Application of TaHSFA6e gene in regulating tolerance of plant to salt stress

Abstract

The invention discloses application of a TaHSFA6e gene in regulation and control of salt stress tolerance of plants, and belongs to the technical field of gene engineering. The nucleotide sequence of the TaHSFA6e gene disclosed by the invention is as shown in at least one of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 8 and SEQ ID NO: 12. According to the invention, four TaHSFA6e family genes are cloned from wheat, and the expression quantity of the four genes in leaves and roots after wheat salt stress is obviously improved. After the four genes are mutated, the salt tolerance of the mutated wheat is obviously reduced. In conclusion, the four TaHSFA6e family genes are related to the salt tolerance of the wheat, can be used for regulating and controlling the salt tolerance of the wheat and enriching wheat germplasm resources, and have important application value in breeding of the salt-tolerant wheat.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to application of TaHSFA6e gene in regulating plant tolerance to salt stress. Background Art

[0002] Wheat (Triticum aestivum l.) is one of the three major food crops in the world. Due to climate change and the influence of human activities, soil salinization is becoming increasingly serious, posing a serious threat to agricultural production and development. Soil salinization can inhibit the growth and development of wheat, reduce reproductive capacity, reduce yield, and even cause food shortages, seriously affecting food production and sustainable agricultural development. Therefore, exploring the molecular mechanism of wheat salt tolerance and identifying the key genes using molecular biology and genetics will have a relatively important theoretical basis and practical value for breeding salt-tolerant varieties. Summary of the invention

[0003] In view of the problems existing in the prior art, the object of the present invention is to provide an application of the TaHSFA6e gene in regulating the tolerance of plants to salt stress.

[0004] In order to achieve the above object, the present invention adopts the following technical solution:

[0005] The use of the TaHSFA6e gene in regulating plant tolerance to salt stress is characterized in that the amino acid sequence encoded by the TaHSFA6e gene is shown in at least one of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:9, and SEQ ID NO:13.

[0006] Based on the above scheme, the nucleic acid sequence of the TaHSFA6e gene is shown in at least one of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:12.

[0007] On the basis of the above scheme, the tolerance of plants to salt stress is improved by increasing the expression level of TaHSFA6e gene in plants; or the tolerance of plants to salt stress is reduced by reducing the expression level of TaHSFA6e gene in plants.

[0008] Based on the above scheme, the plant is wheat.

[0009] A method for regulating plant tolerance to salt stress, comprising increasing the expression level of a TaHSFA6e gene in a plant to increase the plant tolerance to salt stress; or decreasing the expression level of a TaHSFA6e gene in a plant to decrease the plant tolerance to salt stress; wherein the amino acid sequence encoded by the TaHSFA6e gene is shown in at least one of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:9, and SEQ ID NO:13.

[0010] Based on the above scheme, the nucleic acid sequence of the TaHSFA6e gene is shown in at least one of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:12.

[0011] On the basis of the above scheme, a plant expression vector of the TaHSFA6e gene is constructed and transformed into plants to express the gene in the plants, thereby increasing the expression level of the TaHSFA6e gene in the plants.

[0012] On the basis of the above scheme, the TaHSFA6e gene in the plant is mutated or silenced to reduce the expression level of the TaHSFA6e gene in the plant.

[0013] Based on the above scheme, the TaHSFA6e gene in the plant is mutated by using the Crispr / Cas 9 method to mutate at least one sequence among SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:12 in the plant.

[0014] Based on the above scheme, the plant is wheat.

[0015] The beneficial effects of the present invention are:

[0016] The TaHSFA6e gene family is mainly involved in the regulation of plant heat stress response and heat tolerance, and this gene family has multiple homologous genes.

