Potato transcription factor gene bHLH47 and application thereof

Abstract

The invention discloses a potato transcription factor gene bHLH47 and application thereof, and discloses that the potato bHLH47 gene participates in regulating and controlling the drought stress response of potatoes for the first time, and the drought resistance of the potatoes can be improved by reducing the expression quantity of the potato bHLH47 gene; the gene can be used for cultivating new transgenic drought-resistant varieties of potatoes, and has potential application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biological breeding, and in particular relates to a potato transcription factor gene bHLH47 and an application thereof. Background Art

[0002] Potato (Solanum tuberosum L.) is an annual herbaceous plant of the Solanaceae family. Its tubers are edible and it is the fourth largest food crop in the world after wheat, corn and rice. Due to its shallow root system, potato growth is very susceptible to drought stress, which significantly affects potato growth and development and is an important limiting factor in potato production. Plants respond to drought stress through osmotic regulation, antioxidant defense systems, hormone signals and morphological changes.

[0003] Integrating drought-resistant and salt-tolerant genes into the target plant genome through genetic engineering or purposefully inhibiting or inducing the expression of some drought-related genes to obtain transgenic plants has opened up a new way to cultivate new varieties of highly drought-resistant crops. With the understanding of plant drought resistance mechanisms and the increasing maturity of transgenic technology, using transgenic technology to obtain drought-resistant breeding materials or genetically improved new varieties has become the most promising direction for the cultivation of drought-resistant varieties.

[0004] Among them, transcription factors not only regulate various life processes of plants such as metabolism, development, and biosynthesis, but also respond to different adverse stresses. They are also considered to be the best potential direction for exploring stress-resistant plants so far. The bHLH transcription factor family has a large number of genes and a wide range of functions. In addition to participating in multiple processes of growth and development, bHLH transcription factors in plants also play an important role in plant response to adverse stresses. In the process of plant response to abiotic stress, bHLH transcription factors, as regulatory genes, can regulate the expression changes of related stress genes, thereby playing an important role in adverse stress. At present, bHLH genes have not been used to improve potato resistance to drought stress. Summary of the invention

[0005] Based on the above background, the present invention applies the gene bHLH47 and the protein encoded by it to improve the ability of potatoes to cope with drought stress.

[0006] The technical solution of the present invention is:

[0007] One of the purposes of the present invention is to provide: a potato transcription factor gene bHLH47, characterized in that its nucleotide sequence is shown in SEQ ID NO.1.

[0008] The second purpose of the present invention is to use the potato transcription factor gene bHLH47 in improving the ability of potatoes to cope with drought stress.

[0009] The third object of the present invention is to provide a protein encoded by potato transcription factor gene bHLH47, whose amino acid sequence is shown in SEQ ID NO.4.

[0010] The fourth purpose of the present invention is to use the protein in improving the ability of potatoes to cope with drought stress.

[0011] The fifth object of the present invention is to provide a method for improving the ability of potatoes to cope with drought stress, thereby reducing the expression level of the transcription factor gene bHLH47 or the protein encoded by it in potatoes.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1) The present invention introduces a recombinant vector containing the gene bHLH47 into potatoes to construct potato plants that overexpress the gene bHLH47. By comparing the potato plants with the wild-type strains, the potato plants exhibit a trait that is sensitive to drought stress, indicating that the bHLH47 gene plays an important role in potato adaptation to drought stress. The bHLH47 gene can be used to improve potato resistance to drought stress and cultivate transgenic potatoes with high drought resistance.

[0014] The bHLH47 gene and the protein encoded by the present invention can provide valuable resources for breeding new drought-resistant potato varieties, and at the same time lay a theoretical foundation for studying the mechanism of potato response to drought stress and the molecular mechanism of resisting adverse environment.

