AvCHIT1 protein and application thereof in improving salt tolerance of plants

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to an AvCHIT1 protein and application thereof in improving salt tolerance of plants. The invention provides an AvCHIT1 protein, wherein the amino acid sequence of the AvCHIT1 protein is as shown in SEQ ID NO. 2. The AvCHIT1 protein plays an important role in adversity stress response, and can improve the salt tolerance of plants. The result of the embodiment shows that the AvCHIT1 protein is overexpressed in arabidopsis thaliana, the germination rate of arabidopsis thaliana seeds can be remarkably increased, the salt tolerance of the arabidopsis thaliana seeds can be improved, the AvCHIT1 gene is constructed into a plant expression vector, genetic transformation is carried out through an agrobacterium tumefaciens-mediated genetic transformation method, and new germplasm with the salt tolerance characteristic can be effectively obtained. The method is of great significance to cultivation of new varieties of excellent crops and wide application in production.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to an AvCHIT1 protein and an application thereof in improving the salt tolerance of plants. Background Art

[0002] Apocynum venetum is a plant of the genus Apocynum in the family Apocynaceae. It was named after the Lop Nur area in Xinjiang where it was discovered. It has strong adaptability to the environment and can grow in arid areas, saline-alkali land and even coastal tidal flats. It is a halophyte with strong salt tolerance.

[0003] In recent years, the agricultural ecological environment has continued to deteriorate, and high-salt stress has become more and more serious. Soil salinization has become an important problem that needs to be solved in the field of agricultural production worldwide, and has become a key stress factor affecting plant growth, development and yield. Breeding new salt-tolerant crop varieties is an important way to effectively utilize saline soil and increase grain production. In the process of adaptive growth and evolution under long-term salt stress, salt-tolerant plants have established a series of mechanisms to reduce the harm of salt stress, and are good donors for mining excellent salt-tolerant genes. With the continuous improvement of molecular biology technology, improving plant stress resistance through genetic engineering combined with molecular design concepts has become a hot research direction. Therefore, it is crucial to mine and utilize salt-tolerant genes. Summary of the invention

[0004] The purpose of the present invention is to provide an AvCHIT1 protein and its application in improving plant salt tolerance. The AvCHIT1 protein of the present invention can regulate the salt tolerance of plants and can be used to create new salt-tolerant germplasm.

[0005] The present invention provides an AvCHIT1 protein, the amino acid sequence of the AvCHIT1 protein is shown as SEQ ID NO.2.

[0006] The present invention also provides an AvCHIT1 gene, which is a gene encoding the AvCHIT1 protein described in the above scheme; the cDNA sequence of the AvCHIT1 gene is shown in SEQ ID NO.1.

[0007] The present invention also provides the use of the AvCHIT1 protein described in the above scheme or the AvCHIT1 gene described in the above scheme in regulating plant salt tolerance.

[0008] Preferably, the regulation includes: increasing the expression level of AvCHIT1 protein or increasing the expression level of AvCHIT1 gene to improve the salt tolerance of plants.

[0009] Preferably, said improving plant salt tolerance comprises improving plant germination rate.

[0010] Preferably, the plant comprises Apocynum venetum and / or Arabidopsis thaliana.

[0011] The present invention also provides a recombinant expression vector, which comprises the AvCHIT1 gene described in the above scheme.

[0012] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the recombinant expression vector described in the above scheme.

[0013] The present invention also provides the use of the AvCHIT1 protein described in the above scheme, or the AvCHIT1 gene described in the above scheme, or the recombinant expression vector described in the above scheme, or the recombinant bacteria described in the above scheme in cultivating salt-tolerant plant varieties.

[0014] The present invention also provides a method for improving plant salt tolerance, comprising: transferring the recombinant expression vector described in the above scheme or the recombinant bacteria described in the above scheme into the plant.