[0017] The present invention clones and obtains four TaHSFA6e family genes from wheat, namely two TaHSFA6e-A genes, one TaHSFA6e-B gene and one TaHSFA6e-D gene; wherein the TaHSFA6e-A gene has two splicing forms, namely TaHSFA6e-A1 and TaHSFA6e-A2, and the amino acid sequence encoded by TaHSFA6e-A1 lacks a segment at the end compared with the amino acid sequence encoded by TaHSFA6e-A2. The expression levels of these four genes in leaves and roots of wheat after salt stress are significantly increased. The TaHSFA6e-A2 gene overexpression wheat strain and TaHSFA6e-A1, TaHSFA6e-A2, TaHSFA6e-B, TaHSFA6e-D four gene mutation TaHSFA6e Crispr mutant strain were constructed. The results showed that after salt stress treatment, the TaHSFA6e Crispr mutant strain wilted significantly and the leaves turned yellow seriously, the SOD and POD enzyme activities decreased, the soluble sugar content decreased, the MDA content increased, the relative conductivity increased, the seed width became narrower, and the grain length became shorter. It can be seen that the salt tolerance of wheat decreased after the TaHSFA6e-A1, TaHSFA6e-A2, TaHSFA6e-B, and TaHSFA6e-D gene mutations. In summary, the four TaHSFA6e genes cloned by the present invention are related to the salt tolerance of wheat, can be used to regulate the salt tolerance of wheat, enrich the germplasm resources of wheat, and have important application value in salt-tolerant wheat breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The expression analysis of TaHSFA6e family genes in salt-stressed wheat at the one-leaf-one-heart stage (A is the expression analysis of TaHSFA6e genes A, B, and D in leaves, and B is the expression analysis of TaHSFA6e genes A, B, and D in roots);

[0019] Figure 2 Figure 2 is a salt tolerance phenotype diagram of the TaHSFA6e transgenic strain (A is the salt tolerance phenotype of Fielder and TaHSFA6e transgenic strains under normal conditions and salt stress conditions, B is a comparison of seed length and width of Fielder and TaHSFA6e transgenic plants, C is a comparison of plant height of Fielder and TaHSFA6e transgenic strains, and D is a comparison of 1000-grain weight of Fielder and TaHSFA6e transgenic strains);

[0020] Figure 3 The biochemical index determination diagram of Fielder and TaHSFA6e transgenic lines before and after salt stress (where A is the SOD activity determination result, B is the POD activity determination result, C is the MDA content determination result, D is the soluble sugar content determination result, and E is the relative conductivity determination result);

[0021] Figure 4 This is a graph showing the determination of photosynthetic indicators of Fielder and TaHSFA6e transgenic lines before and after salt stress. DETAILED DESCRIPTION

[0022] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below in conjunction with specific examples and with reference to data. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0023] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The experimental materials, reagents, drugs, etc. used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0024] In the following embodiments,

[0025] E. coli Fast-T1 competent cells and 5min TM TA / Blunt-Zero Cloning Kit (containing pCE2TA / Blunt-Zero) was provided by Nanjing Vayme Biotechnology Co., Ltd. (Vayme);

[0026] Chinese Spring (CS) wheat seeds are maintained by the Wheat Genetics and Breeding Laboratory, College of Agriculture, Qingdao Agricultural University;

[0027] The TaHSFA6e-A overexpression wheat line is the one that overexpresses the TaHSFA6e-A2 gene; the TaHSFA6e Crispr mutant wheat line is the one that mutated the TaHSFA6e-A1, TaHSFA6e-A2, TaHSFA6e-B, and TaHSFA6e-D genes of wheat;

[0028] The TaHSFA6e-A overexpression wheat line and the TaHSFA6e Crispr mutant wheat line were gifted by China Agricultural University;

[0029] The transgenic recipient variety Fielder is maintained by the Wheat Genetics and Breeding Laboratory of the College of Agriculture, Qingdao Agricultural University.

[0030] Example 1

[0031] Cloning of TaHSFA6e-A1 gene

[0032] The total RNA in the leaves of Chinese spring (CS) wheat grown in water until one leaf and one heart was extracted using the Trizol method. After purification, the cDNA was obtained by reverse transcription using HiScriptⅢRT SuperMix for qPCR (+gDNA wiper) reverse transcriptase. The cDNA was used as a template and PCR amplification was performed using TaHSFA6e-A1 gene-specific primers. The obtained PCR amplification product was electrophoresed in a 1% agarose gel to recover the target fragment, and then the cDNA was purified by PCR for 5 min. TM The target fragment was connected to pCE2 TA / Blunt-Zero using the TA / Blunt-Zero Cloning Kit and transformed into E. coli Fast-T1 competent cells for sequencing. The nucleic acid sequence of the TaHSFA6e-A1 gene was obtained as shown in SEQ ID NO: 1, and the amino acid sequence encoded by it was shown in SEQ ID NO: 2.

