AvCHIT1 protein and application thereof in improving plant salt tolerance

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to an AvCHIT1 protein and its application in improving plant salt tolerance. This invention provides an AvCHIT1 protein, the amino acid sequence of which is shown in SEQ ID NO.2. The AvCHIT1 protein of this invention plays an important role in the response to abiotic stress and can improve the salt tolerance of plants. The results of the examples show that overexpression of the AvCHIT1 protein in Arabidopsis thaliana can significantly improve the seed germination rate and salt tolerance. Constructing the AvCHIT1 gene into a plant expression vector and performing genetic transformation using Agrobacterium-mediated transformation can effectively obtain new germplasm with salt tolerance characteristics. This invention is of great significance for the breeding of superior new crop varieties and their widespread application in production.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an AvCHIT1 protein and its application in improving plant salt tolerance. Background Technology

[0002] Apocynum venetum is a plant belonging to the genus Apocynum in the family Apocynaceae. It is named after the Lop Nur region in Xinjiang where it was first discovered. It has a strong ability to adapt to different environments and can grow in arid areas, saline-alkali lands, and even coastal mudflats. 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 increasingly severe. Soil salinization has become a critical problem that urgently needs to be solved in agricultural production worldwide, and a key stress factor affecting plant growth, development, and yield. Developing new salt-tolerant crop varieties is an important way to effectively utilize saline soils and increase grain yield. Salt-tolerant plants have established a series of mechanisms to mitigate the harm of salt stress through long-term adaptive growth and evolution, making them excellent donors for discovering superior salt-tolerant genes. With the continuous improvement of molecular biology techniques, improving plant stress resistance through genetic engineering combined with molecular design concepts has become a hot research topic. Therefore, discovering and utilizing salt-tolerant genes is of paramount importance. Summary of the Invention

[0004] The purpose of this invention is to provide an AvCHIT1 protein and its application in improving plant salt tolerance. The AvCHIT1 protein described in this 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 which is shown in SEQ ID NO.2.

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

[0007] This invention also provides the application of the AvCHIT1 protein 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 plant salt tolerance.

[0009] Preferably, improving plant salt tolerance includes increasing plant germination rate.

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

[0011] The present invention also provides a recombinant expression vector comprising 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 application of the AvCHIT1 protein, the AvCHIT1 gene, the recombinant expression vector, or the recombinant bacteria described in the above scheme in the cultivation of salt-tolerant plant varieties.

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

[0015] This invention provides an AvCHIT1 protein, the amino acid sequence of which is shown in SEQ ID NO.2. The AvCHIT1 protein of this invention plays an important role in the response to abiotic stress and can improve the salt tolerance of plants. Results from examples show that overexpression of the AvCHIT1 protein in Arabidopsis thaliana significantly improves seed germination rate and salt tolerance. Constructing the AvCHIT1 gene into a plant expression vector and performing genetic transformation using Agrobacterium-mediated transformation can effectively obtain new germplasm with salt tolerance characteristics. This invention is of great significance for the breeding of superior crop varieties and their widespread application in production. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The diagram shows the salt tolerance characteristics of Apocynum venetum in Example 1; where A represents the morphological phenotype of Apocynum venetum seedlings after a series of salt concentration treatments; B represents the dry-to-fresh mass ratio of Apocynum venetum seedlings; and C represents the Na+ content of Apocynum venetum seedling leaves. + Content; D represents the germination phenology of Apocynum venetum seeds under salt stress; E represents the germination rate of Apocynum venetum seeds under salt stress.

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

[0019] Figure 3This is a diagram showing the upregulation of the AvCHIT1 gene induced by salt stress in Example 3; where A is the qRT-PCR corrected transcriptome data; and B is the semi-quantitative experiment of AvCHIT1 induced by salt stress.

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

[0021] Figure 5 Example 4 shows the protein analysis diagrams for AvCHIT1, HOT2, and PR-3; where A is the sequence alignment diagram of AvCHIT1, HOT2, and PR-3 proteins; and B is the analysis diagram of conserved domains of AvCHIT1, HOT2, and PR-3 proteins.

[0022] Figure 6 This is a tissue expression map of AvCHIT1 protein in Example 5; where A is the subcellular localization map of AvCHIT1 protein; B and C are spatiotemporal expression patterns of AvCHIT1 protein detected by GUS staining, with a scale bar of 2 mm in B and 5 mm in C.

