Application of sweet potato IbbHLH149 gene in improvement of plant stress resistance

Abstract

The invention discloses application of a sweet potato IbbHLH149 gene in improvement of plant stress resistance, and belongs to the technical field of molecular biology. The nucleotide sequence of the sweet potato IbbHLH149 gene is as shown in SEQ ID NO.1, and the coded amino acid sequence of the sweet potato IbbHLH149 gene is as shown in SEQ ID NO.2. The method comprises the following steps: constructing a plant expression vector of the IbbHLH149 gene, and transforming arabidopsis thaliana; the result shows that the morphological development of the arabidopsis thaliana plant into which the IbbHLH149 gene is transferred is normal, and the arabidopsis thaliana seedling into which the IbbHLH149 gene is transferred can at least resist the stress of 125mM NaCl high salt or the stress of 400mM mannitol; the expression of the IbbHLH149 gene in arabidopsis thaliana can significantly improve the salt tolerance and drought resistance of the arabidopsis thaliana. Therefore, the IbbHLH149 gene has an important application value in the cultivation of stress-resistant plants.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and particularly relates to application of sweet potato IbbHLH149 gene in improving plant stress resistance. Background Art

[0002] Organisms often face various adverse conditions (such as drought, high salt, low temperature, etc.) in the natural environment; these adverse conditions can inhibit the growth of organisms and even cause the death of organisms. With the continuous deterioration of the environment, stress such as high salt and drought has become a global problem. Breeding new varieties of organisms with multiple stress resistance has become one of the main research goals of breeders.

[0003] Sweet potatoes are moderately drought-resistant and salt-tolerant crops, but their drought and salt tolerance vary at different growth stages. Drought and salt stress will inhibit the growth of sweet potatoes to a certain extent, affect the development of tubers, and cause a decrease in yield. Plant drought resistance and salt tolerance are controlled by multiple genes. Conventional breeding techniques are inefficient and have a long cycle. The rapidly developing genetic engineering technology provides a new way for biological genetic improvement. Genetic transformation using genes that play an important role in drought and salt stress response is an important means to obtain new drought-resistant and salt-tolerant germplasm. Summary of the invention

[0004] In view of the problems existing in the prior art, the object of the present invention is to provide an application of the sweet potato IbbHLH149 gene in improving plant stress resistance.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] The application of the sweet potato IbbHLH149 gene in improving plant stress resistance, the amino acid sequence encoded by the sweet potato IbbHLH149 gene is shown in SEQ ID NO.2.

[0007] Based on the above scheme, the nucleic acid sequence of the sweet potato IbbHLH149 gene is shown in SEQ ID NO.1.

[0008] On the basis of the above scheme, the stress resistance of plants is improved by increasing the expression level of the IbbHLH149 gene in plants.

[0009] On the basis of the above scheme, the IbbHLH149 gene sequence was constructed into a plant expression vector and transformed into plants to overexpress it in the plants.

[0010] Based on the above scheme, the stress resistance is drought resistance and high salt resistance.

[0011] A method for improving plant stress resistance, which improves the stress resistance of plants by increasing the expression level of the IbbHLH149 gene in the plants, wherein the amino acid sequence encoded by the IbbHLH149 gene is shown in SEQ ID NO.2.

[0012] On the basis of the above scheme, the IbbHLH149 gene sequence was constructed into a plant expression vector and transformed into plants to overexpress it in the plants.

[0013] On the basis of the above scheme, the method of transforming the plant body is one of the following: Agrobacterium-mediated method, gene gun method, electric shock method, PEG method, and liposome method.

[0014] Based on the above scheme, the stress resistance is drought resistance and high salt resistance.

[0015] Based on the above scheme, the plant is sweet potato or Arabidopsis thaliana.

