Application of tomato slahl1 gene in breeding of salt and / or drought tolerance in plants

Abstract

The application discloses application of a tomato SlAHL1 gene in cultivating a salt-resistant and / or drought-resistant plant variety and belongs to the technical field of genetic engineering. The nucleotide sequence of the SlAHL1 gene is shown in SEQ ID NO. 1, or is completely complementary to the sequence shown in SEQ ID NO. 1, or is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2. The application can improve the salt resistance and / or drought resistance of plants by overexpressing the SlAHL1 gene, can be applied to plant genetic engineering breeding, and provides a new method for creating or improving new plant salt-resistant and / or drought-resistant germplasm.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of the tomato SlAHL1 gene in plant salt-tolerant and / or drought-tolerant breeding. Background Technology

[0002] Crops often suffer from abiotic stresses such as drought and high salinity during their growth and development, which in turn affects the quality and grade of agricultural products. Breeding superior salt- and / or drought-resistant crop varieties is an important way to solve the problem of salt and / or drought resistance. However, the genetic regulatory network of plant salt and / or drought resistance is intricate, and the molecular regulatory mechanisms vary considerably among plants with different genetic backgrounds. Although some genes regulating plant salt and / or drought resistance have been reported, discovering and cloning more new genes that regulate plant salt and / or drought resistance in different genetic backgrounds, as well as developing new methods for breeding salt- and / or drought-resistant plant varieties, remain urgent technical problems to be solved. Summary of the Invention

[0003] The purpose of this invention is to provide an application of the tomato SlAHL1 gene in regulating plant salt and / or drought resistance and / or breeding salt and / or drought resistant plant varieties.

[0004] This invention clones the tomato SlAHL1 gene, transforms it into Arabidopsis thaliana, and analyzes the performance of transgenic lines under ABA, drought, and high salt conditions. The results demonstrate that the SlAHL1 gene can be used to simultaneously improve the drought and salt tolerance of crops. The nucleotide sequence of the SlAHL1 gene is shown in SEQ ID NO. 1, or is completely complementary to the sequence shown in SEQ ID NO. 1, or is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2. This gene holds promise for application in plant genetic engineering breeding, providing a theoretical basis for the creation or improvement of new salt- and / or drought-resistant plant germplasm. Considering codon degeneracy, modifications to the bases of the above nucleotide sequence without altering the amino acid sequence also fall within the scope of protection of this invention.

[0005] On the one hand, the present invention provides an application of the tomato SlAHL1 gene in regulating plant salt tolerance and / or drought resistance. The nucleotide sequence of the SlAHL1 gene is shown in SEQ ID NO.1, or is completely complementary to the sequence shown in SEQ ID NO.1, or is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2.

[0006] Furthermore, the regulation of plant salt and / or drought resistance is achieved by overexpressing the SlAHL1 gene to enhance the plant's salt and / or drought resistance.

[0007] In a preferred embodiment, the present invention enhances the salt and / or drought resistance of monocotyledonous plants by overexpressing the SlAHL1 gene.

[0008] Furthermore, this invention enhances the salt and / or drought resistance of Solanaceae plants by overexpressing the SlAHL1 gene.

[0009] Furthermore, this invention enhances the salt and / or drought resistance of Solanum plants by overexpressing the SlAHL1 gene.

[0010] In another preferred embodiment, the present invention enhances the salt and / or drought resistance of dicotyledonous plants by overexpressing the SlAHL1 gene.

[0011] Furthermore, this invention enhances the salt and / or drought resistance of cruciferous plants by overexpressing the SlAHL1 gene.

[0012] On the other hand, the present invention provides the application of a protein encoded by the tomato SlAHL1 gene in regulating plant salt tolerance and / or drought resistance, the amino acid sequence of the SlAHL1 protein being shown in SEQ ID NO.2.

[0013] Furthermore, the regulation of plant salt and / or drought resistance is achieved by overexpressing the protein encoded by the SlAHL1 gene to enhance plant salt and / or drought resistance.

[0014] In a preferred embodiment, the present invention enhances the salt and / or drought resistance of monocotyledonous plants by overexpressing the SlAHL1 protein.

[0015] Furthermore, this invention enhances the salt and / or drought resistance of Solanaceae plants by overexpressing the SlAHL1 protein.