[0015] The present invention provides an AvCHIT1 protein, the amino acid sequence of the AvCHIT1 protein is shown in SEQ ID NO.2. The AvCHIT1 protein of the present invention plays an important role in the response to adverse stress and can improve the salt tolerance of plants. The results of the embodiment show that overexpression of the AvCHIT1 protein in Arabidopsis can significantly increase the germination rate of Arabidopsis seeds and improve its salt tolerance. The AvCHIT1 gene is constructed into a plant expression vector and genetically transformed by an Agrobacterium-mediated genetic transformation method, and new germplasm with salt tolerance can be effectively obtained. The present invention is of great significance for the cultivation of new varieties of excellent crops and their wide application in production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 The figure is a diagram for identifying the salt tolerance characteristics of Apocynum venetum in Example 1; wherein A is the morphological phenotype of Apocynum venetum seedlings after a series of salt concentration treatments; B is the dry-to-fresh matter ratio of Apocynum venetum seedlings; C is the Na content of Apocynum venetum seedling leaves + content; D is the germination phenotype of Apocynum venetum seeds under salt stress; E is the germination rate of Apocynum venetum seeds under salt stress;

[0018] Figure 2 This is a screening diagram of differentially expressed genes in response to salt stress in Apocynum venetum in Example 2, wherein A is the Venn diagram analysis result; B is the gene expression trend analysis result;

[0019] Figure 3The above diagram is the up-regulated expression diagram of AvCHIT1 gene induced by salt stress in Example 3; A is the qRT-PCR corrected transcriptome data; B is the semi-quantitative experiment of AvCHIT1 induced by salt stress;

[0020] Figure 4 AvCHIT1 phylogenetic tree in Example 4;

[0021] Figure 5 Analysis diagram of AvCHIT1, HOT2 and PR-3 proteins in Example 4; wherein A is a sequence alignment diagram of AvCHIT1, HOT2 and PR-3 proteins; B is an analysis diagram of conserved domains of AvCHIT1, HOT2 and PR-3 proteins;

[0022] Figure 6 The tissue expression diagram of AvCHIT1 protein in Example 5; A is the subcellular localization diagram of AvCHIT1 protein; B and C are the spatiotemporal expression patterns of AvCHIT1 protein detected by GUS staining, the scale in B is 2 mm, and the scale in C is 5 mm;

[0023] Figure 7 Figure 6 is a graph showing the tolerance of the AvCHIT1 overexpressing strain to sodium chloride; A is the phenotype of Arabidopsis thaliana under control and different concentrations of sodium chloride stress; B is the statistics of Arabidopsis root length; C is the statistics of Arabidopsis Na + content;

[0024] Figure 8 The figure is a validation diagram of the AvCHIT1 complementation experiment in Example 7; A is the background detection of hot2, pr3, 35Spro:AvCHIT1, 35Spro:AvCHIT1:hot2 and 35Spro:AvCHIT1:pr-3 mutants; B is pH = 6.0, 150 mM Seed germination experiment of WT, hot2, pr3, 35Spro:AvCHIT1, 35Spro:AvCHIT1:hot2 and 35Spro:AvCHIT1:pr-3 under NaCl treatment conditions; C is the seed germination experiment of WT, hot2, pr3, 35Spro:AvCHIT1, 35Spro:AvCHIT1:hot2 and 35Spro:AvCHIT1:pr-3 under pH = 5.0 and 150mM NaCl treatment conditions; D is the order of seeds placed in the culture dish; E is the statistical chart of seed germination rate of WT, hot2, pr3, 35Spro:AvCHIT1, 35Spro:AvCHIT1:hot2 and 35Spro:AvCHIT1:pr-3 under two pH conditions. DETAILED DESCRIPTION

[0025] The present invention provides an AvCHIT1 protein. The amino acid sequence of the AvCHIT1 protein is shown in SEQ ID NO.2: MRFWAVVLCSALCLVGVSAQVGSFVSKSLFDQLLKHRNDANCPAKGFYTYEAFIAAANSFGGFGTTGDTDTRKREIAAFLAQTSHETTGGWASAPDGPYSWGYCFKQEQGNPPSYCVQSQQWPCAPGKKYYGRGPIQISYNYNYGPAGRAIGANLLNNPDLVANDPVISFKTALWFWMTPQQPKPSSHDVMTGRWRPSGADSAAGRVPGFGVVTNIINGGIECGKGNNAQMQSRIGFYRRYCSILGVSPGNNLDCANQRPFA.