[0016] Advantages of the technical solution of the present invention

[0017] The present invention cloned a stress resistance gene from sweet potato, and sequencing results showed that the gene coding sequence contained 624 nucleotides, and the protein encoded by it contained 207 amino acids, which was named IbbHLH149. A plant expression vector of the IbbHLH149 gene was constructed, and Arabidopsis thaliana was transformed; the results showed that the Arabidopsis plants into which the IbbHLH149 gene was transferred had normal morphological development, and the Arabidopsis seedlings transferred with the IbbHLH149 gene could at least resist 125mM NaCl high salt stress or 400mM mannitol stress; the expression of the IbbHLH149 gene in Arabidopsis thaliana could significantly improve its salt tolerance and drought resistance. Therefore, the IbbHLH149 gene has important application value in the cultivation of stress-resistant plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 IbbHLH149 transcriptional activation activity analysis (pGBKT7 empty as negative control; pGAL4 as positive control);

[0019] Figure 2 The expression of IbbHLH149 in the sweet potato variety “Xushu 55-2” after being subjected to 200 mM NaCl, 30% PEG6000 and 100 μM ABA stress;

[0020] Figure 3 Seed salt and drought tolerance germination test of Columbia wild-type Arabidopsis and IbbHLH149 transgenic Arabidopsis (WT refers to the wild type, OE1, OE2, and OE3 refer to the three strains of transgenic Arabidopsis; A is the germination of Arabidopsis, B is the germination rate statistics of Arabidopsis; C is the cotyledon expansion rate statistics of Arabidopsis);

[0021] Figure 4 In vitro identification of salt tolerance and drought resistance of Columbia wild-type Arabidopsis and IbbHLH149 transgenic Arabidopsis (WT refers to the wild type, OE1, OE2, and OE3 refer to the three strains of transgenic Arabidopsis; A is the growth of Arabidopsis, B is the root length statistics of Arabidopsis; C is the fresh weight statistics of Arabidopsis);

[0022] Figure 5 Pot culture identification of salt and drought tolerance of Columbia wild-type Arabidopsis and IbbHLH149 transgenic Arabidopsis (WT refers to the wild type, OE1, OE2, and OE3 refer to the three lines of transgenic Arabidopsis);

[0023] Figure 6 Expression analysis of stress resistance-related genes in Columbia wild-type Arabidopsis and IbbHLH149 transgenic Arabidopsis under high salt and drought stress (WT refers to the wild type, OE1, OE2, and OE3 refer to the three lines of transgenic Arabidopsis);

[0024] Figure 7 Analysis of physiological and biochemical indices of stress resistance of Columbia wild-type Arabidopsis and IbbHLH149 transgenic Arabidopsis under high salt and drought stress (WT refers to the wild type, OE1, OE2, and OE3 refer to the three strains of transgenic Arabidopsis);

[0025] Figure 8 Analysis of reactive oxygen accumulation in leaves of Columbia wild-type Arabidopsis and IbbHLH149 transgenic Arabidopsis under high salt and drought stress (WT refers to the wild type, OE1, OE2, and OE3 refer to the three strains of transgenic Arabidopsis). DETAILED DESCRIPTION

[0026] 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.

[0027] 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.

[0028] In the following embodiments:

[0029] The sweet potato variety "Xushu 55-2" is a drought-tolerant sweet potato strain and is preserved in the Sweet Potato Research Center of Qingdao Agricultural University;

[0030] Agrobacterium tumefaciens strain GV3101 was purchased from Shanghai Weidi Biotechnology Co., Ltd.;

[0031] The transgenic recipient material was Arabidopsis Columbia wild-type variety provided by the Sweet Potato Research Center of Qingdao Agricultural University.

[0032] pGBKT7 vector, deposited in the Sweet Potato Research Center of Qingdao Agricultural University;

[0033] Y2H yeast cells were purchased from Beijing Coolaibo Technology Co., Ltd.;

[0034] pGAL4, deposited in the Sweet Potato Research Center of Qingdao Agricultural University;

[0035] Super1300, stored in the Sweet Potato Research Center of Qingdao Agricultural University.

[0036] Example 1

[0037] Cloning of IbbHLH149 gene

[0038] Using the cDNA obtained by reverse transcription of RNA from sweet potato (Xushu 55-2) as a template, PCR was performed to amplify the sweet potato IbbHLH149 gene using primer pair P1 and P2, and its gene sequence is shown in SEQ ID NO.1; sequencing results showed that the gene coding sequence contained 624 nucleotides, and the protein encoded by it contained 207 amino acids, and its amino acid sequence was shown in SEQ ID NO.2.