[0015] 2) The present invention reveals for the first time that the potato bHLH47 gene is involved in regulating the drought stress response of potatoes. By reducing its expression level, the drought resistance of potatoes can be improved; and the gene can be used to breed new transgenic drought-resistant potato varieties, which has potential application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The morphological indexes, physiological indexes and qRT-PCR analysis of the overexpression plants and wild-type plants using the potted water control stress method of the embodiment of the present invention; wherein a is the growth of WT and OE plants under normal conditions; b is the growth of WT and OE plants after 14 days of water control; c is the relative expression of StbHLH47 gene in WT and OE-28 plants under potted drought conditions; df is the comparison of plant height, leaf area and lateral branch length of WT and OE plants under normal conditions; hi is the content of StbHLH47 gene, chlorophyll content, relative conductivity and MDA of WT and OE-28 plants under potted drought conditions;

[0017] Note: From left to right in Figures a and b are WT, OE-5, OE-6, and OE-28. DETAILED DESCRIPTION

[0018] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0019] Embodiment 1:

[0020] In this example, positive transgenic plants were constructed, and the specific operations were as follows:

[0021] The RNA of potato was extracted and reverse transcribed into cDNA. The CDS sequence of the bHLH47 gene was amplified using the cDNA as a template and F and R as primers. The amplified product was constructed into a plant overexpression vector. The obtained recombinant expression vector was then used to transform Agrobacterium. The transformed Agrobacterium was then used to infect potato callus, and positive transgenic plants were screened to obtain drought-sensitive transgenic potatoes.

[0022] The overexpression vector uses pBWA(V)KS as the starting vector, and the Agrobacterium is GV3101.

[0023] The primers for amplifying the gene bHLH47 are as follows:

[0024] Upstream primer F:

[0025] 5'-ATTTGGAGAGAACACGGGGGACTTTGCAACATGGATACAGAAAATCCTGCTCCA

[0026] A-3' (SEQ ID NO. 2);

[0027] Downstream primer R:

[0028] 5'-GGCCCAGTACTGAAGACAGAGCTAGTTACATTATGCACCATTCTGAACCTCCTGT-3' (SEQ ID NO. 3).

[0029] The nucleotide sequence of the transcription factor gene bHLH47 is shown in SEQ ID NO.1:

[0030] ATGGATACAGAAAATCCTGCTCCAATAGTTGAAAAGGATGCCACAGATGCCGAGACATCATTAGATAGCTCTCATCTCGGGAAGAAGATTCAGAAGAAAGTTCCAAAAAGAATTCACAAAGCTGAGAGAGAGAAAATGAAGAGAGAGCATCTGAATGAGCTTTTTCTTGGTTTGGCTGATGCTCTTGAACTATCTGAGCAGATGAATGGAAAAGCCTCTGTATTGAGTGAAGCTGCTCGATTTGTAAAGGACATGCTTTCTCAGATCAAGCATATGAGGACAGAAAATACAACTTTGCTGTCTGAATCTCAATATCTTAGCGTGGAGAAAAAGGAGCTTGAGGATGAAAATACAGCTCTAGAGGCTGAAATTAGCAAACTGCAGACTGAGGTCAAAGCAAGGGAAGCTGAGACTAGTCTTGACCTAAATCTAGCTCCTCCTGAAATTCATCACACAGAGTTTGCCTCACAAACTAACTATATGAGATTGCCTGCTTCGGAGCACGCATTTCAGCAGTCACAGATGATGAACCCTGTGTACGTCTTTCCCTTGAGTTCTAATCCCCAGGCTTATCCAGCGCCTGATGCTGCAGATTCCATGGCTATGCCCACATCTACTGTGAAGAAACCACAGCCCAGATACCCCACTCCAAATGATGTATGGCCATCCCAAATCCTTGAAAAGAGGCCTCAGTTGTTGCGACAGGAGGTTCAAGATGGTGCATAA。

[0031] The amino acid sequence of the protein encoded by the transcription factor gene bHLH47 is shown in SEQ ID NO.4:

[0032] MDTENPAPIVEKDATDAETSLDSSHLGKKIQKKVPKRIHKAEREKMKREHLNELFLGLADALELSEQMNGKASVLSEAARFVKDMLSQIKHMRTENTTLLSESQYLSVEKKELEDENTALE AEISKLQTEVKAREAETSLDLNLAPPEIHTEFASQTNYMRLPASEHAFQQSQMMNPVYVFPLSSNPQAYPAPDAADSMAMPTSTVKKPQPRYPTPNDVWPSQILEKRPQLLRQEVQDGA.