[0033] SEQ ID NO:1(5'→3')

[0034]

[0035] SEQ ID NO:2

[0036] MDAMPPEGIVKEEEVLLHEERTHPAAAPPQQRQDGALPRPMEGLHEAGPPPFLTKTYDLVEDPATDQVVSWGRAGNTFVVWDPHVFAEALLPRLFKHSNFSSFVRQLNTYGFRKVDPDRWEFANEGFLRGQRHLLKTIKRRKPPSNAPSSQQQALTSCLEVGEFGFEEEIDRLKRDKNLLI TEVVKLRQEQQATKDNVQAMEGRLRAAEQRQAQMMGFLARAMRNPHFFQQLVQKQDKRKELEDAISKKRRRPIDNAPFYGSGATTSQSEQLDSQFLFDSGVLSEPGMNGMENLAQNIQELGQGKTDEEKKDEANGQLDINSDFWAELFSDDFGDEDGSGLSELEGRRPEDIDELGQQLGFS

[0037] The TaHSFA6e-A1 gene-specific primer sequences are as follows:

[0038] TaHSFA6e-A / DF: 5'-ATGGACGCGATGCCGCCGGAGGGC-3' (SEQ ID NO: 3);

[0039] TaHSFA6e-A1-R: 5'-TCAACTAAATCCCAACTGCTGACCCAGTTCA-3' (SEQ ID NO: 4);

[0040] PCR reaction system (25 μL): ddH2O 8 μL, 2×Phanta Max Master Mix (Dye Plus) 12.5 μL, upstream and downstream primers 1 μL each, cDNA template 2.5 μL;

[0041] PCR reaction conditions: 95°C, 3 min; 35 cycles: 95°C, 15 s, 59°C, 15 s, 72°C, 30 s; 72°C extension for 5 min, and finally storage at 4°C.

[0042] Example 2

[0043] Cloning of TaHSFA6e-A2 gene

[0044] The total RNA in the leaves of Chinese spring (CS) wheat cultured with one leaf and one heart was extracted by Trizol method, and then the cDNA was obtained by reverse transcription using HiScriptⅢRT SuperMix for qPCR (+gDNA wiper) reverse transcriptase after purification. The cDNA was used as a template and PCR amplification was performed using TaHSFA6e-A2 gene specific primers. The obtained PCR amplification product was electrophoresed in 1% agarose gel, the target fragment was recovered, and then connected to pCE2 TA / Blunt-Zero through 5min TMTA / Blunt-Zero Cloning Kit, and transformed into Escherichia coli Fast-T1 competent cells for sequencing. The nucleic acid sequence of the TaHSFA6e-A2 gene was obtained as shown in SEQ ID NO:5, and the amino acid sequence encoded by it was shown in SEQ ID NO:6.

[0045] SEQ ID NO:5(5'→3')

[0046]

[0047] SEQ ID NO:6

[0048] MDAMPPEGIVKEEEVLLHEERTHPAAAPPQQRQDGALPRPMEGLHEAGPPPFLTKTYDLVEDPATDQVVSWGRAGNTFVVWDPHVFAEALLPRLFKHSNFSSFVRQLNTYGFRKVDPDRWEFANEGFLRGQRHLLKTIKRRKPPSNAPSSQQQALTSCLEVGEFGFEEEIDRLKRDKN