[0039] SEQ ID NO.1(5'-3')

[0040] ATGGAGTTATCGTCTTCGATAGCGAATCCCGATGTCAACTCCAACCGATCGCGGAAAAAGCGGAGAAAAATCGGCGAGTCTGACCAGAATCAGCCACAACCTACCGGCGTCGATGTACTCAGGTGGAGGACACAGACCGAGCAGCAGATCTACTCGTCGAAGCTTCTGGAGGCTCTCCGTCAGGTCCGCCGGAGAAATGAGGCGCCATCTCCCGCTGTTGCCGGCCGCACTGTTCGGGAGACTGCGGACAAGGTCCTCGCCGTCGCCGCCAAGGGGAGGACGCGCTGGAGCCGAGCGATTCTCACCGGACGGCTTAGTTTGAGACTCAGGCAGATCAACAAAAAGCATAAGAGAGCCAAGGTGGTAGCCTCCGGCGATATCCGGCCGAAGAAACCGGGGACTAAGAAGAGGTTGCCGCCGCTGCAAAGTAAGGCCCGCGTTCTCGGGCGGTTAGTTCCCGGTTGCCGGAAACTCTCGTTCCCGAATCTTCTAGAAGAAGCTACTGATTATATTGCAGCTCTGGAGATGCAAGTTAGAGCCATGAGCGTTCTCACAGGGCTTCTCAACGGTGCCGGAGTCGGATTACCGGCTAATCCCGACCGGCTCGGCTCAGAACAGTCATAA

[0041] SEQ ID NO.2

[0042] MELSSSIANPDVNSNRSRKKRRKIGESDQNQPQPTGVDVLRWRTQTEQQIYSSKLLEALRQVRRRNEAPSPAVAGRTVRETADKVLAVAAKGRTRWSRAILTGRLSLRLRQINKKHKRAKVVASGDIRPKKPGTKKRLPPLQSKARVLGRLVPGCRKLSFPNLLEEATDYIAALEMQVRAMSVLTGLLNGAGVGLPANPDRLGSEQS

[0043] The specific primer sequences are as follows:

[0044] P1: 5'-ATGGAGTTATCGTCTTCGAT-3' (SEQ ID NO.3);

[0045] P2: 5'-TTATGACTGTTCTGAGCCGA-3' (SEQ ID NO.4);

[0046] The PCR reaction system was (50 μL): 25 μL 2× Flash HS PCR Master Mix, 2 μL each of 20 μmol / L forward and reverse primers, 500 ng reverse transcription product, and supplemented with ddH2O;

[0047] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min in each cycle; after the cycle was completed, extension was continued for 10 min.

[0048] Example 2

[0049] Analysis of IbbHLH149 transcriptional activation activity

[0050] (1) Primers were designed based on the IbbHLH149 sequence, restriction sites (NdeⅠ and SalⅠ) and protective bases were added to both ends of the primers, the full-length sequence of the IbbHLH149 gene was amplified, and the primers were inserted into the multiple cloning site of the pGBKT7 vector (Kan resistance) to obtain the pGBKT7-IbbHLH149 recombinant vector.

[0051] The primers for constructing the IbbHLH149 transcriptional activation activity analysis vector are:

[0052] P3 (NdeⅠ): 5'-GGAATTCCATATG ATGGAGTTATCGTCTTCGAT-3' (SEQ ID NO.5);

[0053] P4 (SalⅠ): 5'-ACGCGTCGACTTATGACTGTTCTGAGCCGA-3' (SEQ ID NO. 6);

[0054] (2) The recombinant vector pGBKT7-IbbHLH149 was transferred into Y2H yeast cells. The yeast Y2H Gold was activated on YPDA solid medium. A single clone yeast plaque was picked and placed in a centrifuge tube. YPDA liquid medium was added for shaking culture. 1 mL of bacterial solution was taken and centrifuged at 5000 rpm for 1 min. The supernatant was discarded. 500 mL of ddH2O was used to suspend the precipitate. The centrifuge was centrifuged at 5000 rpm for 1 min. The supernatant was discarded. 50 μL of LiAc (1 M), 20 μL of DTT (1 M), 6.75 μL of Carrier DNA, and 1 μg of recombinant plasmid were added to the centrifuge tube in sequence. Mix well and add 160 μL of 50% PEG4000. Place in a 42°C water bath for 30 min. Place in a centrifuge at 5000 rpm for 1 min. The supernatant was discarded. 500 mL of ddH2O was used to suspend the precipitate. The centrifuge was centrifuged at 5000 rpm for 1 min. The supernatant was discarded. Add 150 μL ddH2O to the centrifuge tube to suspend the precipitate, streak the suspended cells on the solid medium of SD / -Trp / -His / X-α-Gal, and culture in a constant temperature incubator at 30°C for 3-5 days. Observe the growth of the colonies, and use the pGBKT7 empty vector as a negative control (pGBKT7-empty) and the pGAL4 vector as a positive control. The results are shown in Figure 1 This indicates that the full-length IbbHLH149 has no transcriptional activation activity.