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

[0024] Figure 8 The diagram shows the AvCHIT1 complementation experiment verification in Example 7; where A represents the background detection of the hot2, pr3, 35Spro:AvCHIT1, 35Spro:AvCHIT1:hot2, and 35Spro:AvCHIT1:pr-3 mutants; B represents the pH=6.0, 150mM... C shows the seed germination experiments of WT, hot2, pr3, 35Spro:AvCHIT1, 35Spro:AvCHIT1:hot2, and 35Spro:AvCHIT1:pr-3 under NaCl treatment conditions; D shows the seed placement order in the petri dishes; E shows the statistical graph of seed germination rates of WT, hot2, pr3, 35Spro:AvCHIT1, 35Spro:AvCHIT1:hot2, and 35Spro:AvCHIT1:pr-3 under pH=5.0 and 150mM NaCl treatment conditions. Detailed Implementation

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

[0026] This invention also provides an AvCHIT1 gene, which is the encoding gene for the AvCHIT1 protein described in the above-described scheme; the cDNA sequence of the AvCHIT1 gene is shown in SEQ ID NO.1, ending with a stop codon, as follows:

[0027] 5′-ATGAGGTTTTGGGCAGTGGTTTTGTGCTCTGCATTGTGTCTTGTAGG -3′.

[0028] This invention also provides the application of the AvCHIT1 protein or the AvCHIT1 gene described in the above-described scheme in regulating plant salt tolerance. As one embodiment, the regulation described in this invention includes: increasing the expression level of the AvCHIT1 protein or increasing the expression level of the AvCHIT1 gene to improve plant salt tolerance. As one embodiment, improving plant salt tolerance includes increasing plant germination rate.

[0029] As one embodiment, the plant described in this 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 comprising 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 application of the AvCHIT1 protein, the AvCHIT1 gene, the recombinant expression vector, or the recombinant bacteria described in the above scheme in the cultivation of salt-tolerant plant varieties.

[0033] This invention also provides a method for improving the salt tolerance of plants, comprising: transferring the recombinant expression vector or the recombinant bacteria described in the above-described scheme into a plant. As one embodiment, the plant described in this invention includes *Apocynum venetum* and / or *Arabidopsis thaliana*. In a specific embodiment, the plant may be *Apocynum venetum* or *Arabidopsis thaliana*.

[0034] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes an AvCHIT1 protein provided by the present invention and its application in improving plant salt tolerance, but these descriptions 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 Na+ content in the leaves was measured. + The content was determined by measuring the fresh and dry weight of Apocynum venetum and calculating its dry-to-fresh ratio. The results are shown in [reference needed]. Figure 1 In the diagram, A through C represent p < 0.05, and different lowercase letters represent p < 0.05.

[0038] Na +Content determination method: Take 2g of Apocynum venetum leaves and dry them in an oven at 120℃ for 30min, then place them in an oven at 80℃ overnight to achieve 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 of HNO3, and maintain it at 110℃ until a colorless liquid is obtained (about 6h). Then cool the liquid mixture to room temperature and make up to 10mL with deionized water. Determine the Na content of the supernatant using a Perkins-Elmer 360 atomic absorption spectrophotometer. + concentration.

[0039] according to Figure 1 It can be seen that the growth of Apocynum venetum is gradually inhibited with increasing salt concentration. Under 400mM treatment, the growth of Apocynum venetum seedlings almost stops, and they gradually age and die within the next 2-3 weeks. Figure 1 (A) However, its biomass dry-to-fresh ratio gradually increases with increasing salt concentration, reaching its maximum under 200 mM NaCl treatment. Figure 1 (B) As the salt concentration increases, the Na+ in Apocynum venetum leaves... + The content gradually increased ( Figure 1 The presence of C in the sample indicates that a large amount of Na has accumulated in the leaves of Apocynum venetum. + This may be the reason why the plant's external morphology is inhibited during growth.

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

[0041] NaCl was added to the culture medium at concentrations of 0 mM, 10 mM, 20 mM, 50 mM, 100 mM, and 250 mM, respectively. Seeds of *Apocynum venetum* were placed in the culture medium for seed germination experiments. Seed germination was observed and the germination rate was calculated. The results are shown below. Figure 1 D and E in the text.