[0049] LL

[0050] ITEVVKLRQEQQATKDNVQAMEGRLRAAEQRQAQMMGFLARAMRNPHFFQQLVQKQDK

[0051] RKELEDAISKKRRRPIDNAPFYGSGATTSQSEQLDSQFLFDSGVLSEPGMNGMENLAQNIQE

[0052] LGQGKTDEEKKDEANGQLDINSDFWAELFSDDFGDEDGSGLSELEGRRPEDIDELGQQLG

[0053] YLSSTSPQ

[0054] The TaHSFA6e-A2 gene-specific primer sequences are as follows:

[0055] TaHSFA6e-A / DF: 5'-ATGGACGCGATGCCGCCGGAGGGC-3' (SEQ ID NO: 3);

[0056] TaHSFA6e-A2-R: 5'-CTACTGCGGGCTAGTAGAACTCAGATAC-3' (SEQ ID NO: 7);

[0057] The PCR reaction system and reaction conditions were the same as those in Example 1.

[0058] Example 3

[0059] Cloning of TaHSFA6e-B gene

[0060] The total RNA in the leaves of Chinese spring (CS) wheat cultured with one leaf and one heart was extracted by Trizol method, and then the cDNA was obtained by reverse transcription using HiScriptⅢRT SuperMix for qPCR (+gDNA wiper) reverse transcriptase after purification. The cDNA was used as a template and PCR amplification was performed using TaHSFA6e-B gene specific primers. The obtained PCR amplification product was electrophoresed in 1% agarose gel, the target fragment was recovered, and then connected to pCE2 TA / Blunt-Zero through 5min TMTA / Blunt-Zero Cloning Kit, and transformed into Escherichia coli Fast-T1 competent cells for sequencing. The nucleic acid sequence of the TaHSFA6e-B gene was obtained as shown in SEQ ID NO:8, and the amino acid sequence encoded by it was shown in SEQ ID NO:9.

[0061] SEQ ID NO:8(5'→3')

[0062]

[0063] SEQ ID NO:9

[0064] MEAAVAGGVVKEEELLLHEEQPPAGPQQQDGALPRPMEGLHEAGPPPFLTKTYDLVEDPATDQVVSWGRAGNTFVVWDPHVFAEALLPRLFKHSNFSSFVRQLNTYGFRKVDPDRWEFANEEGFLRGQRHLLKMIKRRKPPSNAPSSQQQALTSCLEVGEFGFEEEIDRLKRDKNLLIT

[0065] EV

[0066] VKLRQEQQATKDNVQAMEGRLRAAEQRQAQMMGFLARAMCNPHFFQQLVQKQDKRKE

[0067] LEDAISKKRRRPIDNAPFYGPAATTSQSEQLDSQFLFDSGVLSEPGMNGMENLAQNIQELG

[0068] The sequences of the specific primers for the QGKTDEEKKDEANGQLDINNDFWAELFSDDFGDGDGSGLSELEGRRPEDIDELAQQLGFSTaHSFA6e-B gene are as follows:

[0069] TaHSFA6e-BF: 5'-ATGGAGGCGGCGGTGGCGGGGGGCGTTG-3' (SEQ ID NO: 10); TaHSFA6e-BR: 5'-TCAACTAAATCCCAACTGCTGTGCCAG-3' (SEQ ID NO: 11);

[0070] The PCR reaction system and reaction conditions were the same as those in Example 1.

[0071] Example 4

[0072] Cloning of TaHSFA6e-D gene

[0073] The total RNA in the leaves of Chinese spring (CS) wheat cultured with one leaf and one heart was extracted by Trizol method, and then the cDNA was obtained by reverse transcription using HiScriptⅢRT SuperMix for qPCR (+gDNA wiper) reverse transcriptase after purification. The cDNA was used as a template and PCR amplification was performed using TaHSFA6e-D gene specific primers. The obtained PCR amplification product was electrophoresed in 1% agarose gel, the target fragment was recovered, and then connected to pCE2 TA / Blunt-Zero through 5min TMTA / Blunt-Zero Cloning Kit, and transformed into Escherichia coli Fast-T1 competent cells for sequencing. The nucleic acid sequence of the TaHSFA6e-D gene was obtained as shown in SEQ ID NO:12, and the amino acid sequence encoded by it was shown in SEQ ID NO:13.

