Application of rlck86 gene in improving drought tolerance of tomato

Abstract

The application discloses application of an RLCK86 gene in improving drought resistance of tomatoes and belongs to the technical field of biotechnology.The application comprises the following steps: silencing or deleting a tomato RLCK86 gene by means of a biological technology, and improving the drought stress tolerance of tomato plants; and the nucleotide sequence of the protein coding region of the RLCK86 gene is shown in SEQ ID NO.1.The application discloses, for the first time, the use of the RLCK86 gene in regulating the drought stress resistance of tomatoes, and the drought resistance of the tomatoes can be significantly improved by deleting the RLCK86 gene function through a gene editing technology, thereby providing a reference for cultivating drought-resistant tomato germplasm resources.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to the application of RLCK86 gene in improving the drought tolerance of tomato. BACKGROUND

[0002] Water plays an important role in the growth and development of tomato, especially in the flowering and fruiting stages, more water is needed to promote fruit development and increase yield. It is reported that 300 kg of water is consumed to produce 1 kg of tomato fruit. Drought is one of the most important factors affecting crop yield and quality, and tomato plants will be affected when they encounter high temperature (temperature continuously exceeds 35 degrees) and drought weather and cannot replenish water in time, which will affect plant growth and lead to yield reduction and quality deterioration.

[0003] Therefore, it is very important to study and improve the drought tolerance genes of tomato. It has been a difficult problem in the field to create tomato drought tolerance germplasm to ensure normal plant growth under limited water supply conditions.

[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 there are many studies on plant disease resistance and stress resistance regulation.

[0005] For example, the RLCK protein BIK1 in Arabidopsis is a key signal transduction element in plant basic 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] The RLCK protein OsSAPK8 in rice can phosphorylate the calcium ion channel OsCNGC9 to trigger Ca 2+The inner flow enhances the resistance of the plant to frost (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 stress, the function of RLCK family members in the regulation of plant drought resistance is rarely reported. SUMMARY

[0008] The purpose of the present application is to study and explore the genes involved in the regulation of drought tolerance in tomato, and apply them to the creation and selection of drought-tolerant tomato germplasm.

[0009] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0010] The present application provides the application of RLCK86 gene in improving the drought tolerance of tomato, which includes: using biological technology to silence or delete the function of tomato RLCK86 gene, and improving the drought tolerance of tomato plants.

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

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

[0013] The present application shows that by deleting the function of tomato RLCK86 gene, the resistance of tomato plants to drought stress can be significantly enhanced, which can be applied to drought-tolerant plant breeding.

[0014] Further, the expression of RLCK86 gene in tomato plants is reduced or deleted by using gene mutation, gene knockout, gene interference or gene silencing technology, so as to obtain mutant plants with enhanced drought tolerance.

[0015] The present application uses CRISPR-Cas9 gene editing technology to obtain two tomato rlck86 mutant strains, and the drought tolerance of the mutant plants is significantly enhanced compared with the control wild type plants. It is revealed that tomato RLCK86 plays a negative regulatory role in drought resistance.

[0016] Further mechanism research shows that the tomato RLCK86 gene affects the water loss rate by affecting the stomata movement in the plant leaf epidermis, and regulates the drought resistance of the tomato to drought stress. After the RLCK86 gene function is silenced or deleted in the tomato plant, the closing of the stomata in the leaf epidermis of the tomato plant is promoted, the damage of the lipid membrane in drought is reduced, and the drought tolerance of the tomato plant to drought stress is enhanced.

[0017] The application further provides a method for enhancing the drought resistance of a tomato, comprising the following steps:

[0018] (1) selecting a target fragment containing a PAM structure in the protein coding region of the tomato RLCK86 gene, and designing primers 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 genetic engineering bacterium containing the CRISPR / Cas9 vector in step (1);

[0020] (3) transforming the genetic engineering bacterium in step (2) into a tomato cotyledon, and cultivating and screening to obtain a stable genetic homozygous mutant strain line without foreign Cas9 protein and with a target sequence variation.

[0021] In the above method, a mutant strain line with a RLCK86 gene variation is obtained by using a gene editing technology and a tissue culture technology, and the mutant strain line shows enhanced drought stress resistance.

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

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

[0024] Further, the host bacterium for constructing the genetic engineering bacterium is GV3101 agrobacterium.

