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Brassica napus bntlk1 gene related to fungal diseases and its application

A technology for cabbage rape and fungal diseases, which is applied in the directions of application, genetic engineering, plant genetic improvement, etc., can solve the problems of unclear biological functions, loss of rape yield and quality, etc., and achieve the effect of improving sclerotinia resistance.

Active Publication Date: 2022-02-11
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

[0004] Although the TLP-K fusion gene has been reported in rice, sorghum, wheat, and Arabidopsis, its biological function is not clear. The functional analysis of the TLP-K fusion gene BnTLK1 in rapeseed will help The huge loss of rapeseed yield and quality caused by sclerotinia in production can provide more alternatives for the directional molecular design and genetic improvement of rapeseed and other crops in the future to create new germplasm

Method used

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  • Brassica napus bntlk1 gene related to fungal diseases and its application
  • Brassica napus bntlk1 gene related to fungal diseases and its application
  • Brassica napus bntlk1 gene related to fungal diseases and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: BnTLK1, BnTLP TLK1 Gene cloning and sequencing

[0036] Take the young leaves of Shuang11 in rapeseed, use TriZol Reagent (life technologies company) to extract total RNA of rapeseed, use NANODROP software to detect the content and purity of total RNA, take 2.0 μg of total RNA for reverse transcription, the reverse transcription method used The kit is a product of Takara Company, and the reverse transcription reaction steps refer to the instructions of the kit. Using the cDNA synthesized by reverse transcription reaction as a template, use primers:

[0037] BnTLK1: 5'-ATGTTCGCAGGAGTGTTGTC-3'

[0038] 5'-TAGTGGCTTGGTGTCAAAA-3'

[0039] BnTLP TLK1 : 5'-ATGTTCGCAGGAGTGTTGTC-3'

[0040] 5'-TAATTGGGAATGGACGAGG-3'

[0041] Carry out conventional PCR amplification; PCR reaction system (50 μL) is: 2 μL cDNA, forward / reverse primer (concentration 10 μM) 2 μL each, 2×Phanta Max Master Mix is ​​25 μL and 19 μL ddH 2 O. Mix well after adding the sample on ice. T...

Embodiment 2

[0042] Example 2: Cloning and sequence determination of AtTLK1 specific sequence

[0043] Take the young leaves of wild-type Arabidopsis thaliana, use TriZol Reagent (life technologies company) to extract total RNA, use NANODROP software to detect the content and purity of total RNA, take 2.0 μg of total RNA for reverse transcription, the reverse transcription method used The kit is a product of Takara Company, and the reverse transcription reaction steps refer to the instructions of the kit. Using the cDNA synthesized by reverse transcription reaction as a template, use primers:

[0044] 5'-ATCTCGGCTAGGACGCTATG-3'

[0045] 5'-TTCAACAACGCAACCAGCGC-3'

[0046] Carry out conventional PCR amplification; PCR reaction system (50 μL) is: 2 μL cDNA, forward / reverse primer (concentration 10 μM) 2 μL each, 2×Phanta Max Master Mix is ​​25 μL and 19 μL ddH 2 O. Mix well after adding the sample on ice. The PCR reaction conditions were: 95°C for 3min; 95°C for 15sec, 53°C for 15sec, 7...

Embodiment 3

[0047] Example 3: Prokaryotic expression and purification of BnTLK1 protein

[0048] The expression vector used in the present invention is pGEX-4T-1, with GST tag, and the schematic diagram of GST-BnTLK1 prokaryotic expression vector is as follows figure 1 As shown, the BnTLK1 gene (SEQ ID NO.1) was obtained by PCR reaction cloning, using primers:

[0049] 5'-ATCTGGTTCCGCGTGGATCCATGTTCGCAGGAGTGTTGTC-3'

[0050] 5'-GCTCGAGTCGACCCGGGAATTCCTAGTGGCTTGGTGTCAAAA-3'

[0051] Perform PCR amplification, and the amplification template is the pEASY-BnTLK1 recombinant plasmid in Example 1. PCR reaction system (50 μL): 2 μL cDNA, forward / reverse primer (concentration 10 μM) 2 μL each, 2×Phanta Max Master Mix 25 μL and 19 μL ddH 2 O. Mix well after adding the sample on ice. The PCR reaction conditions are: 95°C for 3min; 95°C for 15sec, 53°C for 15sec, 72°C for 90sec, 32 cycles; 72°C for 10min. After the target fragment was recovered by agarose gel electrophoresis, the pGEX-4T-1 vect...

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Abstract

The present invention clones a gene BnTLK1 naturally fused with a disease course-related protein (Thaumatin-like protein, TLP) and a receptor-like kinase (Receptor-like kinase) in Brassica napus. Experiments have confirmed that BnTLK1 protein can inhibit the growth of Sclerotinia sclerotiorum and Botrytis cinerea hyphae, and positively regulate fungal resistance. The invention lays a foundation for exploring the evolution and function research of disease-resistant genes in the interaction between pathogenic bacteria and plants, helps to solve the huge loss of rapeseed yield and quality caused by Sclerotinia sclerotiorum in production, and can provide orientation for rapeseed and other crops in the future. Molecular design and genetic improvement to create new germplasm offer more options.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to the BnTLK1 gene of Brassica napus related to fungal diseases and its application. Background technique [0002] A new gene is a gene that appears at a site that did not exist before at a certain time during the evolution of a species, and is a segment of DNA with a new function. There are multiple origin mechanisms for new genes, including gene duplication, gene fusion, gene division, de novo synthesis origin, and gene horizontal transfer. In 1978, Gilbert proposed a model for the formation of new genes, that is, new genes can be formed by fusion of unrelated genes. A fusion gene refers to the evolution of the coding regions previously present in different genes joined together to form a single coding gene. Gene fusion events account for about 0.5% of all prokaryotic genes, the vast majority of gene fusions occur in bacteria and fungi, and the study of fusion ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N9/12C07K14/415C12N15/29C12N15/84A01H5/00A01H6/20
CPCC07K14/415C12N9/12C12N15/8282C07K2319/00
Inventor 刘胜毅钟雪白泽涛程晓辉左蓉张园园
Owner OIL CROPS RES INST CHINESE ACAD OF AGRI SCI
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