[0074] SEQ ID NO:12(5'→3')

[0075]

[0076] MDAMPPEGIVKEEEVLLHEEHTHPQAGPQQQQQRQDGALPRPMEGLHEAGPPPFLTKTYDLVEDPATDQVVSWGRAGNTFVVWDPHVFAEALLPRLFKHSNFSSFVRQLNTYGFRKVDPDRWEFANEGFLRGQRHLLKMIKRRKPPSNAPSSQQQALTSCLEVGEFGFEEEIDRLKRDKNLL ITEVVKLRQEQQATKDNVQAMEGRLRAAEQRQAQMMGFLARAMRNPHFFQQLVQKQDKRKELEDAISKKRRRPIDNAPFYGPGATTSQSEQLDSQFLFDSGVLTEPGMNGMENLAQNIQELGQGKTDEEKKDEANGQLDINNDFWAELFSDDFGDGDGSGLSEGRRPEDIDELAQQLGFS

[0077] The TaHSFA6e-D gene-specific primer sequences are as follows:

[0078] TaHSFA6e-A / DF: 5'-ATGGACGCGATGCCGCCGGAGGGC-3' (SEQ ID NO: 3);

[0079] TaHSFA6e-DR: 5'-TCAACTAAATCCCAACTGCTGAGCCAGTTCA3' (SEQ ID NO: 14);

[0080] The PCR reaction system and reaction conditions were the same as those in Example 1.

[0081] Example 5

[0082] Expression analysis of TaHSFA6e gene in wheat under salt stress

[0083] The salt-sensitive wheat variety Chinese Spring (CS) was used as the material and cultured with distilled water to the one-leaf-one-heart stage. Salt stress was simulated with 250 mmol / L NaCl solution. The leaves and roots at 0 min, 10 min, 20 min, 40 min, 60 min and 120 min after salt stress were used as materials to analyze the expression patterns of TaHSFA6e family genes at different time points after salt stress.

[0084] The total RNA of the wheat materials before and after the salt stress treatment was extracted and reverse transcribed into cDNA, which was used as a template and qRT-PCR was performed using fluorescent quantitative PCR primers of the TaHSFA6e gene, and the TaActin gene was used as an internal reference.

[0085] The primer sequences used for real-time fluorescence quantification are as follows:

[0086] TaHSFA6e-qRT-AF (A1, A2): 5'-TCGGGGGCAACAACAAGTCA-3' (SEQ ID NO: 15);

[0087] TaHSFA6e-qRT-AR (A1, A2): 5'-CTGTTTTGCCCTGCCCAAG-3' (SEQ ID NO: 16);

[0088] TaHSFA6e-qRT-BF: 5'-GCTTGGGCAGGGCAAAACA-3' (SEQ ID NO: 17);

[0089] TaHSFA6e-qRT-BR: 5'-TGCTGTGCCAGTTCGTCGATA-3' (SEQ ID NO: 18);

[0090] TaHSFA6e-qRT-DF: 5'-ATTTGAGGAAGAGATCGACAGGCTC-3' (SEQ ID NO: 19);

[0091] TaHSFA6e-qRT-DR: 5'-ATTGTTGGAAGAAGTGGGTTACG-3' (SEQ ID NO: 20);

[0092] TaActin-qRT-F: 5'-TATGCCAGCGGTCGAACAAC-3' (SEQ ID NO: 21);

[0093] TaActin-qRT-R: 5'-GGAACAGCACCTCAGGGCAC-3' (SEQ ID NO: 22);

[0094] qRT-PCR reaction system (10 μL): cDNA 1 μL; F primer 0.2 μL; R primer 0.2 μL; ddH2O 3.6 μL; ChamQ Universal SYBR qPCR Master Mix 5 μL.

[0095] The qRT-PCR amplification procedure uses a three-step approach:

[0096] Pre-denaturation at 95°C for 3 min; reaction at 95°C for 15 s, 59°C for 20 s, 72°C for 20 s, for a total of 40 cycles; 95°C for 1 s, 60°C for 20 s, 95°C for 1 s.

