Application of RLCK86 gene in improvement of drought tolerance of tomatoes

Abstract

The invention discloses application of an RLCK86 gene in improvement of drought tolerance of tomatoes, and belongs to the technical field of biology. According to the application, the tomato RLCK86 gene function is silenced or deleted by utilizing a biological technical means, so that the tolerance of tomato plants to drought stress is improved; the nucleotide sequence of a protein coding region of the RLCK86 gene is as shown in SEQ ID NO. 1. The invention discloses the application of the RLCK86 gene in regulating and controlling the drought stress resistance of the tomato for the first time, the function of the RLCK86 gene is deleted through a gene editing technology, the drought tolerance of the tomato can be remarkably improved, and a reference is provided for cultivating drought-resistant tomato germplasm resources.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of RLCK86 gene in improving drought resistance of tomatoes. Background Art

[0002] Water plays an important role in the growth and development of tomatoes, especially during the flowering and fruiting stages, when more water is needed to promote fruit development and increase yield. It is reported that 300 kg of water is needed to produce 1 kg of tomato fruit. Drought is one of the most important factors affecting crop yield and quality. When tomatoes encounter high temperatures (temperatures continuously exceeding 35 degrees) and drought during their growth, if they cannot replenish water in time, it will affect plant growth, resulting in reduced yield and deterioration of quality.

[0003] Therefore, it is very important to study and improve the drought-resistant genes of tomatoes. Creating drought-resistant tomato germplasm to ensure the normal growth of plants under limited water supply conditions has always been a difficult problem to be solved in this field.

[0004] Receptor-Like Cytoplasmic Kinases (RLCKs) are important environmental signal transduction factors in plants (Xiangxiu Liang et al., Receptor-Like Cytoplasmic Kinases: Central Players in Plant Receptor Kinase–Mediated Signaling. Annual Review of Plant Biology, 2018, 69: 267-299.), and have been widely studied in the regulation of plant disease resistance and stress tolerance.

[0005] For example, the RLCK-like protein BIK1 in Arabidopsis is a key signal transduction element in plant basal immune resistance (LuD et al., A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. Proceedings of the National Academy of Sciences of the United States of America. 2010, 107: 496-501).

[0006] Rice RLCK-like protein OsSAPK8 can phosphorylate calcium channel OsCNGC9 to trigger Ca 2+Inward flow enhances the plant's frost resistance (Wang, J et al., Transcriptional activation and phosphorylation of OsCNGC9 confer enhanced chilling tolerance in rice. Molecular Plant. 2021, 14: 315-329).

[0007] However, compared with other adverse stresses, the functions of RLCK family members in the regulation of plant drought resistance have been rarely reported. Summary of the invention

[0008] The purpose of the present invention is to study and explore genes involved in regulating tomato drought stress resistance and apply them to the creation and breeding of tomato drought-resistant germplasm.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] The present invention provides an application of the RLCK86 gene in improving drought tolerance of tomatoes, the application comprising: utilizing biological technology means to silence or delete the function of the tomato RLCK86 gene, thereby improving the tolerance of tomato plants to drought stress.

[0011] The nucleotide sequence of the protein coding region of the RLCK86 gene is shown in SEQ ID NO.1, and has a length of 2364 bp.

[0012] The protein encoded by the RLCK86 gene is a receptor-like cytoplasmic kinase, consisting of 787 amino acids, and its sequence is shown in SEQ ID NO.2.

[0013] The present invention shows that the tomato RLCK86 gene function can be deleted, which can significantly enhance the resistance of tomato plants to drought stress and can be applied to the breeding of drought-resistant plants.

[0014] Furthermore, gene mutation, gene knockout, gene interference or gene silencing technology is used to reduce or eliminate the expression of the RLCK86 gene in tomato plants, thereby obtaining mutant plants with enhanced drought resistance.

[0015] The present invention uses CRISPR-Cas9 gene editing technology to obtain two tomato rlck86 mutant strains. Compared with the control wild-type plants, the mutant plants have significantly enhanced drought resistance, revealing that tomato RLCK86 plays a negative regulatory role in drought resistance.