[0016] Furthermore, this invention enhances the salt and / or drought resistance of Solanum plants by overexpressing the SlAHL1 protein.

[0017] In another preferred embodiment, the present invention enhances the salt and / or drought resistance of dicotyledonous plants by overexpressing the SlAHL1 protein.

[0018] Furthermore, this invention enhances the salt and / or drought resistance of cruciferous plants by overexpressing the SlAHL1 protein.

[0019] On the other hand, the present invention provides an application of the tomato SlAHL1 gene in the breeding of salt-resistant and / or drought-resistant plant varieties. The SlAHL1 gene sequence is a nucleotide sequence as shown in SEQ ID NO.1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO.1, or a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0020] Furthermore, the regulation of plant flowering time is achieved by overexpressing the SlAHL1 gene to cultivate salt-tolerant and / or drought-resistant plant varieties.

[0021] In a preferred embodiment, the present invention cultivates salt- and / or drought-resistant monocotyledonous plant varieties by overexpressing the SlAHL1 gene.

[0022] Furthermore, this invention cultivates salt- and / or drought-resistant Solanaceae plant varieties by overexpressing the SlAHL1 gene.

[0023] Furthermore, this invention cultivates salt- and / or drought-resistant Solanaceae plant varieties by overexpressing the SlAHL1 gene.

[0024] In another preferred embodiment, the present invention breeds salt- and / or drought-resistant dicotyledonous plant varieties by overexpressing the SlAHL1 gene.

[0025] Furthermore, this invention cultivates salt- and / or drought-resistant cruciferous plant varieties by overexpressing the SlAHL1 gene.

[0026] On the other hand, the present invention provides the application of the protein encoded by the tomato SlAHL1 gene in the cultivation of salt-resistant and / or drought-resistant plant varieties. The SlAHL1 gene sequence is a nucleotide sequence as shown in SEQ ID NO.1, or a nucleotide sequence that is completely complementary to the sequence shown in SEQ ID NO.1, or a nucleotide sequence encoding an amino acid sequence as shown in SEQ ID NO.2.

[0027] Furthermore, the cultivation of salt- and / or drought-resistant plant varieties involves cultivating salt- and / or drought-resistant plant varieties by overexpressing the SlAHL1 protein.

[0028] In a preferred embodiment, the present invention cultivates salt- and / or drought-resistant monocotyledonous plant varieties by overexpressing the SlAHL1 protein.

[0029] Furthermore, this invention cultivates salt- and / or drought-resistant Solanaceae plant varieties by overexpressing the SlAHL1 protein.

[0030] Furthermore, this invention cultivates salt- and / or drought-resistant Solanaceae plant varieties by overexpressing the SlAHL1 protein.

[0031] In another preferred embodiment, the present invention cultivates salt- and / or drought-resistant dicotyledonous plant varieties by overexpressing the SlAHL1 protein.

[0032] Furthermore, this invention cultivates salt- and / or drought-resistant cruciferous plant varieties by overexpressing the SlAHL1 protein.

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] 1) This invention provides a novel method for regulating plant salt tolerance and / or drought resistance, namely, regulating plant salt tolerance and / or drought resistance and / or obtaining salt-tolerant and / or drought-resistant plant varieties by overexpressing the SlAHL1 gene or its encoded protein.

[0035] 2) This invention provides a novel method for breeding salt- and / or drought-resistant plant varieties, namely, regulating the salt and / or drought resistance of plants and / or obtaining salt- and / or drought-resistant plant varieties by overexpressing the SlAHL1 gene or its encoded protein.

[0036] 3) The new method provided by this invention can simultaneously regulate salt resistance and drought resistance.

[0037] 4) The new method provided by this invention can be used in plants with different genetic backgrounds. Attached Figure Description

[0038] The following, in conjunction with the accompanying drawings and specific embodiments, provides a detailed description of the method of the present invention for regulating plant salt tolerance and / or drought tolerance and / or cultivating salt-tolerant and / or drought-tolerant plant varieties, and its beneficial effects.

[0039] Figure 1 The results of the identification of the 35S-SlAHL1-GFP transgenic line are shown.

[0040] Figure 2 Analysis of SlAHL1 expression levels in transgenic Arabidopsis lines;

[0041] WT represents wild-type plants; OE-4, OE-5, OE-7, and OE-8 represent overexpression plants.