[0026] The present invention also provides an AvCHIT1 gene, which is a gene encoding the AvCHIT1 protein described in the above scheme; the cDNA sequence of the AvCHIT1 gene is shown in SEQ ID NO.1, and the end is a stop codon, which is as follows:

[0027] 5′-ATGAGGTTTTGGGCAGTGGTTTTGTGCTCTGCATTGTGTCTTGTAGG -3′.

[0028] The present invention also provides the use of the AvCHIT1 protein described in the above scheme or the AvCHIT1 gene described in the above scheme in regulating plant salt tolerance. As an embodiment, the regulation of the present invention includes: increasing the expression of the AvCHIT1 protein or increasing the expression of the AvCHIT1 gene to improve plant salt tolerance. As an embodiment, the improving plant salt tolerance includes improving plant germination rate.

[0029] As an embodiment, the plant of the present invention includes Apocynum venetum and / or Arabidopsis thaliana. In a specific embodiment, the plant may be Apocynum venetum or Arabidopsis thaliana.

[0030] The present invention also provides a recombinant expression vector, which comprises the AvCHIT1 gene described in the above scheme.

[0031] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the recombinant expression vector described in the above scheme.

[0032] The present invention also provides the use of the AvCHIT1 protein described in the above scheme, or the AvCHIT1 gene described in the above scheme, or the recombinant expression vector described in the above scheme, or the recombinant bacteria described in the above scheme in cultivating salt-tolerant plant varieties.

[0033] The present invention also provides a method for improving plant salt tolerance, comprising: transferring the recombinant expression vector or the recombinant bacteria described in the above scheme into a plant. As an embodiment, the plant of the present invention includes Apocynum venetum and / or Arabidopsis thaliana. In a specific embodiment, the plant can be Apocynum venetum or Arabidopsis thaliana.

[0034] To further illustrate the present invention, an AvCHIT1 protein provided by the present invention and its application in improving plant salt tolerance are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] ① Identification of salt tolerance of Apocynum venetum

[0037] Two-week-old Apocynum venetum seedlings were randomly divided into six groups and treated with five different concentrations of NaCl solution (0 mM, 20 mM, 50 mM, 100 mM, 200 mM and 400 mM) for four weeks. The growth of Apocynum venetum was observed and the NaCl content in Apocynum venetum leaves was determined. + content, determine the fresh weight and dry weight of Apocynum venetum, calculate its fresh-dry ratio, and refer to Figure 1 In A~C, different lowercase letters in the figure represent p < 0.05.

[0038] Na +Content determination method: Take 2g of Apocynum venetum leaves and keep them in an oven at 120℃ for 30min, then place them in an oven at 80℃ overnight to dry them to constant weight. Grind the dried Apocynum venetum leaves into powder and pass them through a 0.5mm sieve to obtain Apocynum venetum powder. Take 0.25g of Apocynum venetum powder, digest it with 5mL HNO3, keep it at 110℃ until it becomes a colorless liquid (about 6h), then cool the liquid mixture to room temperature and make up to 10mL with deionized water. Use a Perkins-Elmer 360 atomic absorption spectrophotometer to determine the Na content of the supernatant. + concentration.

[0039] according to Figure 1 It can be seen that the growth of Apocynum venetum was gradually inhibited with the increase of salt concentration. Under the 400mM treatment, the growth of Apocynum venetum seedlings almost stopped, and they gradually aged and died within the next 2 to 3 weeks ( Figure 1 However, its biomass dry-to-fresh ratio tended to increase gradually with the increase of salt concentration, and reached the maximum under the treatment of 200mM NaCl ( Figure 1 B). As the salt concentration increases, the Na + The content of Figure 1 C), which indicates that a large amount of Na accumulated in the leaves of Apocynum venetum + , which may be the reason why the external morphology of the plant is inhibited during growth.