[0016] Further mechanism studies have shown that the tomato RLCK86 gene affects the water loss rate by affecting the movement of stomata in the plant leaf epidermis, thereby regulating the resistance of tomatoes to drought stress. When the RLCK86 gene function in tomato plants is silenced or missing, it promotes the closure of stomata in the leaf epidermis of tomato plants, reduces the damage of lipid membranes in drought, and enhances the tolerance of tomato plants to drought stress.

[0017] The present invention also provides a method for enhancing drought resistance of tomatoes, comprising the following steps:

[0018] (1) A target fragment containing a PAM structure was selected from the protein coding region of the tomato RLCK86 gene, and primers were designed based on the first 20 bases of the PAM structure of the target fragment to construct a CRISPR / Cas9 vector;

[0019] (2) constructing an Agrobacterium genetically engineered bacterium containing the CRISPR / Cas9 vector described in step (1);

[0020] (3) The genetically engineered bacteria described in step (2) are transformed into tomato cotyledons, and a homozygous mutant strain with stable inheritance and mutation of the target sequence is obtained by cultivation and screening.

[0021] In the above method, gene editing technology and tissue culture technology are used to obtain a mutant strain in which the RLCK86 gene is mutated, and the mutant strain shows enhanced resistance to drought stress.

[0022] Furthermore, the nucleotide sequence of the first 20 bases of the PAM structure of the target fragment is shown in SEQ ID NO.3.

[0023] Furthermore, the nucleotide sequences of the primer pairs for constructing the CRISPR / Cas9 vector are shown in SEQ ID NO.4 and SEQ ID NO.5; PCR amplification is performed using the pBAtC-tRNA plasmid as a template, and the product is connected with the pHEE401 plasmid to construct the CRISPR / Cas9 vector.

[0024] Furthermore, the host bacteria for constructing the genetically engineered bacteria is GV3101 Agrobacterium.

[0025] Further, the variety of the tomato is Condine Red.

[0026] The present invention has the following beneficial effects:

[0027] The present invention discloses for the first time the use of the RLCK86 gene in regulating tomato's resistance to drought stress. By deleting the RLCK86 gene function through gene editing technology, the drought resistance of tomatoes can be significantly improved, providing a reference for cultivating drought-resistant tomato germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the gene editing type of tomato rlck86 mutant.

[0029] Figure 2 This is the drought phenotype of the tomato rlck86 mutant (scale length: 10 cm).

[0030] Figure 3 MDA content in leaves of tomato rlck86 mutant.

[0031] Figure 4 The morphology of tomato guard cells before and after treatment with 50 μM ABA (scale bar: 50 μm).

[0032] Figure 5 The opening degree of guard cells in tomato leaves before and after treatment with 50 μM ABA.

[0033] Figure 6 is the relative water loss rate of detached tomato leaves. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0035] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0036] The tomato variety used in the following examples is the conventional tomato variety Condine Red, and ordinary tomatoes without gene editing are used as controls (Wild type, WT).

[0037] Example 1: Gene editing to construct and screen mutant strains

[0038] 1. Construction of CRISPR / Cas9 vector containing specific sgRNA

[0039] The CDS sequence of tomato RLCK86 (gene ID: Solyc03g025450) was obtained from the Sol Genomics Network website (https: / / solgenomics.net / ), and the nucleotide sequence is shown in SEQ ID NO.1, with a length of 2364 bp. The protein encoded by RLCK86 is a receptor-like cytoplasmic kinase, consisting of 787 amino acids, and its sequence is shown in SEQ ID NO.2.

[0040] CRISPR-P2.0 (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / ) was used to search for PAM sequences, and the result with the highest comprehensive score was selected. The 20 bp sequence before NGG was defined as the target, which specifically targeted the third exon of RLCK86, and its DNA sequence was: 5'-TTGAAGAGTCAGTAAAACGG-3' (SEQ ID NO. 3).

[0041] Design CRISPR primers for the above targets as follows:

[0042] CRISPR / Cas9 vector forward primer:

[0043] 5'-gcGGTCTCTATTGaacaaagcaccagtggtctagtg-3' (SEQ ID NO. 4);

[0044] CRISPR / Cas9 vector rear primer:

[0045] 5'-gcGGTCTCTAAACCCGTTTTACTGACTCTTCAAtgcaccagccgggaat cg-3' (SEQ IDNO.5);

[0046] The above primers were used for PCR amplification with pBAtC-tRNA plasmid as template. After the products were purified by DNA purification kit, the amplified fragments were connected with pHEE401 plasmid using Bsa I enzyme. The products were transformed into Escherichia coli at 42°C and plated for screening. The resistance was kanamycin.