[0042] Figure 3 The results show the sensitivity analysis of SlAHL1 overexpression lines to ABA. SlAHL1 #4 is SlAHL1-OE4, and SlAHL1 #8 is SlAHL1-OE8.

[0043] (A) Phenotypes of wild-type (WT) and SlAHL1 overexpression materials grown on 1 / 2 MS plates for 7 days;

[0044] (B) Phenotypes of wild-type and SlAHL1 overexpression materials grown on 1 / 2 MS plates with added ABA for 7 days;

[0045] (C) Germination rate analysis of wild-type and SlAHL1 overexpression materials on 1 / 2 MS plates with 0.25 μM ABA for 7 consecutive days;

[0046] (D) Statistical analysis of the seedling rate of wild-type and SlAHL1 overexpression materials after 7 days of growth on 1 / 2 MS plates with or without 0.25 μM ABA;

[0047] Figure 4 The results show the tolerance of SlAHL1 overexpression lines to salt and drought. T-test analysis, * indicates p < 0.05 (n=20).

[0048] (A, B) Phenotypic and root length growth data of wild-type and SlAHL1 overexpression materials grown vertically on 1 / 2 MS plates;

[0049] (C, D) Phenotypic and root length data of wild-type and SlAHL1 overexpression materials transferred to 1 / 2 MS plates with 100 mM NaCl and grown for 3 days;

[0050] Phenotypic and root length data analysis of (E, F) wild-type and SlAHL1 overexpression materials after 3 days of growth on 1 / 2 MS plates with 6% PEG. Detailed Implementation

[0051] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0053] The nucleotide sequence of the tomato SlAHL1 gene in the example is shown in SEQ ID NO.1:

[0054]

[0055] The amino acid sequence of the tomato SlAHL1 protein is shown in SEQ ID NO.2:

[0056] MDIREGMALSGSAAYYLNRGISGSGSNVGSGSGTPSGVSTPSGYKSLTNANIAVQSNMGSSSGNVNSGYQVENSSSNFGHGVNISMASSVSPGSDPVKKKRPRKYGPDGTNMSLALSPLSSNPPSGSITPGPKRIRGRPPGSGWKQQLASVGEWMSSSAGLAFTP HVIHIGVGEDVAEKLLAFAQQRPRALCILSANGAVSAITLRPPANSGATVAYEGRFEILSLSGSYLVAETGGPRTRTGGISISVCSPDGHVIGGAIGGRLIAASPVQVVVCSFVYDPKGKSKPESSSTRDEQESAEKSSTPGSGRSVWPPSSRADVRNSQTEIDLTRG

[0057] Example 1

[0058] Construction and identification of genetically modified materials

[0059] 0.5g of tomato seedlings of the variety "Henz 1706" that had germinated and grown for 7 days on 1 / 2 MS solid medium were taken. Total RNA was extracted using a plant RNA extraction kit (Vazyme), reverse transcribed, and then the target gene was amplified. After obtaining the target gene, it was ligated into the plant binary expression vector pBI121 and transformed into Arabidopsis thaliana using Agrobacterium. The specific experiments are as follows:

[0060] Take 1 μg of high-quality (OD) 260 / OD 280 : 1.8-2.0; OD 260 / OD 230RNA with a density of approximately 2.0 μL was reverse transcribed (Vazyme Reverse Transcription Kit) to obtain first-strand cDNA. Using the cDNA as a template, PCR amplification was performed using KOD FX high-fidelity enzyme (ToYoBo). The reaction mixture consisted of: 10 μL 2×PCR buffer, 2 μL 2 mM dNTPs, 0.5 μL F primer (5'-GGACTCTAGAGGATCCATGGATATAAGGGAAGGGATG-3'), 0.5 μL R primer (5'-GACCACCCGGGGATCCTCCACGCGTAAGATCAATCTC-3'), 1 μL cDNA template, 0.4 μL KOD FX (1 U / μL), and water to a final volume of 20 μL. The reaction conditions were: 98℃ for 5 min; 98℃ for 15 sec, 56℃ for 30 sec, 68℃ for 30 sec, 35 cycles; 68℃ for 5 min. After the reaction was complete, the PCR products were recovered using a gel recovery kit (Magen).