[0042] The results showed that no salt treatment concentration was found to promote the germination of Apocynum venetum seeds. Figure 1 Even on a medium containing only 10 mM NaCl, the germination rate of Apocynum venetum seeds (44.03%) was significantly lower than that on a medium without added sodium chloride (75.28%). Figure 1 The value of E in the figure indicates 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, the seedlings can maintain growth at concentrations as high as 200 mM sodium chloride (salt content of about 11.7‰), indicating that Apocynum venetum is an atypical halophyte and can exhibit strong salt tolerance.

[0044] Example 2

[0045] Potted mature Apocynum venetum plants were randomly divided into two groups. One group was treated with 200 mM NaCl for salt stress (treatment group), while the other group was treated with an equal volume of pure water (control group, CK). Samples were taken at 10:00 AM on days 0, 7, and 18. Transcriptome sequencing was performed on leaves from both the control and treatment groups. The control group was designated CK0Z, CK7Z, and CK18Z, respectively, while the treatment group was designated NaCl7Z and NaCl18Z, respectively. Each treatment was performed in triplicate.

[0046] The second-generation transcriptome sequencing yielded a total of 254,005 transcripts, which were spliced ​​to obtain the longest transcript of 133,486. The parameter-free transcriptome used FPKM (Fragments Per Kilobase of transcript per Million mapped reads) as an indicator to measure gene expression levels. Differentially expressed genes (DEGs) were identified 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, which were 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] Venn diagram analysis of the comparison group associated with salt stress yielded 25 common differentially expressed genes. Figure 2 (A in the text); gene expression trend analysis revealed 22 genes that were upregulated under salt stress. Figure 2 (B in the table). The two have 15 differentially expressed genes (see Table 1). Among them, TRINITY_DN39146_c0_g1 encodes a chitinase gene. The gene annotation shows that it is homologous to the Arabidopsis chitinase gene At3g12500 (PR-3) and is named AvCHIT1.

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

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

[0050] Example 3

[0051] The transcriptome data were validated using qRT-PCR. The primer sequences for the AvCHIT1 gene in the qRT-PCR are shown in SEQ ID NO.3 and SEQ ID NO.4. The internal reference gene is the Apocynum venetum EF-α gene, and the primer sequences for the Apocynum venetum EF-α gene are 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 consisted of 20 μL of 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), and cDNA template (1 μL). ddH2O was added to bring the volume to 20 μL, and three technical replicates were set up. The cDNA template in the system was cDNA obtained by reverse RNA inversion (1 μg).

[0061] The qRT-PCR amplification was performed using a two-step method. The reaction program was as follows: pre-denaturation at 95℃ for 30 seconds; then 40 cycles of 95℃ for 10 seconds and 60℃ for 30 seconds; followed by the melting curve stage: 95℃ for 15 seconds, 60℃ for 1 min, and 95℃ for 15 seconds. After the reaction was completed, the melting curve was analyzed.

[0062] Transcriptome and qRT-PCR data were analyzed; results are shown in [link to results]. Figure 3 In the diagram, A represents CK0, which 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 the qRT-PCR data and transcriptome data in each sample were basically consistent, and the expression level of the AvCHIT1 gene in *Apocynum venetum* was significantly upregulated under salt stress compared to the control.

[0063] Four-week-old Apocynum venetum seedlings were treated with 0 mM, 50 mM, 100 mM, and 200 mM NaCl stress. The expression level of the AvCHIT1 gene was then detected using a semi-quantitative assay. The results are shown below. Figure 3 B in the text. According to Figure 3 As shown in B, compared with the control, the expression level of AvCHIT1 gene was significantly upregulated when subjected to 100mM and 200mM NaCl stress.

[0064] Example 4

[0065] A phylogenetic tree was constructed using Mega 6.0 to compare the chitinase genes related to Arabidopsis thaliana with those reported in the literature to enhance plant resistance under salt stress regulation or overexpression. (See [link to Mega 6.0]). Figure 4 .