[0097] The results are as follows Figure 1 As shown in the figure, the expression levels of TaHSFA6e-A (A1, A2), TaHSFA6e-B and TaHSFA6e-D in both roots and leaves were the highest at 40 minutes after salt stress, and the expression levels in leaves were significantly higher than those in roots. Among them, the expression level of TaHSFA6e-A increased by 28 times at 40 minutes compared with 0 minutes, and the increase was significantly higher than that of TaHSFA6e-B and TaHSFA6e-D. This indicates that the three partial homologous genes of TaHSFA6e may all be involved in the response to salt stress in wheat, and the expression level of TaHSFA6e-A increased the most at 40 minutes compared with 0 minutes ( Figure 1 ).

[0098] Example 6

[0099] 1. Effects of salt stress on transgenic wheat phenotypes

[0100] 40 to 50 seeds of the receptor variety Fielder, TaHSFA6e-A overexpression strain, and TaHSFA6eCrispr mutant strain of the same size were selected, disinfected with 1% NaClO for 10 minutes, and then rinsed with distilled water for 4 to 5 times. After culturing in a petri dish for 1 to 2 days, they were transplanted into a 20 cm diameter pot for soil culture. Two treatments and three replicates were set for each variety. After transplanting, when the plants grew to the two-leaf and one-heart stage, they were treated with salt stress with 500 mmol / L NaCl solution, and the concentration was increased every 5 days until the phenotype appeared, and the plant height was measured. After harvesting, the grain length, grain width, and thousand-grain weight were measured.

[0101] The results showed that compared with the control Fielder, the plant height of the TaHSFA6e-A overexpression strain was higher than that of the control Fielder and TaHSFA6e Crispr mutant strains without salt stress. After salt stress, the wilting and yellowing of leaves of the TaHSFA6e-A overexpression strains were milder, among which the OE-1 overexpression transgenic strain grew the best. The wilting degree of the TaHSFA6e Crispr mutant strains hsfa6e-1 and hsfa6e-2 increased and the leaves turned yellow seriously, among which the hsfa6e-2 mutant strain grew the worst ( Figure 2 (A).

[0102] The statistical results showed that under salt stress, the seeds of the TaHSFA6eA overexpressing strains were longer and wider than those of the Fielder receptor varieties ( Figure 2 Middle B), taller plants ( Figure 2 Medium C) and heavier thousand-grain weight ( Figure 2 The grain length, grain width, plant height and 1000-grain weight of the TaHSFA6e Crispr mutant strain were not as good as Fielder. The above results show that under salt stress conditions, the TaHSFA6e gene can affect the grain shape, plant height and 1000-grain weight of wheat.

[0103] 2. Effects of salt stress on biochemical parameters of transgenic wheat

[0104] After the salt stress phenotype of the recipient variety Fielder, TaHSFA6e-A overexpression strain and TaHSFA6e Crispr mutant strain emerges, take leaves and measure the physiological indicators of plant conductivity, SOD, POD, MDA and soluble sugar content. The measurement method is as follows:

[0105] (1) Plant electrical conductivity

[0106] ① Select three leaves of different strains under different treatments as experimental materials, wash them twice with distilled water, dry them with clean filter paper, and cut the leaves into small pieces of 1 to 2 cm long;

[0107] ② Place the cut materials into large test tubes with labels, and add 20 mL of distilled water to each test tube to submerge the leaves;

[0108] ③Put the three prepared materials into a vacuum dryer and use a vacuum pump to extract air from the intercellular spaces for about 15 minutes;

[0109] ④ Slowly release air again, the water will be pressed into the tissue and the leaves will sink (if there is no vacuum dryer, you can also soak the leaves in distilled water for 4 to 6 hours);

[0110] ⑤ Under room temperature, measure the conductivity of the solution with a conductivity meter;

[0111] ⑥Put the solution in a 100℃ boiling water bath for 15-20 minutes to kill the plant tissue, bring it to room temperature, and measure its boiling conductivity;

[0112]