[0047] Pick a single clone and perform PCR verification using the verification primers designed for the pHEE401 vector. The verification primers are as follows:

[0048] 401-F: 5'-GTAAAACGACGGCCAGT-3' (SEQ ID NO. 6);

[0049] 401-R: 5'-GGTATTGGTTTATCTCATCGGAACTGCA-3' (SEQ ID NO. 7).

[0050] The bacterial solution with the correct band size was sent to a sequencing company for sequencing. The sequencing results showed that the vector contained the above-mentioned sgRNA sequence. The plasmid was extracted and introduced into the competent Agrobacterium GV3101 by heat shock. After culturing at 28°C for 48 hours, the bacterial solution was picked for PCR verification to obtain the Agrobacterium strain that can be used for genetic transformation.

[0051] 2. Preparation and identification of rlck86 mutant plant materials

[0052] The Agrobacterium strain prepared in step 1 was transformed into the cotyledons of wild-type tomato Condine Red using the Agrobacterium infection method, and the T0 generation gene-edited tomatoes were obtained by utilizing the totipotency of plant cells.

[0053] T0 generation gene-edited tomato seedling detection: The genomic DNA of T0 generation plants was extracted using the CTAB method and used as a template. The following primers were designed about 100-250 bp before and after the DNA sequence containing sgRNA for PCR amplification and sequencing verification:

[0054] Tomato rlck86 mutant material verification front primer:

[0055] 5'-CTGGCTCTAGAATTTCCTCAAC-3' (SEQ ID NO. 8);

[0056] Primers after verification of tomato rlck86 mutant material

[0057] 5'-CATACATACAAGTGTGTCG-3' (SEQ ID NO. 9);

[0058] The obtained PCR products were sent to a sequencing company for sequencing. The sequencing results were compared with the original sequence of the gene using Snapgene software, and plants with base deletions in the sgRNA sequence and its upstream and downstream sequences and single peaks in sequencing were selected for self-pollination to obtain T0 generation seeds.

[0059] The T0 generation seeds were planted in a growth chamber to obtain T1 generation plants. The sgRNA sequence base editing of the T1 generation plants was detected using the same method as above. At the same time, the DNA of the T1 generation plants was PCR amplified using Cas9 gene primers to detect whether it contained the Cas9 sequence.

[0060] Cas9 vector front primer: 5'-GTTAAGCAGCTCAAGGAGGACT-3' (SEQ ID NO. 10);

[0061] Cas9 vector rear primer: 5'-GTCTGCAGAATCCCCTTCTTGA-3' (SEQ ID NO. 11);

[0062] T1 plants with sgRNA mutation and no Cas9 protein were selected to identify two gene-edited plant lines, named rlck86#6 and rlck86#14, respectively. Figure 1As shown in Figure 2, compared with the control plant, rlck86#6 lacked 14 bp and rlck86#14 lacked 2 bp. Both types of editing resulted in frameshift mutations in RLCK86 and premature termination.

[0063] After sowing the T1 generation seeds of the above two strains, stable T2 generation plants without the exogenous gene Cas9 and with mutated sgRNA were obtained.

[0064] The following examples all used the T2 generation plants of the above two homozygous strains as materials for experiments.

[0065] Example 2: Functional analysis of gene-edited tomatoes

[0066] The wild-type tomato and the tomato rlck86 mutant plants were subjected to drought treatment as follows:

[0067] Tomato seeds were soaked in 55°C warm water for 15 min and then placed in a 28°C, 200 rpm shaker for germination for 2-3 days, with water changed every 12 hours. After the seeds turned white, they were sown in 36-hole trays and placed in an artificial climate chamber for cultivation. The temperature in the artificial climate chamber was 25 / 20°C (day / night), the photoperiod was 12 h light / 12 h dark, and the light intensity was 200 μmol m -2 s -1 When the tomato seedlings grow one true leaf, transplant them into planting pots and water them with an appropriate amount of Hoagland nutrient solution every 3 days.

