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Cloning and function of Chinese cabbage LRR disease-resistant protein gene BcLRR

A technology of disease-resistant protein and Chinese cabbage, which is applied in the field of plant disease-resistant genetic engineering, can solve the problems of lack and difficulty in resisting soft rot

Inactive Publication Date: 2009-07-22
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of sources of resistance to this disease in existing breeding materials, it is very difficult to further improve the resistance potential of existing varieties to soft rot through conventional breeding

Method used

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  • Cloning and function of Chinese cabbage LRR disease-resistant protein gene BcLRR
  • Cloning and function of Chinese cabbage LRR disease-resistant protein gene BcLRR
  • Cloning and function of Chinese cabbage LRR disease-resistant protein gene BcLRR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1: Cloning and sequence analysis of cDNA fragment of BcLRR gene

[0079] 1) Extraction of total RNA from leaves of Chinese cabbage inoculated with soft rot bacteria at different stages

[0080] Chinese cabbage is grown in the greenhouse to 6-8 true leaves and placed in an artificial climate box. After cultivating for 3 to 4 days under the conditions of 28℃, relative humidity above 90%, light for 13 hours, and light intensity of 9000 Lux, EccBC1 is inoculated in vivo by acupuncture. The inoculation concentration is 2×10 8 Pcs / ml, the inoculation amount for each wound is about 5ul, each plant is inoculated with 3 leaves, and samples are taken at 0min (control, no inoculation with acupuncture), 15min, 30min, 1h, 3h, and 6h after inoculation, and extract the seedling stage Total RNA. Electrophoresis and measuring OD value to detect RNA concentration and integrity, store at -70°C for later use.

[0081] 2) Northern hybridization of BcLRR gene expression

[0082] Use Olig...

Embodiment 2

[0090] Example 2 Construction of BcLRR gene expression vector and analysis of soft rot resistance of transgenic strains

[0091] 1) Construction of BcLRR gene expression vector

[0092] Design a pair of primers 5’-GC containing the complete coding region of the gene GGA TCC ATG GCT TCT CGG TGT GAG-3’ and 5’-CG CCC GGG T CA CCA CAC AGC AAA ATC-3' (underlined are the BamHI and Sma I restriction sites introduced respectively). Using the 3h reverse transcribed cDNA in Example 1 as the PCR reaction template, the reaction procedure was 94°C pre-denaturation for 2 minutes, 94°C denaturation for 30 seconds, 55°C renaturation for 30 seconds, 72°C extension for 1 minute, 30 cycles after 72°C extension for 7 minutes, and recovery The PCR product with a size of about 1kb was cloned into the pGEM-T Easy vector, named pBcLRR, and transformed into E. coli DH5α for sequencing. After sequencing and verification, the BcLRR gene in pBcLRR was cut with BamHI and SmaI, and then ligated with the 35S-c...

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Abstract

The invention relates to the field of plant disease resistance in gene engineering, in particular to a vegetable LRR resistance protein gene BcLRR, an encoding protein of the gene, as well as a carrier and a plant cell strain that contain the gene. The gene has the nucleotide sequences that are shown in SEQ ID NO.1 or SEQ ID NO.2, and the gene encoding contains a protein that has the amino acid sequence shown in SEQ ID NO.3. The vegetable LRR resistance protein gene BcLRR has wide application prospect in the field of plant carotovora resistance, can be used for cultivating carotovora-resistant horticultural plants, and has enormous economic benefits and potentials.

Description

Technical field [0001] The present invention relates to the field of plant disease resistance genetic engineering, in particular to a cabbage LRR disease resistance protein gene BcLRR and its encoded protein, as well as a vector and plant cell line containing the gene. Background technique [0002] The cloning of plant resistance genes (Rgene) is not only the basis for in-depth understanding of plant disease resistance mechanisms, but also a very important gene resource in plant disease resistance genetic improvement projects. At present, more than 40 R genes have been cloned from 11 different plants. Analysis of the protein structure encoded by these R genes found that most of them have Leucine-Rich Repeats (LRRs) domains. The LRR domain is a typical structure of protein-protein interaction, and it may perform the function of receptor recognition in disease-resistant proteins. According to the cellular location of the LRR region of the encoded protein, R genes containing the LRR...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/63C12N5/10C07K14/415
Inventor 马荣才谢华姚磊徐勇张艳秋贾云贺
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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