[0113] (2) SOD enzyme activity assay

[0114] ① Weigh 0.2g wheat leaves in a pre-cooled mortar, add 1mL of pre-cooled extract and grind to homogenate, 4℃, 10000rpm, 15min, take the supernatant as SOD crude solution;

[0115] ② Add 1.5 mL of 0.05 mol / L PBS solution, 0.3 mL of 130 mmol / L Met solution, 750 μmol / L NBT solution, 20 μmol / L riboflavin solution and 100 μmol / L EDTA-Na2 solution to the control tube and the assay tube in sequence, add 0.1 mL of enzyme solution to the assay tube, and do not add enzyme solution to the control tube, but replace it with 0.1 mL of buffer solution, and fill the total volume to 3.3 mL with distilled water;

[0116] ③ Set up three replicates, shield one of the control tubes from light and place it under a 4000lx fluorescent lamp with the other tubes for 20-30 minutes at 25-35℃, then compare the color at 560nm;

[0117]

[0118] Ao: light absorption value of the illumination control tube

[0119] As: light absorption value of the sample tube

[0120] Vt: Total volume of sample solution (mL)

[0121] V1: Sample volume during measurement (mL)

[0122] FW: fresh weight of sample (g)

[0123] (3) POD enzyme activity assay

[0124] ① Weigh 0.25 g of leaves, add 5 times the amount of (M / V) pH 7.0 PBS, grind at 15000 rpm on ice for 15 min, and the supernatant is the crude POD extract;

[0125] ② Add 1 mL of 0.3% H2O2, 0.95 mL of 0.2% guaiacol, and 1 mL of PBS (pH 7.0) to the reaction tube in sequence, and finally add 0.05 mL of enzyme solution to start the reaction, and record the increasing rate at 470 nm (an increase of 0.01 OD per minute is defined as 1 activity unit);

[0126]

[0127] ΔA 470 : Change in absorbance during reaction time

[0128] W: plant fresh weight (g)

[0129] Vt: Total volume of the extracted enzyme solution (mL)

[0130] Vs: Volume of enzyme solution used in the assay (mL)

[0131] t: reaction time (min)

[0132] (4) Determination of MDA content

[0133] ① Weigh 1g of wheat leaves, add 2mL 10% TCA and grind in a mortar, then add 8mL TCA and continue grinding until homogenized, 4000rpm, 10min, the supernatant is the crude MDA extract;

[0134] ②Take 2 mL of the supernatant, add 2 mL of 0.6% TBA, mix well, react in boiling water for 15 min, take the supernatant, and measure the absorbance at 450, 532 and 600 nm;

[0135] MDA concentration = 6.45*(D 532 -D 600 )-0.56*D 450

[0136] MDA content = 2MDA concentration * extract volume (mL) / fresh weight (g) / 1000

[0137] (5) Determination of soluble sugar content (anthrone method)

[0138] ① Preparation of standard curve

[0139] Take the standard sugar solution and dilute it into a series of solutions with different concentrations, 10 mL each, with concentrations of 0, 25, 50, 75, 100, 120, 150, and 200 μg of sugar per mL. Number the test tubes, add 1 mL of the above glucose standard solution and 5 mL of anthrone reagent to each tube in turn, shake to mix them completely, boil them in a boiling water bath for 10 minutes, take them out and cool them, then use a spectrophotometer at a wavelength of 620 nm to measure the optical density of each solution, and draw a standard curve on the coordinate paper with the optical density as the ordinate and the sugar solution concentration as the abscissa.

[0140] ② Determine the soluble sugar content of the sample

[0141] Weigh 1 g of the chopped and mixed fresh sample, put it into a test tube, add 10 mL of distilled water, and extract it in a boiling water bath for 20 min. After cooling, filter it into a 100 mL volumetric flask, rinse the residue with hot water 2 to 3 times, filter it into the volumetric flask, cool it to room temperature, and make up to the scale to obtain the sample solution.