[0040] ② Determination of germination rate of Apocynum venetum seeds

[0041] 0mM, 10mM, 20mM, 50mM, 100mM and 250mM NaCl were added to the culture medium, and the seeds of Apocynum venetum were placed in the culture medium for seed germination experiment. The germination of seeds was observed and the germination rate was calculated. The results are shown in Figure 1 D and E in .

[0042] The results showed that there was no salt treatment concentration that could promote the germination of Apocynum venetum seeds ( Figure 1 D), even in the medium containing only 10mMNaCl, the germination rate of Apocynum venetum seeds (44.03%) was significantly lower than that of the medium without sodium chloride (75.28%). Figure 1 E), indicating that salt stress can inhibit the germination of Apocynum venetum seeds.

[0043] Although there is no optimal salt concentration to promote the growth of Apocynum venetum during seed germination and seedling growth stages, the seedlings can maintain growth at a concentration of up to 200 mM sodium chloride (salt content of about 11.7‰), indicating that Apocynum venetum is an atypical halophyte that can exhibit strong salt tolerance.

[0044] Example 2

[0045] Potted adult Apocynum venetum was randomly divided into two groups. One group was treated with 200 mM NaCl for salt stress, which was recorded as the treatment group, NaCl, and the other group was treated with an equal amount of pure water as the control group CK. Samples were taken at 10 am on the 0th, 7th and 18th days, and the Apocynum venetum leaves of the control group and the treatment group were respectively taken for transcriptome sequencing, where the control group was recorded as CK0Z, CK7Z, CK18Z, and the treatment group was recorded as NaCL7Z and NaCl18Z, respectively, and each treatment had 3 biological replicates.

[0046] A total of 254,005 transcripts were obtained by next-generation transcriptome sequencing, and 133,486 longest transcripts were obtained after splicing. The reference-free transcriptome used FPKM (Fragments Per Kilobase of transcript per Million mapped reads) as an indicator to measure gene expression levels, and differentially expressed genes (DEGs) were determined using two parameters: (1) |log2Fold Change (expected count)|>=1; (2) FDR<0.05. After removing duplicate genes, a total of 9134 differentially expressed genes were identified in the 10 comparison groups, including CK0Z_vs_CK7Z(3235), CK0Z_vs_CK18Z(2932), CK7Z_vs_CK18Z(1497), CK0Z_vs_NaCl7Z(2430), CK0Z_vs_NaCl18Z(1848), CK7Z_vs_NaCl7Z(814), CK7Z_vs_NaCl18Z(2727), CK18Z_vs_NaCl7Z(2636), CK18Z_vs_NaCl18Z(2775), and NaCl7Z_vs_NaCl18Z(814).

[0047] A total of 25 common differentially expressed genes ( Figure 2 A in the figure); Gene expression trend analysis revealed 22 genes that were upregulated under salt stress ( Figure 2 B). There were 15 differentially expressed genes in both strains (see Table 1), among which TRINITY_DN39146_c0_g1 encoded a chitinase gene, which was homologous to the Arabidopsis chitinase gene At3g12500 (PR-3) and named AvCHIT1.

[0048] Table 1. Common differentially expressed genes in Venn diagram analysis and gene expression trend analysis

[0049] GeneID Arabidopsis thaliana homologous gene Adjust up and down Gene annotation TRINITY_DN42650_c1_g1 At1g20380 Increase POQR TRINITY_DN38959_c0_g2 At1g65800 Increase ATARK2 TRINITY_DN44625_c1_g1 At2g40300 Increase FER4 TRINITY_DN41289_c3_g1 At2g47760 Increase ALG3 TRINITY_DN39146_c0_g1 At3g12500 Increase PR-3 TRINITY_DN40614_c3_g5 At3g22640 Increase PAP85 TRINITY_DN44188_c0_g4 At3g57270 Increase BG1 TRINITY_DN33945_c0_g2 At4g09670 Increase Oxidoreductase TRINITY_DN42941_c3_g8 At5g19040 Increase IPT5 TRINITY_DN42308_c0_g2 At5g66400 Increase ATDI8 TRINITY_DN29716_c0_g1 At4g11650 Increase ATOSM34 TRINITY_DN32094_c0_g1 At1g53540 Increase HSP20 TRINITY_DN28411_c1_g1 At4g27670 Increase HSP21 TRINITY_DN28657_c0_g1 At4g10250 Increase HSP22.0 TRINITY_DN31702_c0_g1 At4g25200 Increase HSP23.6