[0061] The pBI121-GFP vector (modified from the pBI121 vector, containing a 35S promoter and GFP tag) was linearized using the restriction endonuclease BamHI (NEB). The digestion system consisted of 2 μg of vector, 2 μL of BamHI, 10 μL of 10× buffer, and water to a final volume of 100 μL. The digestion conditions were 25°C for 2 hours. After purification and recovery of the vector digestion products and PCR products, the DNA concentration was determined using NanoDrop 2000. Recombination was then performed using a recombinant kit (Novizan). The recombination system consisted of 14 ng of target fragment, 130 ng of vector fragment, 2 μL of 5× buffer, 1 μL of Exnase II, and water to a final volume of 10 μL. The recombination conditions were 37°C for 30 min. All product was added to 100 μL of *E. coli* DH5α competent cells, and the transformation product was plated on LB agar (containing kanamycin resistance, kanamycin concentration 50 mg / L). The culture was incubated overnight at 37°C, and four single clones were selected for colony PCR identification. Two positive clones were selected for sequencing to obtain positive clones containing the SlAHL1 gene sequence, ultimately yielding an overexpression vector containing the SlAHL1 target gene.

[0062] The Agrobacterium GV3101-mediated genetic transformation method was used to transform the overexpression vector constructed in Example 1 into Arabidopsis thaliana Col-0 via the flower-dipping method. The experimental method reference is: Research on Agrobacterium-mediated Transgenic Technology; Zhang Zhaoyichun; Agricultural Science and Technology, 2017, No. 9. After flower dipping, Arabidopsis seeds were harvested, surface-sterilized with 2% sodium hypochlorite, and sown on 1 / 2 MS solid plates containing 25 μg / L hygromycin. Resistant seedlings (long roots and the ability to develop true leaves) were selected and identified as positive transgenic seedlings. Ten positive T1 generation transgenic seedlings were propagated to obtain the T2 generation. T2 generation seeds were germinated on a medium containing hygromycin. If all seeds grew normally, the line was considered homozygous.

[0063] We performed RCR testing on the selected transgenic lines and identified nine 35S-SlAHL1-GFP transgenic lines (1 to 9) through electrophoresis experiments. Figure 1 ).

[0064] Example 2

[0065] Analysis of SlAHL1 expression levels in transgenic Arabidopsis lines

[0066] The process of identifying the overexpression effect of the SlAHL1 gene in transgenic Arabidopsis lines using qRT-RCR is as follows:

[0067] 1. Total RNA was extracted from 7-day-old Arabidopsis thaliana seedlings using a plant RNA extraction kit (Vazyme). 1 μg of high-quality (OD) RNA was then extracted. 260 / OD 280 : 1.8-2.0; OD 260 / OD 230 RNA with a density of ≈ 2.0 was reverse transcribed (using the Vazyme reverse transcription kit) to obtain first-strand cDNA.

[0068] 2. Using the cDNA from step 1 above as a template, the expression of the SlAHL1 gene was detected using primer pairs SlAHL1-qF (TTGCTTATGAGGGCCGTTTC) and SlAHL1-qR (GCTGCTATAAGTCTACCTGG). The expression of the tomato AtUBQ5 gene was detected using primer pairs AtUBQ5-qF (GACGCTTCATCTCGTCC) and AtUBQ5-qR (CCACAGGTTGCGTTAG) as internal controls. The quantitative PCR reagent was SYBR® Premix Ex Taq™ (TAKARA), and the quantitative PCR instrument was a CFX 96 (Bio RAD). The reaction mixture consisted of: 5 μL 2×PCR buffer, 0.4 μL qF primers, 0.4 μL qR primers, 1 μL cDNA template, and 3.2 μL sterile water, for a total reaction volume of 10 μL. Reaction program: 95℃ for 30 sec; 95℃ for 5 sec, 68℃ for 30 sec, 45 cycles.

[0069] The results are as follows Figure 2 As shown, the expression level of the SlAHL1 gene was significantly increased in all four selected lines (OE4, 5, 7, 8). Subsequently, OE4 and OE8 overexpression lines were selected for further experiments.

[0070] The expression level of SlAHL1 in transgenic Arabidopsis thaliana lines was analyzed using qRT-PCR. The results showed that these lines were all SlAHL1 overexpression lines. Figure 2 ).