[0142] ③ Sugar content determination

[0143] Pipette 0.2mL of the test solution (sugar content 30-80μg), add it to the test tube, add 2.3mL of distilled water, and shake well. Then quickly add 6.5mL of anthrone reagent along the wall of the test tube, shake and mix immediately, place it on a test tube rack for color development, cool to room temperature, use a blank tube as a control, and determine the sugar content of the extract in the test tube at a wavelength of 620nm by multi-point calibration quantitative method.

[0144] ④Calculation

[0145] According to the optical density value, the corresponding sugar content is found from the standard curve, and the sugar content of the sample is calculated according to the following formula:

[0146]

[0147] Through observation, it was found that under salt stress conditions, compared with Fielder, the SOD activity and POD activity of OE1 and OE2 of the TaHSFA6e-A overexpression transgenic strain were increased, the soluble sugar content was higher, the MDA content was lower, and the relative conductivity was reduced, while the TaHSFA6e Crispr strain was the opposite ( Figure 3 ).

[0148] 3. Determination of photosynthetic indexes of transgenic wheat under salt stress

[0149] The portable photosynthetic meter LI-6800 was used to measure the transpiration rate, net photosynthetic rate, stomatal conductance and intercellular CO2 concentration of the recipient variety Fielder, TTaHSFA6e-A overexpression line and TaHSFA6e Crispr mutant line before and after salt stress treatment; the relative chlorophyll content of wheat was determined using a SPAD-502 chlorophyll meter.

[0150] Through observation, it was found that under salt stress conditions, the net photosynthetic rate, stomatal conductance, transpiration rate, and relative chlorophyll content of OE1 and OE2 of the TaHSFA6e-A overexpressing transgenic lines increased, and the intercellular CO2 concentration decreased compared with Fielder, while the situation of the TaHSFA6eCrispr line was the opposite ( Figure 4 ), which is consistent with the phenotypic identification results of the TaHSFA6e transgenic strain mentioned above.

[0151] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. Application of the TaHSFA6e gene in regulating plant tolerance to salt stress, characterized in that: The amino acid sequence encoded by the TaHSFA6e gene is shown in at least one of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:9, and SEQ ID NO:

13.

2. The use of the TaHSFA6e gene according to claim 1 in regulating plant tolerance to salt stress, characterized in that: The nucleic acid sequence of the TaHSFA6e gene is shown in at least one of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:

12.

3. Use of the TaHSFA6e gene according to claim 1 or 2 in regulating plant tolerance to salt stress, characterized in that: By increasing the expression level of the TaHSFA6e gene in the plant, the tolerance of the plant to salt stress is improved; or by decreasing the expression level of the TaHSFA6e gene in the plant, the tolerance of the plant to salt stress is reduced.

4. The use of the TaHSFA6e gene in regulating plant tolerance to salt stress according to claim 3, characterized in that: The plant is wheat.

5. A method for regulating plant tolerance to salt stress, characterized in that: By increasing the expression level of the TaHSFA6e gene in the plant, the tolerance of the plant to salt stress is improved; or by reducing the expression level of the TaHSFA6e gene in the plant, the tolerance of the plant to salt stress is reduced; the amino acid sequence encoded by the TaHSFA6e gene is shown in at least one of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 9, and SEQ ID NO:

13.

6. The method for regulating plant tolerance to salt stress according to claim 5, characterized in that: The nucleic acid sequence of the TaHSFA6e gene is shown in at least one of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:

12.

7. The method for regulating plant tolerance to salt stress according to claim 5, characterized in that: By constructing a plant expression vector of the TaHSFA6e gene and transforming the plant, the TaHSFA6e gene is expressed in the plant, thereby increasing the expression level of the TaHSFA6e gene in the plant.

8. The method for regulating plant tolerance to salt stress according to claim 5, characterized in that: The TaHSFA6e gene in the plant is mutated or silenced to reduce the expression level of the TaHSFA6e gene in the plant.

9. The method for regulating plant tolerance to salt stress according to claim 8, characterized in that: The TaHSFA6e gene mutation in the plant is achieved by using the Crispr / Cas 9 method to mutate at least one sequence among SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:12 in the plant.

10. The method for regulating plant tolerance to salt stress according to any one of claims 5 to 9, characterized in that: The plant is wheat.

Images