[0050] Example 3

[0051] The transcriptome data were verified by qRT-PCR, wherein the primer sequences of the AvCHIT1 gene in the qRT-PCR were shown in SEQ ID NO.3 and SEQ ID NO.4, and the internal reference gene was the Apocynum venetum EF-α gene, wherein the primer sequences of the Apocynum venetum EF-α gene were shown in SEQ ID NO.5 and SEQ ID NO.6.

[0052] AvCHIT1-qRTPCR-F (SEQ ID NO.3):

[0053] 5′-TGGTGCAAATCTGCTAAACAATC-3′;

[0054] AvCHIT1-qRTPCR-R(SEQ ID NO.4):

[0055] 5′-TGCGGTGTCATCCAAAACCATA-3′;

[0056] AvEF-α-qRTPCR-F (SEQ ID NO.5):

[0057] 5′-TGAGAGGTCTACCAACCTTGA-3′;

[0058] AvEF-α-qRTPCR-R (SEQ ID NO.6):

[0059] 5′-TTGGGCTCAGAAATCAGGTCAA-3′.

[0060] The qRT-PCR system is 20 μL, including 2×ChamQ SYBR qPCRMasterMix 10 μL, 10 μM forward primer 0.4 μL, 10 μM reverse primer (R) 0.4 μL, 50×ROX Reference Dye 10.4 μL, cDNA template 1 μL, ddH2O is added to 20 μL, and 3 technical replicates are set; the cDNA template in the system is cDNA obtained by reversing 1 μg RNA.

[0061] The qRT-PCR amplification adopts a two-step method, and the reaction procedure is pre-denaturation at 95°C for 30 seconds; then 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds; then run the melting curve stage: 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds. After the reaction is completed, a melting curve analysis is performed.

[0062] The transcriptome data and qRT-PCR data were analyzed, and the results are shown in Figure 3 A in Example 2, wherein CK0 corresponds to CK0Z in Example 2, CK7 corresponds to CK7Z in Example 2, CK18 corresponds to CK18Z in Example 2, NaCl7 corresponds to NaCl7Z in Example 2, and NaCl18 corresponds to NaCl18Z in Example 2. The results showed that the expression trends of qRT-PCR data and transcriptome data in each sample were basically consistent, and the expression level of AvCHIT1 gene in Apocynum venetum under salt stress was significantly upregulated compared with the control.

[0063] After the 4-week-old Apocynum venetum seedlings were treated with 0mM, 50mM, 100mM and 200mM NaCl stress, the expression level of AvCHIT1 gene was detected by semi-quantitative experiment. The test results are shown in Figure 3 According to B. Figure 3 As shown in B, compared with the control, the expression level of AvCHIT1 gene was significantly upregulated under 100mM and 200mMNaCl stress.

[0064] Example 4

[0065] The phylogenetic tree of Arabidopsis chitinase genes and chitinase genes reported in the literature to be regulated by salt stress or overexpressed to enhance plant resistance was constructed using Mega6.0. Figure 4 .