[0055] Example 3

[0056] Expression of IbbHLH149 in sweet potato after abiotic stress

[0057] (1) The seedlings of Shangshu No. 19 were treated with Hoagland solution containing 30% PEG6000, 200 mM NaCl or 100 μM ABA. Samples were taken at 0 h, 1 h, 3 h, 6 h, 12 h and 24 h after treatment, frozen in liquid nitrogen and ground into powder. RNA was extracted using an RNA extraction kit. The extracted total RNA was treated with DNase I, purified and then reverse transcribed into cDNA.

[0058] (2) The reaction was performed on a QuantStudio 3 fluorescent quantitative PCR instrument to detect the expression of IbbHLH149.

[0059] The 20 μL reaction system included: 10 μL 2×SybrGreen qPCR Master Mix, 0.25 μL each of 20 μmol / L forward and reverse primers, and 20 ng of reverse transcription product.

[0060] The amplification procedure was as follows: pre-denaturation at 94°C for 2 min; followed by 40 cycles of reaction, with denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s in each cycle; after the cycle, the temperature was slowly raised to 94°C to prepare a melting curve.

[0061] Each reaction was performed in triplicate.

[0062] The quantitative PCR primers for IbbHLH149 gene were

[0063] P5: 5'-GGTTGCCGGAAACTCTCGTTC-3' (SEQ ID NO.7);

[0064] P6: 5'-AAGCCCTGTGAGAACGCTCAT-3' (SEQ ID NO. 8).

[0065] The primer sequence of the internal standard gene Actin is

[0066] P7: 5'-AGCAGCATGAAGATTAAGGTTGTAGCAC-3' (SEQ ID NO.9);

[0067] P8: 5'-TGGAAAATTAGAAGCACTTCCTGTGAAC-3' (SEQ ID NO. 10).

[0068] The results are as follows Figure 2 As shown, the IbbHLH149 gene was significantly upregulated after 30% PEG6000 and 200 mM NaCl stress treatments and was induced by 100 μM ABA, indicating that it is involved in the ABA-mediated response of sweet potato to drought and salt stress.

[0069] Example 4

[0070] Construction of plant expression vector of IbbHLH149 gene

[0071] Using the cDNA of the drought-tolerant sweet potato line "Xu 55-2" as a template, KpnI and SalI restriction sites were added to the upstream and downstream primers, respectively, and PCR amplification was performed to obtain the coding region sequence of the IbbHLH149 gene containing KpnI and SalI restriction sites.

[0072] P9(KpnI): 5'-GGGGTACCATGGAGTTATCGTCTTCGAT-3' (SEQ ID NO. 11);

[0073] P10(SalI): 5'-ACGCGTCGACTGACTGTTCTGAGCCGA-3' (SEQ ID NO. 12);

[0074] The PCR product was recovered, and the plant expression vector Super1300 was digested with KpnI and SalI. The coding region sequence of the IbbHLH149 gene containing the KpnI and SalI restriction sites was connected to the corresponding restriction sites of the plant expression vector Super1300 to obtain the plant expression vector Super1300-IbbHLH149 of the gene.

[0075] Example 5

[0076] Obtaining Transgenic Arabidopsis thaliana with IbbHLH149 Gene

[0077] (1) Preparation, activation and bacterial liquid preparation of Agrobacterium recombinant strains: The Super1300-IbbHLH149 recombinant plasmid was transformed into Agrobacterium strain GV3101 competent cells by liquid nitrogen freeze-thaw method, and the recombinant strain (IbbHLH149-GV3101) containing the recombinant plasmid was screened. A single colony of the recombinant strain was picked and inoculated into LB (containing 50 mg / L rifampicin and 50 mg / L kanamycin) liquid medium, and cultured at 28°C and 180 rpm until OD600 = 0.5-0.8, then 2 mL of bacterial liquid was transferred to 50 mL LB (containing 50 mg / L rifampicin and 50 mg / L kanamycin) medium and cultured until OD600 = 0.6-0.8. After the bacterial liquid was centrifuged at 5000 rpm for 15 min, it was suspended with the same volume of liquid 1 / 2MS (0.02% silwet L-77) to obtain the impregnation liquid for use.