[0033] The overexpression vector used in the following examples is the pBWA(V)KS-ccDB plasmid, and the bacterial resistance is kanamycin (Kana+);

[0034] E. coli DH5α

[0035] The competent cells (DL1001) and the competent cells of Agrobacterium tumefaciens GV3101 (AC1001) are both resistant to gentamicin (Gent).

[0036] The main reagents used in the following examples are: TRNzol Reagent; FastQuant RT Kit; D2000 DNA Marker; 2× Taq PCR Master Mix; GeneGreen Nucleic Acid Dye; TIANgel Midi Purification Kit; TIANprep Mini Plasmid Kit; 2× miRcute Plus miRNA Premix (with SYBR Green); 6× DNA Loading Buffer; LD 1kb Ladder; 1kb DNA LadderⅠ; 2× PfuPCR Master Mix; NovoRec○R PCR Seamless Cloning Kit; KpnⅠ; XhoⅠ; XbaⅠ; Agarose; TransDirect○R Plant Tissue PCR Kit; 6-BA; IAA; Timentin; Kana+; Gent; Str; Spe; DMSO; As; MES; Chloroform; Glycerol. All other chemical reagents used in the examples are imported or domestic analytical reagents.

[0037] The primers used in the examples were synthesized by Shanghai Bioengineering Co., Ltd., and related sequencing was performed.

[0038] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are based on conventional experimental conditions or the conditions recommended by the manufacturer's instructions.

[0039] 1) Construction and detection of bHLH47 gene overexpression vector

[0040] To understand the molecular mechanism of potato response to drought, the bHLH47 gene was cloned from the potato transcriptome. Based on the sequence analysis of the coding region, primers F and R were designed to amplify the coding region of the gene and ligated to the expression vector pBWA(V)KS with a 35S promoter. The upstream primer F used was (SEQ ID NO.3); the downstream primer R was (SEQ ID NO.4).

[0041] The specific method of connecting the bHLH47 gene to the vector pBWA(V)KS with a 35S promoter is as follows: extract total potato RNA, reverse transcribe it into cDNA, then use the cDNA as a template, use upstream and downstream primers to amplify bHLH47, connect the PCR product to the vector pBWA(V)KS, and name the connection product pBWA(V)KS-bHLH47. Sequencing verifies the correctness. (Reaction connection system):

[0042]

[0043] 2) Construction and detection of potato overexpressing bHLH47 gene

[0044] The pBWA(V)KS vector containing bHLH47 constructed in 1) was transformed into Agrobacterium GV3101, and the potato callus tissue was infected to obtain transgenic seedlings. The specific method is as follows: the Agrobacterium containing the target vector was inoculated into a 50mL shaking tube containing LB liquid culture medium (50mg / mL Kan+50mg / mL Rif) containing antibiotics, and cultured overnight at 28°C and 240rpm until the absorbance of the bacterial solution at 600nm was above 0.5; the bacterial cells were collected by centrifugation at 5000rpm for 6min, and then 30mL1 / 2MS liquid culture medium was added to resuspend the bacterial cells; the pre-cultured potato explants were placed in the Agrobacterium suspension and infected for 10-15min; the infected explants were placed on filter paper to dry, placed on co-culture medium, and cultured at 23°C for 2 days under dark conditions; the explants after co-culture were placed in the screening culture medium for rooting screening. The obtained positive plants were subjected to drought stress treatment.

[0045] The overexpression strains isolated and screened were used to detect the expression of genes by PCR amplification.