[0066] according to Figure 4 It can be seen that AvCHIT1 and Arabidopsis type III chitinase gene PR-3 (At3g12500) are in the same evolutionary branch, indicating that the two have high homology and may have similar functions. The pepper chitinase gene CAChi2, which is significantly induced by salt stress and can improve the salt resistance of Arabidopsis by overexpression, is also in the same evolutionary branch as AvCHIT1, indicating that this type of chitinase gene has functional similarities. The protein sequences and domains of AvCHIT1, HOT2 and PR-3 were predicted using DNAMAN. The results are shown in Figure 5 , where PR-3 protein is the protein encoded by the Arabidopsis thaliana type III chitinase gene At3g12500. Figure 5 From the A in the figure, we can see that the primary sequence similarities of AvCHIT1, HOT2 and PR-3 are 30.22% and 50.75% respectively. In addition, AvCHIT1 has the same glycosyl hydrolase domain as HOT2 and PR-3 ( Figure 5 Therefore, it can be inferred that AvCHIT1 and HOT2 / PR-3 are likely to have similar functions. However, whether the transgenic AvCHIT1 gene of Apocynum venetum can improve the salt tolerance of plants still needs further experimental verification.

[0067] Example 5

[0068] The p35S:AvCHIT1-GFP fusion protein expression vector and pro:AvCHIT1-GUS expression vector were constructed to study the subcellular localization of AvCHIT1 protein and the spatiotemporal expression pattern of AvCHIT1 gene.

[0069] The construction process of the pro:AvCHIT1-GUS expression vector is as follows: using pBI101-GUS vector as a basic vector (purchased from BioVector NTCC typical culture collection center, item number: pBI101 vetor), using Xba I single enzyme to cut the linearized vector; using cDNA of leaves of Apocynum venetum seedlings as a template, using amplification primers pAvCHIT1-GUS-F (SEQ ID NO.7) and pAvCHIT1-GUS-R (SEQ ID NO.8) to obtain an AvCHIT1 gene fragment containing a promoter, and under the action of a homologous recombinase, connecting the AvCHIT1 gene fragment containing a promoter with the linearized vector to obtain a pro:AvCHIT1-GUS recombinant vector.

[0070] pAvCHIT1-GUS-F (SEQ ID NO.7):

[0071] 5′-TGCATGCCTGCAGGTCGACTCTAGAATGAGGTTTTGGGCAGTGGTT T-3′;

[0072] pAvCHIT1-GUS-R (SEQ ID NO.8):

[0073] 5′-ACTGACCACCCGGGGATCCTCTAGATGCAAATGGCCTTTGGTTGGC A-3′.

[0074] The pro:AvCHIT1-GUS recombinant vector was transformed into Agrobacterium GV3101 to obtain Agrobacterium GV3101 containing the p35S:AvCHIT1-GFP vector. The Agrobacterium GV3101 containing the p35S:AvCHIT1-GFP vector was used to transiently transform Nicotiana benthamiana, and the fluorescence signal was observed under a laser confocal microscope. The results are shown in Figure 6 A in. According to Figure 6 As can be seen from A in the figure, the fluorescent signal is mainly concentrated on the cell membrane, indicating that the AvCHIT1 protein is mainly located on the cell membrane and plays a role.

[0075] The construction process of the p35S:AvCHIT1-GFP fusion protein expression vector is as follows: using pCAMBIA1301-EGFP vector as a basic vector, using Kpn I and BamHI to double-digest the linearized vector; using cDNA of leaves of Apocynum venetum seedlings as a template, using amplification primers 35S-AvCHIT1-GFP-F (SEQ ID NO.9) and 35S-AvCHIT1-GFP-R (SEQ ID NO.10) to obtain an AvCHIT1 gene fragment, and under the action of a homologous recombinase, connecting the AvCHIT1 gene fragment with the linearized vector to obtain a p35S:AvCHIT1-GFP recombinant vector.

[0076] 35S-AvCHIT1-GFP-F (SEQ ID NO.9):

[0077] 5′-CGAACGATAGCCATGGTACCAATGAGGTTTTGGGCAGTGGTTT-3′;

[0078] 35S-AvCHIT1-GFP-R (SEQ ID NO.10):

[0079] 5′-CATGCCTGCGGCCGCGCCGGATCCTGCAAATGGCCTTTGGTTGGCA-3′.