[0078] (2) Planting of Arabidopsis thaliana: Select appropriate Arabidopsis thaliana seeds, soak them in 1% NaClO for 5 min, rinse them with sterile water for 4-6 times, and plant them on the substrate soil.

[0079] (3) Agrobacterium-mediated genetic transformation: Select healthy plants in the early fruiting stage, place them upside down with the pots and bowls on top of a container containing Agrobacterium-infected liquid, and immerse the entire inflorescence in the Agrobacterium suspension for about 20-30 seconds. Be careful not to let the leaves come into contact with the infecting liquid. Remove the pots and bowls and place them horizontally in a dark box for about 24 hours. Keep a certain humidity. After 24 hours, place the treated Arabidopsis plants under light conditions of 22-25°C to allow them to grow normally. Mature seeds are collected after about 3 weeks.

[0080] The harvested transgenic IbbHLH149 Arabidopsis seeds were inoculated into 20 mL MS (hygromycin 50 mg / L) medium and cultured at 22°C for about 1 week. Bright green and strong Arabidopsis seedlings were selected and transplanted into substrate soil. Transgenic plants were purified to the T3 generation.

[0081] The PCR method was used to detect the positive plants of Arabidopsis thaliana transgenic with IbbHLH149 gene, and the steps were as follows:

[0082] The genomic DNA of the transgenic plants was extracted, and the primers were designed using the above vector sequence for PCR amplification. The PCR reaction program was: 95°C, 5min; 95°C, 50s, 55°C, 50s, 72°C, 1min, 32 cycles; 72°C, 10min. The correct amplified band was the positive plant of Arabidopsis transgenic with IbbHLH149 gene.

[0083] The primers for identifying transgenic plants are:

[0084] P11: 5'-CGCCATTTCGCCTTTTCAGAAATGG-3' (SEQ ID NO. 13);

[0085] P12: 5'-TGGTACAAACGTAGGGCTAGCTG-3' (SEQ ID NO. 14).

[0086] Example 6

[0087] Identification of Drought and Salt Tolerance in Transgenic Arabidopsis Plants with IbbHLH149 Gene

[0088] (1) Salt tolerance and drought resistance of transgenic Arabidopsis thaliana with IbbHLH149 gene during germination

[0089] The transgenic Arabidopsis thaliana of IbbHLH149 gene obtained in Example 5 and the wild-type Arabidopsis thaliana of Columbia were inoculated in normal 1 / 2MS medium containing 130 mM NaCl or 300 mM mannitol, respectively. The medium was cultured at 22°C for about 1 week. The germination of the Arabidopsis seeds was observed, and the germination rate and cotyledon expansion rate were measured. The results are shown in FIG. Figure 3 As shown, under high salt or drought stress conditions, the germination rate and cotyledon expansion of the transgenic IbbHLH149 Arabidopsis plants were better than those of the Columbia wild-type Arabidopsis, so the salt resistance concentration of the transgenic IbbHLH149 Arabidopsis seeds was 130 mM NaCl or above, and the drought resistance concentration was 300 mM mannitol or above.

[0090] (2) Salt tolerance and drought resistance of transgenic Arabidopsis thaliana with IbbHLH149 gene at seedling stage

[0091] The transgenic Arabidopsis thaliana of IbbHLH149 gene obtained in Example 5 and the wild-type Arabidopsis thaliana of Columbia were inoculated in 1 / 2MS medium for germination. One week later, the seedlings were transferred to normal 1 / 2MS medium containing 125 mM NaCl or 400 mM mannitol. The seedlings were cultured at 22°C for about 10 days, and the growth and rooting under high salt or mannitol stress conditions were observed. The root length and fresh weight were measured. The results are as follows: Figure 4As shown. Under high salt or mannitol stress conditions, the growth and rooting of Arabidopsis seedlings transgenic for IbbHLH149 gene were better than those of Columbia wild-type Arabidopsis seedlings ( Figure 4 ), so the salt resistance concentration of Arabidopsis thaliana seedlings transformed with IbbHLH149 gene is 125mM or above, and the drought resistance concentration is 400mM mannitol or above.

