Application of sweet potato IbbHLH149 gene in improving plant stress resistance

Abstract

The present invention discloses the application of the sweet potato IbbHLH149 gene in improving plant stress resistance, and belongs to the technical field of molecular biology. The nucleic acid sequence of the sweet potato IbbHLH149 gene of the present invention is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2. A plant expression vector of the IbbHLH149 gene is constructed, and Arabidopsis thaliana is transformed; the results show that: the morphology and development of Arabidopsis plants into which the IbbHLH149 gene is transferred are normal, and the Arabidopsis seedlings into which the IbbHLH149 gene is transferred can at least resist 125mM NaCl high salt stress or 400mM mannitol stress; the expression of the IbbHLH149 gene in Arabidopsis thaliana can significantly improve its salt tolerance and drought resistance. Therefore, the IbbHLH149 gene has 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 the sweet potato IbbHLH149 gene in improving plant stress resistance. Background Art

[0002] Organisms in their natural environments often face various adverse conditions (such as drought, high salinity, and low temperatures); these conditions can inhibit their growth and even lead to their death. With the continuous deterioration of the environment, stresses such as high salinity and drought have become a global problem. Breeding new varieties with multiple stress tolerances has become a major research goal for plant breeders.

[0003] Sweet potatoes are moderately drought- and salt-tolerant crops, but their tolerance varies across different growth stages. Drought and salt stress can inhibit sweet potato growth, affect tuber development, and reduce yield. Drought and salt tolerance in plants are controlled by multiple genes, and conventional breeding techniques are inefficient and time-consuming. Rapidly developing genetic engineering technologies are providing new avenues for genetic improvement. Genetic transformation using genes that play a key role in drought and salt stress responses is an important means of obtaining new drought- 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 solutions:

[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] Based on the above scheme, the IbbHLH149 gene sequence was constructed into a plant expression vector and transformed into plants to overexpress it in 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, comprising increasing the expression level of the IbbHLH149 gene in the plant to improve the stress resistance of the plant, wherein the amino acid sequence encoded by the IbbHLH149 gene is shown in SEQ ID NO.2.

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

[0013] On the basis of the above scheme, the method of transforming the plant body is one of the following methods: 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-resistant gene from sweet potato. Sequencing results showed that the gene coding sequence contained 624 nucleotides and the protein it encoded contained 207 amino acids. The gene was named IbbHLH149. A plant expression vector of the IbbHLH149 gene was constructed and transformed into Arabidopsis thaliana. The results showed that the Arabidopsis plants into which the IbbHLH149 gene was introduced had normal morphological development, and the Arabidopsis seedlings transformed with the IbbHLH149 gene could at least withstand 125mM NaCl high salt stress or 400mM mannitol stress. The expression of the IbbHLH149 gene in Arabidopsis thaliana significantly improved 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 transgenic Arabidopsis lines; A is the germination status of Arabidopsis, B is the germination rate statistics of Arabidopsis; C is the cotyledon expansion rate statistics of Arabidopsis);

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

[0022] Figure 5 Pot culture evaluation 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 transgenic Arabidopsis lines);

[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 transgenic Arabidopsis lines);

[0024] Figure 7 Analysis of physiological and biochemical indicators 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 transgenic Arabidopsis lines);

[0025] Figure 8 Analysis of reactive oxygen species accumulation in leaves of Columbia wild-type Arabidopsis thaliana and IbbHLH149 transgenic Arabidopsis thaliana under high salt and drought stress (WT refers to the wild type, OE1, OE2, and OE3 refer to the three transgenic Arabidopsis lines). 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. Below, in conjunction with specific examples, the present invention will be further described in detail with reference to data. The following examples are merely for illustration of 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 this field or according to the product instructions. The experimental materials, reagents, and drugs 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 variety 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 the wild-type Arabidopsis thaliana Columbia variety provided by the Sweet Potato Research Center of Qingdao Agricultural University.