[0046] ① Extract DNA from transgenic strains;

[0047] ② The primers used for detection were F: gaatcgggagcggcgatacc (SEQ ID NO.5); R: ccaccaagcgaaacatcgcat (SEQ ID NO.6);

[0048] ③The reaction system used includes: DNA template, 1 mL of upstream and downstream primers, 10 mL of 2*Mix, and 8 mL of ddH2O; the reaction conditions are:

[0049] Pre-denaturation at 95°C for 10 min, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 5 min, extension at 72°C for another 10 min, cooling at 14°C for 10 min. The denaturation-extension process requires 30 cycles.

[0050] 3) Drought resistance testing of potato lines overexpressing the bHLH47 gene

[0051] The WT and OE plants were potted and covered with soil until they reached a height of 30 cm. Watering was stopped and drought stress treatment was started. On the 14th day after watering was stopped, Figure 1 a~b. By measuring the plant height, leaf area and lateral branch length of overexpression plants and wild-type plants, we found that under normal plant growth conditions, there were significant differences in the growth between OE plants and WT plants. The plant height of OE plants was significantly higher than that of WT, and they had larger leaf areas and longer lateral branches (such as Figure 1 ). After 14 days without watering, more leaf wilting symptoms were observed in OE-bHLH47 in response to drought stress compared with WT. Figure 1 b. RT-qPCR was used to detect the expression level of StbHLH47 in OE-bHLH47. Total RNA was extracted from the control and drought-treated WT and OE plants and reversed into cDNA. The cDNA obtained by reverse transcription was diluted 10 times and then subjected to real-time quantitative PCR using Monad's kit. The reaction system used included: MonAmp TM Green qPCRMix, 10mL; 0.4mL each of upstream and downstream primers; 1.5mL of DNA template, add ddH2O to 20mL, mix thoroughly and put into real-time fluorescence quantitative PCR instrument for three-step PCR amplification, the reaction conditions are: 95℃30s, 95℃10s, 60℃10s, 72℃30s, 40 cycles. After the PCR reaction is completed, according to 2 -Δ(ΔCt)The relative expression between the wild type and overexpression was calculated and analyzed graphically according to the principle of PCR. Each sample was amplified simultaneously with U6 as the internal reference gene. The results showed that the expression of StbHLH47 showed a continuous upward trend and was much higher than that of the WT plant. At the same time, an appropriate amount of WT and OE plant leaves were weighed, cut into small pieces, placed in a ddH2O centrifuge tube, soaked in distilled water at room temperature for 2h, and the conductivity of R1 was measured using a DDS-307A conductivity meter. Then, the leaves were mixed and heated in a 95℃ boiling water bath for 25-30min, cooled to room temperature, and the final conductivity of R2 was determined. Relative conductivity (%) = (R1 / R2) × 100 (%). The malondialdehyde (MDA) content was determined according to the MDA kit of Nanjing Jiancheng Bioengineering Institute, and the chlorophyll content was determined using a SPAD instrument. It was found that the MDA content in OE was significantly higher than that in WT as the drought time increased, indicating that drought stress aggravated the damage to transgenic plants. and contained higher electrical conductivity and lower chlorophyll content, which proved that the tolerance of transgenic plants to drought stress was greatly affected, e.g. Figure 1 . According to the results of chlorophyll determination, the chlorophyll content of OE plants was significantly lower than that of WT plants, indicating that overexpression of StbHLH47 had a negative regulatory effect on chlorophyll accumulation. These results indicate that overexpression of StbHLH47 reduces drought tolerance in potatoes. In summary, these results indicate that StbHLH47 acts as a negative regulator of drought tolerance.

[0052] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Potato transcription factor gene bHLH47, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

1.

2. Use of the potato transcription factor gene bHLH47 according to claim 1 in improving the ability of potatoes to cope with drought stress.

3. A protein encoded by potato transcription factor gene bHLH47, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

4.

4. Use of the protein according to claim 3 in improving the ability of potatoes to cope with drought stress.

5. A method for improving the drought stress resistance of potatoes, characterized in that: Reducing the expression level of the transcription factor gene bHLH47 or the protein encoded by it according to claim 1 in potatoes.

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