[0092] (3) Salt tolerance and drought resistance of transgenic Arabidopsis thaliana under soil culture conditions

[0093] The transgenic Arabidopsis thaliana of IbbHLH149 gene obtained in Example 5 and the wild-type Arabidopsis thaliana of Columbia were inoculated in 1 / 2MS culture medium for germination. After 1 week, the seedlings were transferred to culture soil and cultured for 10 days. The following treatments were performed: the salt-treated plants were watered with a 300 mM NaCl aqueous solution once every 3 days, and the growth of Arabidopsis thaliana was observed after 2 weeks; the natural drought-treated plants were not watered, and after 2 weeks of natural drought, they were rehydrated for 3 days and the growth of Arabidopsis thaliana was observed. The results are shown in FIG. Figure 5 As shown, under high salt or natural drought stress conditions, the growth of transgenic IbbHLH149 Arabidopsis seedlings was better than that of Columbia wild-type Arabidopsis seedlings.

[0094] Example 7

[0095] Mechanism of IbbHLH149 improving salt tolerance and drought resistance in transgenic plants

[0096] 1. Analysis of stress resistance gene expression in transgenic Arabidopsis thaliana with IbbHLH149 gene

[0097] To further analyze the mechanism by which IbbHLH149 improves salt tolerance and drought resistance in transgenic plants, the induced expression of stress-resistant genes in potted Arabidopsis thaliana with the IbbHLH149 gene and Columbia wild-type Arabidopsis thaliana plants that were irrigated with normal or saline water (300 mM NaCl solution) for 2 weeks or subjected to natural drought for 2 weeks was determined.

[0098] IbbHLH149 transgenic Arabidopsis and Columbia wild-type Arabidopsis potted plant samples were taken under normal, high salt or natural drought stress. After total RNA was extracted, it was treated with DNase I, purified, reverse transcribed into cDNA and used as a template. Fluorescent quantitative primers were designed according to the specific sequences of Arabidopsis stress resistance genes (AtCAT, AtSOD, AtAOS, AtLOX2, AtZEP and AtP5CR genes), and the samples were reacted on a QuantStudio 3 fluorescent quantitative PCR instrument. The reaction system was the same as in Example 3, and the data were analyzed.

[0099] AtCAT gene quantitative PCR primers are:

[0100] P13: 5'-GCAACTACCCCGAGTGGAAA-3' (SEQ ID NO. 15);

[0101] P14: 5'-TGTTCAGAACCAAGCGACCA-3' (SEQ ID NO. 16);

[0102] AtSOD gene quantitative PCR primers are:

[0103] P15: 5'-ATGAGAAGTTCTATGAAGAG-3' (SEQ ID NO. 17);

[0104] P16: 5'-GTCTTTATGTAATCTGGT-3' (SEQ ID NO. 18);

[0105] AtAOS gene quantitative PCR primers are:

[0106] P17: 5'-CGATTTCTCTCCACCCAAAAAC-3' (SEQ ID NO. 19);

[0107] P18: 5'-GGTCTTTGATTGGTCCTACGATT-3' (SEQ ID NO. 20);.

[0108] AtLOX2 gene quantitative PCR primers are:

[0109] P19: 5'-CAAACCTCAGAAGACGATGTAAGG-3' (SEQ ID NO. 21);

[0110] P20: 5'-GACCTCTCGACCAAGTTATGCC-3' (SEQ ID NO. 22);

[0111] AtZEP gene quantitative PCR primers are:

[0112] P21: 5'-CGGAGCTTTCTTCTTGATGG-3' (SEQ ID NO. 23);

[0113] P22: 5'-TCGATTTCGGAGTTTTCCTG-3' (SEQ ID NO. 24);

[0114] AtP5CR gene quantitative PCR primers are:

[0115] P23: 5'-AGTTTAGCTTCACAGACCGTTC-3' (SEQ ID NO. 25);

[0116] P24: 5'-GCTCTGTGAGAGCTCGCGGCTTC-3' (SEQ ID NO. 26).

[0117] The primer sequence of the internal standard gene AtActin is

[0118] P25: 5'-GCACCCTGTTCTTCTTACCGA-3' (SEQ ID NO. 27);

[0119] P26: 5'-AGTAAGGTCACGTCCAGCAAGG-3' (SEQ ID NO. 28).