[0080] The pro:AvCHIT1-GUS expression vector was transformed into Arabidopsis wild type by the flower drop method, and GUS staining was performed on the transgenic offspring at various growth stages. The staining results are shown in Figure 6 The results showed that GUS was expressed in the entire vascular tissue and mesophyll cells of the rosette leaves of the seedlings, and GUS was also detected in the hypocotyls of the seedlings ( Figure 6 In the B of Figure 2, there was no GUS signal in the leaves of mature plants, which indicates that the AvCHIT1 gene has a high expression level in the early stage of growth and development. In addition, GUS signals were also detected in the base of the pods and calyx of mature plants ( Figure 6 C).

[0081] Example 6

[0082] Construction of a 35Spro:AvCHIT1 overexpression vector: using pCAMBIA1301-EGFP vector as a basic vector, using KpnI and Xba I to double-digest the linearized vector; using cDNA of leaves of Apocynum venetum seedlings as a template, using amplification primers 35S-AvCHIT1-GFP-F (SEQ ID NO.9) and AvCHIT1-R1 (SEQ ID NO.11) to obtain an AvCHIT1 gene fragment; under the action of a homologous recombinase, the AvCHIT1 gene fragment was connected to the linearized vector to obtain a 35Spro:AvCHIT1 overexpression vector.

[0083] AvCHIT1-R1 (SEQ ID NO.11):

[0084] 5′-ACCGGCGCTCAGTTGGAATTCTAGATGCAAATGGCCTTTGGTTGGC A-3′.

[0085] The 35Spro:AvCHIT1 overexpression vector obtained in the above step was transformed into Agrobacterium GV3101 to obtain the GV3101 Agrobacterium strain carrying the 35Spro:AvCHIT1 overexpression vector. The GV3101 Agrobacterium strain carrying the 35Spro:AvCHIT1 overexpression vector was streaked on the LB medium, and a single clone was selected for shaking. When the OD value of the bacterial solution reached about 0.5, it was centrifuged, the supernatant was discarded, and the bacterial body was resuspended in a resuspension solution to obtain a resuspension solution, wherein the resuspension solution contained a final concentration of 5% (w / v) sucrose and 0.02% (v / v) Silwet. After soaking the blooming Arabidopsis inflorescence in the resuspension solution for 30 seconds, it was cultured in the dark for 12 hours, and the seeds were harvested and screened to obtain transgenic lines OE1 and OE2.

[0086] The salt tolerance phenotype of the positive plants OE1 and OE2 screened was identified, the root length of Arabidopsis seedlings was counted, and the Na content of leaves was measured. + Content, determination method is the same as in Example 1, results refer to Figure 7 , ** in the figure represents p<0.05.

[0087] according to Figure 7 It can be seen that the root length of transgenic plants OE1 and OE2 on the medium containing 100mM and 150mM NaCl is significantly longer than that of the control. + The results showed that under salt stress, Na + The content was also significantly lower than that of the wild type. These results indicate that the AvCHIT1 gene can enhance the salt resistance of plants.

[0088] Example 8

[0089] Verification of the complementarity of AvCHIT1 with PR-3 and HOT2 functions:

[0090] Arabidopsis pr-3 and hot2 mutants (article numbers: SALKseq_081718.1 and SALKseq_058663.1) were ordered from the European Arabidopsis Stock Center (https: / / arabidopsis.info / StockInfo?NASC_id=625057), and the 35Spro:AvCHIT1 overexpression vector obtained in Example 7 was transferred into Arabidopsis pr-3 and hot2 mutants by the drop flower method to obtain 35Spro:AvCHIT1:pr-3 and 35Spro:AvCHIT1:hot2 complementation lines. Arabidopsis AtTUBULIN was used as an internal reference gene, and semi-quantitative PCR was used to identify the background of the obtained mutant strains. The primer sequences used for the semi-quantitative PCR are shown in Table 2.

