspension solution into the empty dish and infect for 8 to 10 minutes;

③ Co-cultivation: Transfer the infected leaves to a culture dish containing sterile filter paper to absorb moisture (not for too long), transfer them to the original pre-culture medium after drying, transfer them to the screening medium in a dark place for about 2 days, culture at 28°C, and replace the screening medium every 2 to 3 weeks.

(6) Rooting: Cut the seedlings differentiated from the callus and insert them into the rooting medium for rooting.

(7) Transplantation: Slowly pull out the rooted seedlings from the culture medium, carefully clean the culture medium with tap water, and then transplant them into a nutrient pot filled with vermiculite to grow for about a week (pay attention to cover with film to keep moisture), and then transfer them to a nutrient pot containing a matrix: vermiculite: perlite = 3:1:1 for growth. When conditions are suitable, transfer them to the outdoor net room of Southwest University for seed breeding (Figure 5).

The genomic DNA of transgenic tomato plants was extracted by CTAB method for detection. Wild-type AC plants were used as controls. The overexpression lines were amplified with NPTII primers to detect positive plants. A total of 30 transgenic plants were obtained, and 18 of them were positive plants after detection (Figure 6).

Example 5 Screening of expression levels in transgenic overexpressing tomato plants

In order to detect the expression level of transgenic offspring and screen plants with large expression differences for functional analysis, total RNA was extracted from leaves of wild-type control AC (WT) and SlBBX5 transgenic _T2 plants, and cDNA was obtained by reverse transcription. Fluorescence quantitative PCR was used to detect the relative expression level of each transgenic strain, and transgenic strains with significant expression differences were screened for the next functional analysis. The conditions of the primers used for fluorescence quantitative PCR were the same as those in Example 2.

As shown in Figure 7 , the expression levels of SlBBX5 transgenic lines OE13, OE14, and OE5 were increased by about 6-fold, 5-fold, and 4-fold, respectively, compared with the wild-type control WT, and were used for subsequent analysis.

Example 6 Morphological identification and phenotypic observation of S1BBX5 overexpressing transgenic strains

To further understand the morphological characteristics of SlBBX5 overexpression transgenic lines in tomato growth and development, under the same growth conditions (40 days), it can be observed that these overexpression lines show the same phenotype during vegetative growth and development (Figure 8A), and the plant height and internode length of wild-type and transgenic plants were measured. As can be seen from Figures 8B and C, compared with wild-type plants, transgenic plants are significantly shorter and have more and shorter internodes, indicating that overexpression of the SlBBX5 gene affects the plant type of tomatoes.

To further understand and determine its phenotype, plants at the same growth stage (about 2 months old) were selected to measure their leaf length, width and petiole length. The results showed that compared with wild-type plants, the leaf length, width and petiole length of the transgenic lines were significantly reduced (Figure 9). This indicates that SlBBX5 plays an important role in the morphological characteristics of tomato growth and development.

In order to analyze the function of the SlBBX5 gene, the overexpression lines (OE14, OE5 and OE13) that had been screened were subjected to drought treatment.

The results are shown in Figure 10: before drought treatment (0 day), wild-type and transgenic lines at the same sowing period were selected for treatment, and it can be clearly observed that the overexpression plants were dwarfed relative to the wild-type control plants; after 6 days of drought treatment, the leaves of the wild-type plants began to wilt, but the SlBBX5 overexpression line grew well; when the drought lasted for 12 days, the leaves of the wild-type plants wilted severely, while the leaves of the SlBBX5 overexpression line began to wilt partially; then all plants were rehydrated, and after 3 days of rehydration, the leaves of the wild-type plants were still in a wilted and dried state and could not recover, while the leaves of the upper part of the SlBBX5 overexpression line resumed growth.

The experimental results showed that overexpression of the SlBBX5 gene could improve the drought resistance of tomato compared with the wild type WT.

In order to further analyze its stress resistance, mannitol was used to simulate drought treatment on the culture medium. As can be seen from Figure 11A, the wild-type and transgenic plants grew well on the MS culture medium and the growth potential was basically the same, while in the group with mannitol to simulate drought, the overall growth potential was lower than that of the control group, and the growth potential and root system of the wild-type were weaker than those of the SlBBX5 overexpression strain (Figure 11B and C). This further shows that overexpression of the SlBBX5 gene can improve the drought resistance of tomatoes.

Although the present invention has been described in detail above with general descriptions and specific embodiments, some modifications or improvements can be made on the basis of the present invention, which is easy for those skilled in the art. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims (10)

1. Tomato SlBBX5 protein, which is: 1) a protein consisting of the amino acids shown in SEQ ID No. 2; or 2) A protein derived from 1) with equivalent activity, wherein one or more amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID No. 2. 2. A gene encoding the tomato S1BBX5 protein according to claim 1. 3. The gene according to claim 2, wherein the sequence is as shown in SEQ ID No.1. 4. A vector containing the gene according to claim 2 or 3. 5. A host cell containing the vector according to claim 4. 6. An engineered bacterium containing the gene according to claim 2 or 3. 7. Use of the gene according to claim 2 or 3 in improving drought resistance of plants. 8. The use according to claim 7, characterized in that the gene is transferred into a plant genome so that the gene is overexpressed in the plant to improve the drought resistance of the plant. 9. Use of the gene according to claim 2 or 3 in regulating plant type development. 10. The use according to claim 9, characterized in that after the gene is constructed into an overexpression vector and overexpressed in plants, the plants become dwarfed.