[0120] The expression of different stress resistance related genes Figure 6 As shown in the figure, under normal growth conditions, there was no significant difference in the expression levels of stress-related genes between transgenic lines and wild-type lines. Under high salt or drought stress, the expression levels of active oxygen scavenging system-related genes AtCAT and AtSOD, JA signaling pathway key genes AtAOS and AtLOX2, ABA signaling pathway key gene AtZEP, and proline synthesis pathway-related gene AtP5CR in transgenic Arabidopsis were significantly higher than those in wild-type plants. This indicates that IbbHLH149 activated the expression of stress-resistant genes under high salt or drought stress.

[0121] 2. Physiological and biochemical indicators related to stress resistance

[0122] (1) IbbHLH149 transgenic Arabidopsis and Columbia wild-type Arabidopsis plant samples were taken under normal, high salt and drought stress, and the enzyme activities of key enzymes SOD and POD in the active oxygen scavenging system and the malondialdehyde (MDA) and proline (Pro) contents of Arabidopsis plants under different conditions were determined. Figure 7 As shown). The related indicators of the normally grown IbbHLH149 transgenic strains and the wild-type strains did not show significant differences. Under high salt or drought stress, the MDA content of the IbbHLH149 transgenic strains was significantly lower than that of the wild-type strains, and the Pro content and the enzyme activities of SOD and POD were significantly higher than those of the wild-type plants. The results showed that the overexpression of IbbHLH149 could activate the active oxygen scavenging system of transgenic plants under high salt or drought stress, enhance the osmotic adjustment ability of transgenic plants, and reduce the damage of high salt stress to the membrane system.

[0123] (2) Leaves of Arabidopsis thaliana with IbbHLH149 gene and wild-type Arabidopsis thaliana under normal, high salt and drought stress were taken for NBT staining and DAB staining to analyze the leaf O 2 -2 and H2O accumulation (e.g. Figure 8). Under high salt stress, Arabidopsis transgenic plants overexpressing IbbHLH149 2- The accumulation of H2O2 in Arabidopsis thaliana was significantly lower than that in wild-type Arabidopsis thaliana. Overexpression of the IbbHLH149 gene reduced the excessive accumulation of reactive oxygen species under high salt or drought stress, and alleviated the damage of high salt stress to the physiological activities of Arabidopsis thaliana.

[0124] 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 sweet potato IbbHLH149 gene in improving plant stress resistance, characterized in that: The amino acid sequence encoded by the sweet potato IbbHLH149 gene is shown in SEQ ID NO.

2.

2. The use of the sweet potato IbbHLH149 gene in improving plant stress resistance according to claim 1, characterized in that: The nucleic acid sequence of the sweet potato IbbHLH149 gene is shown in SEQ ID NO.

1.

3. The use of the sweet potato IbbHLH149 gene in improving plant stress resistance according to claim 1, characterized in that: By increasing the expression level of the IbbHLH149 gene in plants, the stress resistance of plants is improved.

4. The use of the sweet potato IbbHLH149 gene in improving plant stress resistance according to claim 3, characterized in that: The IbbHLH149 gene sequence is constructed into a plant expression vector and transformed into a plant body to overexpress it in the plant.

5. The use of the sweet potato IbbHLH149 gene in improving plant stress resistance according to any one of claims 1 to 4, characterized in that: The stress resistance is drought resistance and high salt resistance.

6. A method for improving plant stress resistance, characterized in that: By increasing the expression level of the IbbHLH149 gene in the plant, the stress resistance of the plant is improved. The amino acid sequence encoded by the IbbHLH149 gene is shown in SEQ ID NO.

2.

7. The method for improving plant stress resistance according to claim 6, characterized in that: The IbbHLH149 gene sequence is constructed into a plant expression vector and transformed into a plant body to overexpress it in the plant.

8. The method for improving plant stress resistance according to claim 7, characterized in that: The method of transforming the plant body is one of the following: Agrobacterium-mediated method, gene gun method, electric shock method, PEG method, and liposome method.

9. The method for improving plant stress resistance according to claim 6, characterized in that: The stress resistance is drought resistance and high salt resistance.

10. The method for improving plant stress resistance according to any one of claims 6 to 9, characterized in that: The plant is sweet potato or Arabidopsis thaliana.

Images