[0066] according to Figure 4 It was found that AvCHIT1 and the Arabidopsis type III chitinase gene PR-3 (At3g12500) belong to the same clade, indicating high homology and possible similar functions. The chitinase gene CAChi2, which is significantly induced by salt stress and whose overexpression enhances salt tolerance in Arabidopsis, also belongs to the same clade as AvCHIT1, suggesting functional similarity among these chitinase genes. The protein sequences and domains of AvCHIT1, HOT2, and PR-3 were predicted using DNAMAN; the results are shown in [link to DNAMAN]. Figure 5 The PR-3 protein is encoded by the Arabidopsis thaliana type III chitinase gene At3g12500. According to... Figure 5 As shown in A, the primary sequence similarity of AvCHIT1 with HOT2 and PR-3 proteins is 30.22% and 50.75%, respectively. Furthermore, AvCHIT1 shares the same glycosyl hydrolase domain with both HOT2 and PR-3. Figure 5 Therefore, based on the information in section B), it can be inferred that AvCHIT1 and HOT2 / PR-3 likely have similar functions. However, whether transgenic AvCHIT1 gene in Apocynum venetum can improve the plant's salt tolerance still requires further experimental verification.

[0067] Example 5

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

[0069] Construction of the pro:AvCHIT1-GUS expression vector: Using the pBI101-GUS vector as the base vector (purchased from BioVector NTCC Type Culture Collection Center, catalog number: pBI101 vetor), the vector was linearized by Xba I single enzyme digestion; using cDNA from Apocynum venetum seedling leaves as a template, the AvCHIT1 gene fragment containing the promoter was obtained using amplification primers pAvCHIT1-GUS-F (SEQ ID NO.7) and pAvCHIT1-GUS-R (SEQ ID NO.8), and then the AvCHIT1 gene fragment containing the promoter was ligated to the linearized vector under the action of homologous recombinase to obtain the 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] Agrobacterium GV3101 was transformed with the pro:AvCHIT1-GUS recombinant vector to obtain Agrobacterium GV3101 containing the p35S:AvCHIT1-GFP vector. Then, Agrobacterium GV3101 containing the p35S:AvCHIT1-GFP vector was transiently transformed with *Nicotiana benthamiana*, and the fluorescence signal was observed under a laser confocal microscope. The results are shown in [link to results]. Figure 6 A in the middle. According to Figure 6 As can be seen from A, the fluorescence signal is mainly concentrated on the cell membrane, indicating that the AvCHIT1 protein is mainly located on the cell membrane to perform its function.

[0075] Construction of the p35S:AvCHIT1-GFP fusion protein expression vector: Using the pCAMBIA1301-EGFP vector as the base vector, the vector was linearized by double digestion with Kpn I and BamHI. Using cDNA from Apocynum venetum seedling leaves as a template, the AvCHIT1 gene fragment was obtained using amplification primers 35S-AvCHIT1-GFP-F (SEQ ID NO.9) and 35S-AvCHIT1-GFP-R (SEQ ID NO.10). The AvCHIT1 gene fragment was then ligated to the linearized vector under the action of homologous recombinase to obtain the 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 wild-type Arabidopsis thaliana using the droplet method. GUS staining was performed on the transgenic progeny at various growth stages. The staining results are shown in [link to relevant documentation]. Figure 6 B and C in the results. The results showed that GUS was expressed in the entire vascular tissue and mesophyll cells of the rosette leaves of seedlings, and GUS was also detected in the hypocotyl of seedlings. Figure 6 While GUS signaling was detected in the leaves of mature plants, no GUS signal was observed in the pods (B) of mature plants, indicating that the AvCHIT1 gene has a high expression level in the early stages of growth and development. Furthermore, GUS signaling was also detected in the base of the pods and calyx of mature plants. Figure 6 (C in the middle).

[0081] Example 6

[0082] Construction of the 35Spro:AvCHIT1 overexpression vector: The pCAMBIA1301-EGFP vector was used as the base vector, and the vector was linearized by double digestion with KpnI and XbaI. Using cDNA from Apocynum venetum seedling leaves as a template, the AvCHIT1 gene fragment was obtained using amplification primers 35S-AvCHIT1-GFP-F (SEQ ID NO. 9) and AvCHIT1-R1 (SEQ ID NO. 11). The AvCHIT1 gene fragment was ligated to the linearized vector under the action of homologous recombinase to obtain the 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 steps was transformed into Agrobacterium GV3101 to obtain Agrobacterium GV3101 strain carrying the 35Spro:AvCHIT1 overexpression vector. The Agrobacterium GV3101 strain carrying the 35Spro:AvCHIT1 overexpression vector was streaked on LB agar, and single colonies were picked and shaken. When the OD value of the bacterial suspension reached approximately 0.5, it was centrifuged, the supernatant was discarded, and the bacterial cells were resuspended in a resuspension solution containing a final concentration of 5% (w / v) sucrose and 0.02% (v / v) Silwet. Open Arabidopsis inflorescences were soaked in the resuspension solution for 30 seconds, then cultured in the dark for 12 hours. After harvesting the seeds, they were screened to obtain transgenic lines OE1 and OE2.