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

[0026] The application has the following beneficial effects:

[0027] The application first discloses the use of the RLCK86 gene in regulating the drought stress resistance of the tomato, and the drought tolerance of the tomato can be significantly improved by deleting the RLCK86 gene function through a gene editing technology, thereby providing a reference for cultivating drought-resistant tomato germplasm resources. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Tomato rlck86 mutant gene editing type.

[0029] Figure 2 Tomato rlck86 mutant drought phenotype (scale length: 10 cm).

[0030] Figure 3 Tomato rlck86 mutant leaf MDA content.

[0031] Figure 4 Tomato guard cell morphology before and after 50 μM ABA treatment (scale length: 50 μm).

[0032] Figure 5 Tomato leaf guard cell opening before and after 50 μM ABA treatment.

[0033] Figure 6 Relative water loss rate of tomato in vitro leaf. DETAILED DESCRIPTION

[0034] The application will be further described below in conjunction with specific examples. The following examples are only used to illustrate the application and are not used to limit the application. Any modification or replacement of the method, step or condition of the application without departing from the spirit and essence of the application shall fall within the scope of the application.

[0035] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0036] The tomato variety used in the following examples is the conventional tomato variety Condine Red, and the ordinary tomato without gene editing is used as a control (Wildtype, WT).

[0037] Example 1: Construction of gene editing to screen mutant lines

[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. RLCK86 encodes a receptor-like cytoplasmic kinase composed of 787 amino acids, and its sequence is shown in SEQ ID NO. 2.

[0040] The PAM sequence was searched using CRISPR-P2.0 (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / ), the result with the first comprehensive score was selected, and the sequence of 20 bp before NGG was defined as a target (Target), which specifically targets the third exon of RLCK86, and the DNA sequence is: 5'-TTGAAGAGTCAGTAAAACGG-3'(SEQ ID NO. 3).

[0041] The CRISPR primer was designed for the above target, as follows:

[0042] CRISPR / Cas9 vector forward primer:

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

[0044] CRISPR / Cas9 vector reverse primer:

[0045] 5'-gcGGTCTCTAAACCCGTTTTACTGACTCTTCAAtgcaccagccgggaat cg-3'(SEQ ID NO. 5);

[0046] The above primers were respectively used for PCR amplification with pBAtC-tRNA plasmid as a template, and the product was purified by a DNA purification kit, and the amplified fragment was connected with pHEE401 plasmid by Bsa I enzyme, and the product was transformed into E. coli at 42°C, and plated and screened, and the resistance was kanamycin.

[0047] A single colony was selected, and a verification primer designed for the corresponding pHEE401 vector was used for PCR verification, and the verification primer was 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 liquid with correct band size was sent to a sequencing company for sequencing, and the sequencing result showed that the vector contained the sgRNA sequence, and the plasmid was extracted and introduced into Agrobacterium GV3101 competent cells by heat shock, and the colony was selected for bacterial liquid PCR verification after 48 h of culture at 28°C, and the Agrobacterium strain that could be used for genetic transformation was obtained.

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

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

[0053] T0 generation of gene edited tomato seedling detection: the genomic DNA of T0 generation plant was extracted by CTAB method, and was used as a template to design the following primers about 100-250 bp before and after the DNA sequence containing sgRNA, and PCR amplification and sequencing verification were carried out:

[0054] Tomato rlck86 mutant material verification primer before:

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

[0056] Tomato rlck86 mutant material verification primer after

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

[0058] The obtained PCR product was sent to a sequencing company for sequencing. The sequencing results were compared with the original sequence of the gene by using Snapgene software, and the plants with sgRNA sequence and its upstream and downstream base deletion, and single peak displayed by sequencing were selected for self-crossing and breeding to obtain T0 generation seeds.

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

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

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

[0062] The T1 generation plants with sgRNA variation and without Cas9 protein were selected as two strains of gene edited plants, which were named as rlck86#6 and rlck86#14, respectively, and the gene editing sites were as follows: Figure 1The rlck86#6 deletion is 14 bp and the rlck86#14 deletion is 2 bp compared to the control plants. Both types of editing result in a frame shift mutation of RLCK86 and premature termination.