[0068] When the tomato seedlings grew to four leaves and one heart, healthy plants of similar size were selected and randomly divided into two groups. The control group was watered normally, while the experimental group began to control water for natural drought treatment and placed in an artificial climate chamber for 7 days to observe the drought damage of the plants.

[0069] The results are as follows Figure 2 As shown, the wild-type plants suffered from severe leaf water loss, while the tomato rlck86 mutant plants showed tolerance to drought conditions.

[0070] The malondialdehyde (MDA) content in tomato leaves was further determined. Figure 3 As shown, it was found that the MDA content of tomato rlck86 mutant plants under drought conditions was significantly lower than that of wild-type plants (P<0.01). It is speculated that the mutant plants can improve the drought tolerance of tomatoes by reducing the damage of lipid membrane during drought.

[0071] Example 3: RLCK86 regulates stomatal movement to improve drought resistance in tomatoes

[0072] After treating the wild type tomato and the rlck86 mutant with 50 μM abscisic acid (ABA), the stomatal opening and closing were observed and counted. The specific method is as follows:

[0073] Prepare stomatal buffer: 30 mM KCl, 10 mM MES, 50 μM CaCl2, pH = 6.15.

[0074] The epidermis on the back of the leaves of the tomato rlck86 mutant and wild-type control cultured in the artificial climate chamber was torn off with tweezers and floated in stomatal buffer at 100 μmol m -2 s -1 The leaves were then randomly divided into two groups and transferred to stomatal buffer containing or without 50 μM ABA. -2 s -1 The cells were incubated at room temperature for 15 min under light intensity. The final stomatal conductance was then measured using an optical microscope (Leica, Wetzlar, Germany) equipped with a digital camera and the image analysis software ImageJ.

[0075] The results are as follows Figure 4 As shown, stomata in the tomato rlck86 mutant are more sensitive to ABA.

[0076] Further analysis of the relative water loss rate of detached leaves showed that Figure 5 As shown, the relative water loss rate of tomato rlck86 mutant plants was significantly slower than that of the control wild-type plants.

[0077] The above results suggest that RLCK86 affects the plant water loss rate by regulating ABA-mediated stomatal movement, thereby changing the plant's drought resistance.

Claims

1. The application of RLCK86 gene in improving drought tolerance of tomatoes is characterized by: The application includes: using biological technology to silence or delete the function of the tomato RLCK86 gene, thereby improving the tolerance of tomato plants to drought stress; the nucleotide sequence of the protein coding region of the RLCK86 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The amino acid sequence of the protein encoded by the RLCK86 gene is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that Gene mutation, gene knockout, gene interference or gene silencing technology is used to reduce or eliminate the expression of the RLCK86 gene in tomato plants, thereby obtaining mutant plants with enhanced drought resistance.

4. The use according to claim 1, characterized in that When the RLCK86 gene function in tomato plants is silenced or lost, it promotes the closure of stomata in the leaf epidermis of tomato plants and enhances the tolerance of tomato plants to drought stress.

5. A method for enhancing drought resistance of tomatoes, characterized in that: The following steps are involved: (1) A target fragment containing a PAM structure was selected from the protein coding region of the tomato RLCK86 gene, and primers were designed based on the first 20 bases of the PAM structure of the target fragment to construct a CRISPR / Cas9 vector; (2) constructing an Agrobacterium genetically engineered bacterium containing the CRISPR / Cas9 vector described in step (1); (3) The genetically engineered bacteria described in step (2) are transformed into tomato cotyledons, and a homozygous mutant strain with stable inheritance and mutation of the target sequence is obtained by cultivation and screening.

6. The method according to claim 5, characterized in that The nucleotide sequence of the first 20 bases of the PAM structure of the target fragment is shown in SEQ ID NO.

3.

7. The method according to claim 6, characterized in that The nucleotide sequences of the primer pairs for constructing the CRISPR / Cas9 vector are shown in SEQ ID NO.4 and SEQ ID NO.

5.

8. The method according to claim 5, characterized in that The host bacteria for constructing genetically engineered bacteria is GV3101 Agrobacterium.

9. The method according to claim 5, characterized in that The tomato variety is Condine Red.

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