[0086] Salt tolerance phenotypes were assessed in the selected positive plants OE1 and OE2. Root length of Arabidopsis seedlings was recorded, and leaf Na+ content was measured. + The content was determined using the same method as in Example 1, and the results are shown in [reference needed]. Figure 7 In the figure, ** represents p < 0.05.

[0087] according to Figure 7 It was found that the root lengths of transgenic plants OE1 and OE2 were significantly longer than those of the control on culture media containing 100 mM and 150 mM NaCl. + The results showed that Na+ content in the leaves of AvCHIT1 overexpression lines under salt stress was significantly higher than that in other strains. + The content was also significantly lower than that of the wild type. These results indicate that the AvCHIT1 gene can enhance the salt tolerance of plants.

[0088] Example 8

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

[0090] Arabidopsis pr-3 and hot2 mutants (catalog numbers: SALKseq_081718.1 and SALKseq_058663.1) were ordered from the European Arabidopsis Stock Centre (https: / / arabidopsis.info / StockInfo?NASC_id=625057). The 35Spro:AvCHIT1 overexpression vector obtained in Example 7 was transformed into the Arabidopsis pr-3 and hot2 mutants using the droplet method to obtain complementary lines of 35Spro:AvCHIT1:pr-3 and 35Spro:AvCHIT1:hot2. Using Arabidopsis AtTUBULIN as an internal control gene, semi-quantitative PCR was used to identify the background of the obtained mutant lines. 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] Semi-quantitative PCR results are as follows Figure 8 As shown in Figure A, the exogenous AvCHIT1 gene did not amplify bands in Arabidopsis thaliana WT, hot2, and pr-3, but it did amplify bands in 35Spro:AvCHIT1, 35Spro:AvCHIT1:hot2, and 35Spro:AvCHIT1:pr-3, indicating that AvCHIT1 was expressed in all overexpression lines. Meanwhile, the AtHOT2 and AtPR3 genes did not amplify bands in their respective mutants, but amplified bands of similar brightness in other Arabidopsis thaliana lines, indicating a clean mutant background and successful construction of all mutant materials.

[0094] Salt tolerance at the seed germination and seedling stages was assessed in six lines: WT, 35Spro:AvCHIT1, hot2, pr-3, 35Spro:AvCHIT1:hot2, and 35Spro:AvCHIT1:pr-3. The results are shown in [link to results]. Figure 8 B to E in the sequence. According to... Figure 8As shown in B-E, under normal conditions of pH=6.0, after treatment with 150mM NaCl, the seed germination rate of Arabidopsis hot2 and pr-3 mutants was significantly reduced, while WT was slightly higher than the two, but the germination rate was still around 70%. In contrast, the germination rate of AvCHIT1 overexpressing plants was close to 100%. 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 the salt tolerance of plants.

[0095] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

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

2.

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

1.

3. The application of the AvCHIT1 protein of claim 1 or the AvCHIT1 gene of claim 2 in regulating plant salt tolerance; The regulation is as follows: increasing the expression level of AvCHIT1 protein or increasing the expression level of AvCHIT1 gene to improve plant salt tolerance; The plant in question is Arabidopsis thaliana.

4. The application according to claim 3, characterized in that, Improving plant salt tolerance includes increasing plant germination rate.

5. A recombinant expression vector, characterized in that, The recombinant expression vector contains the AvCHIT1 gene as described in claim 2.

6. A recombinant bacterium, characterized in that, The recombinant bacteria comprises the recombinant expression vector as described in claim 5.

7. The application of the AvCHIT1 protein of claim 1, the AvCHIT1 gene of claim 2, the recombinant expression vector of claim 5, or the recombinant bacteria of claim 6 in the cultivation of salt-tolerant plant varieties; wherein the plant is Arabidopsis thaliana.

8. A method for improving the salt tolerance of plants, characterized in that, include: Transform the recombinant expression vector of claim 5 or the recombinant bacteria of claim 6 into a plant; The plant in question is Arabidopsis thaliana.

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