[0071] Example 3: Sensitivity analysis of SlAHL1 transgenic lines to ABA

[0072] Seeds of the wild-type WT and SlAHL1 transgenic lines OE4 and OE8 were simultaneously sown on 1 / 2 MS medium plates containing 0.25 μM ABA or without ABA. Seed germination rate and seedling emergence rate were observed and statistically analyzed over seven consecutive days. The results showed that on medium without ABA, there was no significant difference in germination and growth between SlAHL1-OE4 and SlAHL1-OE8 and the wild type. Figure 3 A); however, at an ABA concentration of 0.25 μM, the germination rate of the SlAHL1 overexpression line was lower compared to the wild type. Figure 3 B, C), and the seedling survival rate was also significantly lower than that of the control ( Figure 3 (D) This indicates that SlAHL1 is involved in the plant's response to ABA signaling and may affect the plant's tolerance to stress.

[0073] Example 4: Phenotypic analysis of SlAHL1 transgenic lines to drought and salt stress

[0074] Further analysis was conducted on the tolerance of SlAHL1 Arabidopsis transgenic lines to drought and salt stress. First, we observed the taproot growth of wild-type controls and SlAHL1 overexpression lines vertically cultured on 1 / 2 MS plates. The results showed that after 6 days of growth, there was no significant difference in taproot length among the plant materials (…). Figure 4 A, B). Next, we transferred the seedlings grown on 1 / 2 MS medium for 3 days to 1 / 2 MS plates supplemented with 100 mM NaCl. After 3 days of growth, we found that the root length of the SlAHL1 overexpression line was significantly longer than that of the control. Figure 4 (C, D) indicates that the SlAHL1 overexpression line has strong salt tolerance.

[0075] In addition, we also examined the drought tolerance of the SlAHL1 overexpression lines. We simulated drought by adding PEG to 1 / 2 MS medium. Seedlings grown on 1 / 2 MS medium for 3 days were transferred to 1 / 2 MS plates supplemented with 6% PEG. After 3 days of growth, the root length of the primary root of the SlAHL1 overexpression lines was significantly longer than that of the control. Figure 4 (E, F) This result indicates that SlAHL1 overexpression can enhance the drought resistance of Arabidopsis thaliana. In summary, these results confirm that the SlAHL1 gene can improve plant tolerance to salt and drought, and therefore can be used in genetic breeding to enhance the salt tolerance and drought resistance of crops.

[0076] >SEQ ID NO.1

[0077]

[0078] >SEQ ID NO.2

[0079] MDIREGMALSGSAAYYLNRGISGSGSNVGSGSGTPSGVSTPSGYKSLTNANIAVQSNMGSSSGNVNSGYQVENSSSNFGHGVNISMASSVSPGSDPVKKKRPRKYGPDGTNMSLALSPLSSNPPSGSITPGPKRIRGRPPGSGWKQQLASVGEWMSSSAGLAFTP HVIHIGVGEDVAEKLLAFAQQRPRALCILSANGAVSAITLRPPANSGATVAYEGRFEILSLSGSYLVAETGGPRTRTGGISISVCSPDGHVIGGAIGGRLIAASPVQVVVCSFVYDPKGKSKPESSTRDEQESAEKSSTPGSGRSVWPPSSRADVRNSQTEIDLTRG.

[0080] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of the tomato SlAHL1 gene in improving plant salt and / or drought resistance, characterized in that, The SlAHL1 gene sequence encodes the amino acid sequence shown in SEQ ID NO.2, and the plant is Arabidopsis thaliana or tomato.

2. The application of the protein encoded by the tomato SlAHL1 gene in improving plant salt tolerance and / or drought resistance, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.2, and the plant is Arabidopsis thaliana or tomato.

3. The application of the tomato SlAHL1 gene in the breeding of salt-tolerant and / or drought-resistant plant varieties, characterized in that, The SlAHL1 gene sequence encodes the amino acid sequence shown in SEQ ID NO.2, and the plant is Arabidopsis thaliana or tomato.

4. The application of the protein encoded by the tomato SlAHL1 gene in the breeding of salt-tolerant and / or drought-resistant plant varieties, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.2, and the plant is Arabidopsis thaliana or tomato.

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