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Myrothamnus flabellifolia gene MfbHLH15 and application thereof

A kind of milo wood and genetic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as lack of research

Active Publication Date: 2020-05-15
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, the research on the bHLH transcription factors of Miromea milofolia under abiotic stress is very scarce, so it is very important to carry out gene cloning and functional verification of such transcription factors

Method used

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  • Myrothamnus flabellifolia gene MfbHLH15 and application thereof
  • Myrothamnus flabellifolia gene MfbHLH15 and application thereof
  • Myrothamnus flabellifolia gene MfbHLH15 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Cloning of the Mirowood MfbHLH15 gene

[0035] 1. Take the leaves of A. milo, quick-frozen in liquid nitrogen, and store them in a -80°C refrigerator for extraction of total RNA; the total RNA was extracted using the Plant Total RNA Isolation Kit purchased by Chengdu Lambo Biological Company; the cDNA of A. milo The synthesis of the first chain was performed using Reverse Transcriptase M-MLV (RNaseH-) from Dalian Bao Biotechnology Co., Ltd., according to its product manual.

[0036] The first strand of cDNA synthesized by the above kit was used as the amplification template, and the designed F: 5'-TCCCCCGGGATGAATCACTGCGTTCCCGA-3' (SEQ ID NO.3) and R: 5'-GACTAGTTTAAAATTTCTCATACCTGTG-3' (SEQ ID NO.4 ) as primers, for subsequent enzyme digestion and recombination connection, when designing primers, add these two enzyme digestion sites, SmaI and SpeI, and use RT-PCR to amplify cDNA. The amplification system is shown in Table 1, and the amplification conditions are...

Embodiment 2

[0040] Example 2 Subcellular localization of MfbHLH15

[0041] According to the bases and restriction sites on the subcellular carrier pHB-YFP, as well as the instructions of the ClonExpressII Homologous Recombination Kit, two restriction sites, HindIII and BamHI, were selected to design primers; the primers were provided by Chengdu Qingke Biological Co., Ltd. synthesis.

[0042] The primers with homology arms that have been synthesized are used to amplify with Miroma cDNA as a template, and the specific sequences of the primers are as follows:

[0043] F: 5'-ACCAGTCTCTCTCTCAAAGCTTATGAATCACTGCGTTCCCGA-3';

[0044] (SEQ ID NO.5)

[0045] R: 5'-GCTCACCATACTAGTGGATCCAAATTTCTCATACCTGTG-3';

[0046] (SEQ ID NO.6)

[0047] After the amplified PCR product was recovered, it was connected to the pHB vector excised by HindIII and BamHI endonucleases to construct the fusion vector 35S::MfbHLH15-pHB-YFP, by transforming the wild-type Ben's into the pHB-YFP plasmid Nicotiana benthamia...

Embodiment 3

[0048] Example 3: Genetic transformation of the MfbHLH15 gene

[0049] Thaw Agrobacterium LBA4404 competent cells on ice, take 5 μL of recombinant plasmid 35S::pGSA1403-MfbHLH15 in 100 μL of competent cells, mix gently with a pipette tip, ice bath for 30 minutes, liquid nitrogen quick-freeze for 1 minute, and 37 ° C water bath for 5 minutes , put on ice for 2 minutes, add 800 μL LB liquid medium, and incubate in a constant temperature incubator at 29° C. and 120 r / min for 2 to 4 hours. Spread the above bacterial solution evenly in LB solid medium containing 10 μg / mL chloramphenicol, culture in a constant temperature incubator at 28°C, then pick a single plaque and inoculate it in 5mL LB liquid medium, at 28°C, 250r / min shaking culture for 24h. Take 1mL of the above bacterial solution and transfer it into 50mL LB liquid medium containing 10μg / mL chloramphenicol, and culture it with shaking at 28°C and 250r / min until OD 600 ≈1.5; centrifuge the above-mentioned bacterial soluti...

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Abstract

The invention discloses a myrothamnus flabellifolia gene MfbHLH15 and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO.1; and the amino acid sequence of a protein encodedby the gene is shown as SEQ ID NO.2. Through the research on drought-resistant bHLH transcription factors of myrothamnus flabellifolia and improvement on the drought-resistant and salt-tolerant performance of plants, the drought-resistant and salt-tolerant performance of the plants is improved.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a milowood gene MfbHLH15 and its application. Background technique [0002] Drought stress and salt stress are key environmental factors affecting plant growth and development, geographical distribution and crop productivity. For a long time, improving the drought tolerance and salt tolerance of plants has been a research topic that has attracted wide attention. As a supplement to traditional breeding, genetic engineering has been proven to be a powerful way to breed new germplasm with strong drought and salt tolerance. Therefore, it is particularly important to study genes with drought tolerance and salt tolerance. These high-quality genes can be genetically engineered to help plants cope with the increasingly severe natural growth environment. [0003] Basic helix-loop-helix (bHLH) transcription factors (TFs) are a large gene family in plant geno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/70C12N1/21C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/70C12N15/8273
Inventor 黄卓邱嘉睿蒋才忠向香盈徐文欣王嘉彤朱培蕾杨丽蔡仕珍
Owner SICHUAN AGRI UNIV
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