The bntlp1 gene regulating the resistance to Sclerotinia sclerotiorum in Brassica napus and its application

A technology for cabbage rape and sclerotinia, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the loss of rape yield and quality, the lack of understanding of genes and gene regulatory networks, and the lack of comprehensive and in-depth biological functions. Earth analysis and other issues to achieve the effect of improving resistance

Active Publication Date: 2022-07-29
OIL CROPS RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The fungal disease caused by Sclerotinia sclerotiorum is an important disease in the production of rapeseed in my country. At present, the loss of yield and quality of rapeseed caused by Sclerotinia sclerotiorum is huge, which makes it particularly important to breed disease-resistant varieties. Genes and gene regulatory networks for disease defense are still unclear
TLP, as a kind of defense protein family, has a considerable number, and its biological function in the defense of Sclerotinia sclerotiorum has not been fully and deeply analyzed

Method used

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  • The bntlp1 gene regulating the resistance to Sclerotinia sclerotiorum in Brassica napus and its application
  • The bntlp1 gene regulating the resistance to Sclerotinia sclerotiorum in Brassica napus and its application
  • The bntlp1 gene regulating the resistance to Sclerotinia sclerotiorum in Brassica napus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: TA cloning and sequencing of BnTLP1 gene

[0039] Take the young leaves of Brassica napus "Zhongshuang 11", extract total RNA with TriZol Reagent (Invitrogen, Item No. 15596026), use Nanodrop to detect the content and purity of total RNA, take 2.0 μg of total RNA for reverse transcription reaction, the adopted The reverse transcriptase is M-MLV (Promega company, product number M1701), and the steps of reverse transcription reaction refer to the instructions for use of the reverse transcriptase. Using the first-strand cDNA synthesized by reverse transcription reaction as a template, use primers:

[0040] 5'-ATGATTTATCAAAAAACACTTCTC-3';

[0041] 5'-TCACGGACAGAAAATGACAT-3';

[0042] Perform conventional PCR amplification; PCR reaction system is (20 μL): 1 μL cDNA, 2 μL 10×Buffer, 1.6 μL dNTP (2.5 mM), 1 μL each of forward / reverse primers (10 μM), 0.4 μL Taq enzyme (5 U / μL) and 13μL ddH 2 O (set up an 8-tube system to amplify the target gene in large quantiti...

Embodiment 2

[0043] Example 2: Construction of BnTLP1 gene overexpression vector

[0044] Using the BnTLP1 gene TA cloning plasmid of Example 1 as a template, the BnTLP1 gene was cloned with primers BnTLP1-F / R. The primers are as follows:

[0045] BnTLP1-F:

[0046] 5'-CCATCGATAGTACTGTCGACATGATTTATCAAAAAACACTTCTC-3';

[0047] BnTLP1-R:

[0048] 5'-TCCATCCCGGGAGCGGTACCCGGACAGAAAATGACAT-3';

[0049] The BnTLP1 gene was connected to the laboratory modified vector PGTV II-3FLAG by homologous recombination. The ligation system (20 μL): 12 μL of PGTV II-3FLAG linear vector, 1 μL of BnTLP1 purified product, 2 μL of enzyme (Exnase II), and 5 μL of 5×CE II Buffer . In this example, the PGTV II-3FLAG vector is used, and other vectors can also be used to construct the recombinant expression vector of BnTLP1. PCR to verify the size of the target gene fragment in the recombinant vector, such as figure 1 As shown, the lane "M" in the figure is the DL2000 DNA marker, and the lane "1" is the BnTLP1...

Embodiment 3

[0050] Example 3: Genetic transformation of the BnTLP1 gene

[0051]By alkaline lysis method, the DH5α recombinant strain plasmid of BnTLP1 gene was extracted and named 35S::BnTLP1. Transfect Agrobacterium GV3101 competent cells: add 10 μL of recombinant plasmids after freezing and thawing of competent cells, mix well, ice bath for 5 minutes; freeze in liquid nitrogen for 5 minutes; water bath in a constant temperature water bath at 37°C for 5 minutes; add fresh liquid LB medium without antibiotics , After mixing, activate for 2h at 28°C, 220rpm / min. Spread on LB plates containing kanamycin and rifampicin, cultivate in the dark for 2 days at 28°C, and use the BnTLP1-F / R primer in Example 2 to detect positive monoclonal clones. For the genetic transformation of wild-type Arabidopsis Col-0 by expansion culture in liquid LB medium with resistance to fulminant.

[0052] Agrobacterium-mediated genetic transformation method of wild-type Arabidopsis thaliana Col-0: amplify the cult...

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Abstract

The invention discloses a BnTLP1 gene for regulating the resistance of Brassica napus sclerotiorum and its application. The present invention isolates and clones a gene BnTLP1 for regulating cabbage sclerotinia sclerotinia from the high resistance variety "Zhongshuang 11" of rapeseed sclerotinia, and its nucleotide sequence is shown in SEQ ID NO: 1. The gene The encoded protein sequence is shown in SEQ ID NO:2. The present invention carries out functional verification on the BnTLP1 gene. Through genetic engineering technology, it is verified that the defense-related gene BnTLP1 of Brassica napus of the present invention positively regulates Sclerotinia sclerotiorum, which provides a new gene source for the mining of functional genes of Brassica napus and molecular breeding, and is useful for enhancing Brassica napus resistance to Sclerotinia sclerotiorum. Resistance has important application prospects.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to the BnTLP1 gene for regulating the resistance of Brassica napus sclerotiorum and its application. Background technique [0002] Rapeseed is the oil crop with the largest planting area in my country, providing nearly 50% of the domestic edible oil in China. Sclerotinia sclerotiorum is a fungal disease caused by Sclerotiniasclerotiorum (Lib.) de Bary, a member of the genus Sclerotiorum (Lib.) The first major disease of production (Boland G J, Hall R. Index of plant hosts of Sclerotinia sclerotiorum, 1994, Canadian Journal of Plant Pathology, 16(2):93-108). How to effectively prevent and control the occurrence of sclerotinia is an urgent problem to be solved in the current rape industry. At present, the control measures for rape sclerotinia are mainly chemical control, but chemical control is not only costly, but also easily causes secondary pollution. Physical co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00A01H6/20
CPCC07K14/415C12N15/8282
Inventor 刘胜毅石美娟白泽涛程晓辉何贻洲张园园
Owner OIL CROPS RES INST CHINESE ACAD OF AGRI SCI
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