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

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

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

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

[0036] Example 1

[0037] Cloning of the IbbHLH149 gene

[0038] Using cDNA obtained by reverse transcription of sweet potato (Xushu 55-2) RNA as a template, the sweet potato IbbHLH149 gene was amplified by PCR using primer pair P1 and P2. The gene sequence is shown in SEQ ID NO.1; sequencing results showed that the gene coding sequence contains 624 nucleotides, and the encoded protein contains 207 amino acids, and its amino acid sequence is 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 ddH2O supplementation;

[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; 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 to amplify the full-length sequence of the IbbHLH149 gene and insert it 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 transformed into Y2H yeast cells. Yeast Y2H Gold was activated on YPDA solid medium. A single clone of yeast plaque was picked and placed in a centrifuge tube. YPDA liquid medium was added and shaken for culture. 1 mL of bacterial solution was taken and centrifuged at 5000 rpm for 1 min. The supernatant was discarded. The precipitate was suspended in 500 mL of ddH2O and 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. The tube was placed in a 42°C water bath for 30 min. The tube was centrifuged at 5000 rpm for 1 min. The supernatant was discarded. The precipitate was suspended in 500 mL of ddH2O and centrifuged at 5000 rpm for 1 min. The supernatant was discarded. Add 150 μL ddH2O to the centrifuge tube to suspend the pellet, streak the suspended cells on SD / -Trp / -His / X-α-Gal solid medium, and culture in a 30°C constant temperature incubator 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 2. 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 treatment

[0057] (1) Seedlings of "Shangshu No. 19" were treated with Hoagland's solution containing 30% PEG6000, 200 mM NaCl, or 100 μM ABA. Samples were collected at 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h after treatment, quickly frozen in liquid nitrogen, and ground into a 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 includes: 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 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 the melting curve.

[0061] Each reaction was performed with 3 replicate wells.

[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 in the results, the expression of 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 ligated into 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 Carrying the IbbHLH149 Gene

[0077] (1) Preparation, activation, and bacterial liquid preparation of recombinant Agrobacterium strains: The Super1300-IbbHLH149 recombinant plasmid was transformed into competent cells of the Agrobacterium strain GV3101 using the 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 liquid medium (containing 50 mg / L rifampicin and 50 mg / L kanamycin) and cultured at 28°C and 180 rpm until OD600 = 0.5-0.8. 2 mL of the bacterial liquid was then transferred to 50 mL of LB medium (containing 50 mg / L rifampicin and 50 mg / L kanamycin) and cultured to OD600 = 0.6-0.8. The bacterial liquid was centrifuged at 5000 rpm for 15 min and then suspended in the same volume of liquid 1 / 2 MS (0.02% Silwet L-77) to obtain the inoculum solution for later use.

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

[0079] (3) Agrobacterium-mediated genetic transformation: Select healthy plants in the early fruiting stage and place them upside down with their pots on top of a container containing Agrobacterium-infected solution. Immerse the entire inflorescence in the Agrobacterium suspension for about 20-30 seconds, taking care to minimize contact of the leaves with the solution. Remove the pots and place them horizontally in a dark box for about 24 hours. Maintain 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 of MS (hygromycin 50 mg / L) culture 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 positive Arabidopsis plants transfected with the IbbHLH149 gene. The steps are as follows:

[0082] Genomic DNA from transgenic plants was extracted, and primers designed using the aforementioned vector sequences were used for PCR amplification. The PCR reaction procedure was: 95°C for 5 minutes; 95°C for 50 seconds, 55°C for 50 seconds, 72°C for 1 minute, 32 cycles; and 72°C for 10 minutes. Plants expressing the correct amplified band were positive for the 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 wild-type Arabidopsis thaliana of Columbia were inoculated into normal 1 / 2 MS medium containing 130 mM NaCl or 300 mM mannitol, respectively. The culture was carried out at 22°C for about one 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 in the data, the germination rate and cotyledon expansion of transgenic IbbHLH149 Arabidopsis plants were better than those of Columbia wild-type Arabidopsis under high salt or drought stress conditions. Therefore, the salt resistance concentration of transgenic IbbHLH149 Arabidopsis seeds is 130 mM NaCl or above, and the drought resistance concentration is 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 the IbbHLH149 gene obtained in Example 5 and wild-type Arabidopsis thaliana of Columbia were inoculated in 1 / 2 MS medium for germination. One week later, the seedlings were transferred to normal 1 / 2 MS medium containing 125 mM NaCl or 400 mM mannitol. The seedlings were cultured at 22°C for about 10 days. The growth and rooting under high salt or mannitol stress conditions were observed, and 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 transgenic IbbHLH149 Arabidopsis seedlings were better than those of Columbia wild-type Arabidopsis seedlings ( Figure 4 ), so the salt tolerance of Arabidopsis seedlings transgenic for IbbHLH149 gene is 125 mM or above, and the drought tolerance is 400 mM mannitol or above.