[0091] Table 2 Semi-quantitative PCR primer sequence information

[0092] Gene Primers Serial Number Sequence information AvCHIT1 AvCHIT1-F SEQ ID NO.3 5′-TGGTGCAAATCTGCTAAACAATC-3′ AvCHIT1 AvCHIT1-R SEQ ID NO.4 5′-TGCGGTGTCATCCAAAACCATA-3′ AtHOT2 AtHOT2-F SEQ ID NO.12 5′-ATGGTGACAATCAGGAGTGGTT-3′ AtHOT2 AtHOT2-R SEQ ID NO.13 5′-TTACGAAGAGGAAGAGGAAGG-3′ AtPR3 AtPR3-F SEQ ID NO.14 5′-ATGCCTCCACAAAAAGAAAACC-3′ AtPR3 AtPR3-R SEQ ID NO.15 5′-CTAAATAGCAGCTTCGAGGAGG-3′ AtTUBULIN AtTUBULIN-F SEQ ID NO.16 5′-ATCCGTGAAGAGTACCCAGAT-3′ AtTUBULIN AtTUBULIN-R SEQ ID NO.17 5′-AAGAACCATGCACTCATCAGC-3′

[0093] The semi-quantitative PCR results were as follows Figure 8 As shown in A, the exogenous AvCHIT1 gene did not amplify bands in Arabidopsis WT, hot2 and pr-3, but amplified bands in 35Spro:AvCHIT1, 35Spro:AvCHIT1:hot2 and 35Spro:AvCHIT1:pr-3, indicating that AvCHIT1 is expressed in all overexpression strains; while the AtHOT2 and AtPR3 genes did not amplify bands in their respective mutants, but amplified bands with basically the same brightness in other Arabidopsis strains, indicating that the mutant background is clean and each mutant material is successfully constructed.

[0094] The salt tolerance of six strains, WT, 35Spro:AvCHIT1, hot2, pr-3, 35Spro:AvCHIT1:hot2 and 35Spro:AvCHIT1:pr-3, was evaluated at the seed germination and seedling stages. The results are shown in Figure 8 According to B~E. Figure 8From Figures B to E, we can see that under normal conditions of pH = 6.0, after treatment with 150 mM NaCl, the seed germination rates of Arabidopsis hot2 and pr-3 mutants were significantly reduced, and the WT was slightly higher than the other two, but the germination rate was also around 70%; while the germination rate of AvCHIT1 overexpressing plants was close to 100%, and the germination rates of the two complementary lines 35Spro:AvCHIT1:hot2 and 35Spro:AvCHIT1:pr-3 were significantly higher than their respective controls, indicating that AvCHIT1 plays a role in improving plant salt tolerance.

[0095] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An AvCHIT1 protein, the amino acid sequence of the AvCHIT1 protein is shown in SEQ ID NO.

2.

2. An AvCHIT1 gene, wherein the AvCHIT1 gene is a gene encoding the AvCHIT1 protein according to claim 1; the cDNA sequence of the AvCHIT1 gene is shown in SEQ ID NO.

1.

3. Use of the AvCHIT1 protein according to claim 1 or the AvCHIT1 gene according to claim 2 in regulating plant salt tolerance.

4. The use according to claim 3, characterized in that: The regulation includes: increasing the expression level of AvCHIT1 protein or increasing the expression level of AvCHIT1 gene to improve plant salt tolerance.

5. The use according to claim 4, characterized in that: The improving of plant salt tolerance includes improving plant germination rate.

6. The use according to any one of claims 3 to 5, wherein the plant comprises Apocynum venetum and / or Arabidopsis thaliana.

7. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the AvCHIT1 gene according to claim 2.

8. A recombinant bacterium, characterized in that: The recombinant bacteria comprises the recombinant expression vector according to claim 6.

9. Use of the AvCHIT1 protein according to claim 1, the AvCHIT1 gene according to claim 2, the recombinant expression vector according to claim 7, or the recombinant bacteria according to claim 8 in cultivating salt-tolerant plant varieties.

10. A method for improving plant salt tolerance, characterized in that: include: The recombinant expression vector according to claim 7 or the recombinant bacteria according to claim 8 is introduced into a plant.

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