[0063] After sowing the T1 generation seeds of the above two lines, T2 generation plants with stable inheritance of the absence of the exogenous gene Cas9 and variation of sgRNA were obtained.

[0064] The following examples were all carried out using the T2 generation plants of the above two homozygous lines as materials.

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

[0066] Tomato wild type and 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 2-3 d, with water changed every 12 h. After germination, the seeds were sown in 36-hole plug trays and placed in a phytotron. The temperature in the phytotron was 25 / 20°C (day / night), the light cycle was 12 h light / 12 h dark, and the light intensity was 200 μmol m -2 s -1 When the tomato seedlings grew to one true leaf, the seedlings were transplanted into pots, and Hoagland nutrient solution was poured into the pots every 3 d.

[0068] When the tomato seedlings grew to four leaves with a central stem, healthy plants of similar size were selected and randomly divided into two groups. The control group was normally watered, and the experimental group was subjected to natural drought treatment by withholding water. After 7 d in the phytotron, the plants were observed for drought damage.

[0069] The results are shown in Figure 2 The wild type plants showed severe water loss from the leaves, while the tomato rlck86 mutant plants showed tolerance to the drought environment.

[0070] The malondialdehyde (MDA) content of the tomato leaves was further determined, and the results are shown in Figure 3 It was found that the MDA content of the tomato rlck86 mutant plants was significantly lower than that of the wild type plants under drought conditions (P < 0.01), suggesting that the mutant plants could improve the drought tolerance of the tomato by reducing the damage to the lipid membrane under drought conditions.

[0071] Example 3: RLCK86 regulates stomatal movement to improve drought resistance of tomato

[0072] Tomato wild type and rlck86 mutant plants were treated with 50 μM abscisic acid (ABA), and the opening and closing of the stomata were observed and counted, as follows:

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

[0074] From tomato rlck86 mutant and wild type control plants grown in a phytotron, leaves at the same leaf position were taken and the abaxial epidermis was peeled off with forceps and floated on stomatal buffer at 100 μmol m -2 s -1 Incubation at room temperature for 1 h under light intensity allowed stomata to fully open. Then the epidermis was randomly divided into two groups and transferred to stomatal buffer with and without 50 μM ABA at 100 μmol m -2 s -1 Incubation at room temperature for 15 min under light intensity. The final stomatal conductance was measured with an optical microscope (Leica, Wetzlar, Germany) equipped with a digital camera and the image analysis software ImageJ.

[0075] The results are shown in Figure 2. Figure 4 The stomata in tomato rlck86 mutant are more sensitive to ABA.

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

[0077] From the above results, it is speculated that RLCK86 affects the water loss rate of plants by regulating ABA-mediated stomatal movement, thereby changing the drought resistance of plants.​

Claims

1. Silence or absence RLCK86 The application of a gene reagent in improving drought tolerance of tomatoes is characterized in that: The application includes: using biological technology to make tomatoes RLCK86 Gene function silencing or deletion improves the tolerance of tomato plants to drought stress; RLCK86 The nucleotide sequence of the protein coding region of the gene is shown in SEQ ID NO.

1.

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

2.

3. The use according to claim 1, characterized in that Tomato plants RLCK86 When the gene function 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.

4. A method for enhancing drought resistance of tomatoes, characterized in that: The following steps are involved: (1) In tomato RLCK86 A target fragment containing a PAM structure was selected from the protein coding region of the gene. Primers were designed based on the first 20 bases of the PAM structure of the target fragment to construct the CRISPR / Cas9 vector. (2) constructing an Agrobacterium genetically engineered bacterium containing the CRISPR / Cas9 vector described in step (1); (3) Transforming the genetically engineered bacteria described in step (2) into tomato cotyledons, and cultivating and screening to obtain a stably inherited homozygous mutant strain that does not contain exogenous Cas9 protein and has a mutation in the target sequence; described RLCK86 The nucleotide sequence of the protein coding region of the gene is shown in SEQ ID NO.1; The nucleotide sequence of the first 20 bases of the PAM structure of the target fragment is shown in SEQ ID NO.

3.

5. The method according to claim 4, wherein 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.

6. The method according to claim 4, wherein The host bacteria for constructing genetically engineered bacteria is Agrobacterium GV3101.

7. The method according to claim 4, wherein The tomato variety is Condine Red.

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