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

[0093] The transgenic Arabidopsis thaliana of the IbbHLH149 gene obtained in Example 5 and the wild-type Arabidopsis thaliana of Columbia were inoculated in 1 / 2 MS 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 every 3 days, and the growth of the Arabidopsis thaliana was observed after 2 weeks; the natural drought-treated plants were not watered. After 2 weeks of natural drought, they were rehydrated for 3 days and the growth of the Arabidopsis thaliana was observed. The results are shown in FIG. Figure 5 As shown in the results, the growth of transgenic Arabidopsis thaliana seedlings with the IbbHLH149 gene was better than that of Columbia wild-type Arabidopsis seedlings under high salt or natural drought stress conditions.

[0094] Example 7

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

[0096] 1. Analysis of stress-resistant gene expression in transgenic Arabidopsis thaliana containing the 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 IbbHLH149 gene and Columbia wild-type Arabidopsis thaliana plants that were irrigated with normal or saline solution (300 mM NaCl solution) for 2 weeks or naturally drought-stricken for 2 weeks was determined.

[0098] Potted plants of IbbHLH149 transgenic Arabidopsis thaliana and Columbia wild-type Arabidopsis thaliana were sampled under normal, high-salt, or natural drought stress. Total RNA was extracted, treated with DNase I, purified, and reverse transcribed into cDNA, which was used as a template. Fluorescent quantitative primers were designed based on the specific sequences of Arabidopsis stress resistance genes (AtCAT, AtSOD, AtAOS, AtLOX2, AtZEP, and AtP5CR genes). The samples were reacted on a QuantStudio 3 fluorescent quantitative PCR instrument using the same reaction system 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, under normal growth conditions, there was no significant difference in the expression levels of stress-resistance genes between transgenic and wild-type lines. However, under high salt or drought stress, the expression levels of AtCAT and AtSOD, genes involved in the reactive oxygen species scavenging system; AtAOS and AtLOX2, key genes in the JA signaling pathway; AtZEP, a key gene in the ABA signaling pathway; and AtP5CR, a gene involved in the proline biosynthesis pathway, were significantly higher in transgenic Arabidopsis than in wild-type plants. This suggests that IbbHLH149 activates the expression of stress-resistance genes under high salt or drought stress.

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

[0122] (1) IbbHLH149 transgenic Arabidopsis thaliana and Columbia wild-type Arabidopsis thaliana plant samples were taken under normal, high salt and drought stress conditions, 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 thaliana plants under different conditions were determined (e.g. Figure 7 (As shown). Normally grown IbbHLH149 transgenic lines showed no significant differences in related parameters compared to wild-type lines. However, under high-salt or drought stress, the MDA content of the IbbHLH149 transgenic lines was significantly lower than that of the wild-type lines, while the Pro content and SOD and POD enzyme activities were significantly higher than those of the wild-type plants. These results suggest that overexpression of IbbHLH149 can activate the reactive oxygen species scavenging system in transgenic plants under high-salt or drought stress, enhance their osmotic regulation ability, and reduce damage to the membrane system caused by high-salt stress.

[0123] (2) Leaves of Arabidopsis thaliana with IbbHLH149 gene and wild-type Arabidopsis thaliana under normal, high salt and drought stress were taken and stained with NBT and DAB to analyze the leaf O 2 -2 and H2O accumulation (e.g. Figure 8As shown). 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. Overexpression of the IbbHLH149 gene reduced the excessive accumulation of reactive oxygen species under high salt or drought stress, alleviating the damage of high salt stress to the physiological activities of Arabidopsis thaliana.

[0124] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. Sweet potato IbbHLH149 The application of a gene in improving plant stress resistance is characterized in that: The sweet potato IbbHLH149 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2; the stress resistance is drought resistance and high salt resistance, and the plant is Arabidopsis thaliana.

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

1.

3. sweet potato according to claim 1 IbbHLH149 The application of a gene in improving plant stress resistance is characterized in that: By increasing the IbbHLH149 Increase the expression of genes and improve the stress resistance of plants.

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

5. A method for improving plant stress resistance, characterized in that: By increasing the IbbHLH149 The expression level of the gene improves the stress resistance of the plant. IbbHLH149 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2; the stress resistance is drought resistance and high salt resistance, and the plant is Arabidopsis thaliana.

6. The method for improving plant stress resistance according to claim 5, characterized in that: Will IbbHLH149 The gene sequence is constructed into a plant expression vector and transformed into a plant body to overexpress it in the plant.

7. The method for improving plant stress resistance according to claim 6, wherein: 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.

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