A gene gmlecrk-r for improving plant disease resistance and its application

A plant and gene technology, applied in the fields of plant molecular biology and plant genetic engineering, to achieve the effect of improving disease resistance and enhancing disease resistance

Active Publication Date: 2022-06-03
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the recognition of NBS-LRR disease resistance proteins and pathogen effector molecules in plants follows the "gene-to-gene" hypothesis, that is, NBS-LRR disease resistance proteins can only recognize specific pathogen effector molecules.

Method used

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  • A gene gmlecrk-r for improving plant disease resistance and its application
  • A gene gmlecrk-r for improving plant disease resistance and its application
  • A gene gmlecrk-r for improving plant disease resistance and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. Cloning and sequence structure analysis of GmLecRK-R gene

[0047] Seeds of soybean and Feng-47 were directly sown in pots filled with nutrient soil, cultivated in a greenhouse (21-23°C, 14h light / 10h dark), and 2-week-old plants were used for RNA extraction.

[0048] Total RNA extraction: using soybean leaves as materials, the total RNA was extracted using the RNA extraction kit from Omega Company in accordance with the instructions, and the RNA content and quality were detected with a spectrophotometer.

[0049] Reverse transcription to generate the first strand: take 0.7 μg RNA as a template, perform cDNA synthesis according to the instructions of Takara’s PrimeScript reverse transcriptase reagent kit, and dilute to 20uL for reaction. Take an appropriate amount of reverse transcription product for subsequent gene cloning PCR.

[0050] Use the first strand of cDNA as an RT-PCR template, and perform PCR in a conventional method to amplify the GmLecRK-R gene...

Embodiment 2

[0058] Example 2. Expression of GmLecRK-R gene in soybean root hairs:

[0059] The specific steps are briefly described as follows:

[0060] 1) Planting of soybean Hefeng-47:

[0061] Sow the seeds of soybean Hefeng-47 evenly in flower pots with vermiculite, cover the flower pots with black plastic film at the beginning or place the flower pots in a dark place for cultivation, and after the yellowed seedlings grow evenly, uncover Black plastic film, remove the seed coat, water (about 3 days to 4 days), and place under suitable light to cultivate (about 1 and a half days to 2 days), that is, the cotyledon of the six-day-old plant can be used for the experiment.

[0062] 2) Agrobacterium culture

[0063] Single colonies of Agrobacterium rhizogenes K599 transfected with pBin::GmLecRK-R-eGFP vector and pBin::eGFP vector were picked from the plate, respectively, and inoculated into 2mL LB liquid medium (Kana 50ug / mL, Streptomyces 50ug / mL) on a constant temperature shaker at 30°C...

Embodiment 3

[0074] Embodiment 3. the cultivation of Phytophthora

[0075] Inoculate the transgenic Phytophthora sojae with red fluorescence on the solid V8 medium plate (G418 50ug / mL), or inoculate the Phytophthora nicotiana on the solid V8 medium plate. The inoculated V8 medium plate was cultured at 25°C in the dark for 5 days and then used for subsequent experiments.

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Abstract

The invention provides a gene for improving plant disease resistance and its application, and belongs to the field of plant molecular biology and plant genetic engineering. The invention relates to a plant-derived disease resistance gene GmLecRK‑R, its recombinant expression vector and its application. The gene is derived from soybean, has the nucleotide sequence shown in SEQ ID NO.1, and the amino acid sequence of the encoded product is SEQ ID NO.2. The present invention also provides a gene silencing and recombinant expression vector, including the gene GmLecRK‑R described in the present invention. This gene plays a key role in plant disease resistance, especially Phytophthora resistance. Overexpression of this gene significantly promotes the disease resistance of soybean and tobacco to different Phytophthora, and is an ideal gene for enhancing plant disease resistance. The gene is expressed in tobacco through the method of genetic transformation so that it can acquire resistance to various pathogenic bacteria, and can also be used in the improvement of disease resistance in crop breeding.

Description

technical field [0001] The invention belongs to the field of plant molecular biology and plant genetic engineering, in particular, the invention relates to a gene for improving plant disease resistance and its application. Background technique [0002] At present, research on disease resistance genes in plants mainly focuses on a class of receptor protein genes encoding nucleotide binding sites (NBS) and leucine-rich repeats (LRR). [1-3] . The NBS-LRR disease-resistant protein encoded by this type of gene acts in plant cells, and can recognize the effector molecules secreted by pathogenic bacteria to activate the defense response of plants, thereby inhibiting the infection of pathogenic bacteria. However, the identification of NBS-LRR disease resistance proteins and pathogen effector molecules in plants follows the "gene-to-gene" hypothesis, that is, NBS-LRR disease resistance proteins can only recognize specific pathogen effector molecules. Once the effector molecules sec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82C12N15/84A01H5/00A01H6/54A01H6/82
CPCC12N15/8261C12N15/8279C07K14/415
Inventor 王燕万博闻王源超
Owner NANJING AGRICULTURAL UNIVERSITY
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