A gene for controlling Sclerotinia sclerotiorum and its application

A rapeseed sclerotinia and genetic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., achieves the effect of fast speed, high efficiency and simple operation

Inactive Publication Date: 2016-05-25
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As for the role of BnWRKY33 in rapeseed, there are no literature and patent reports

Method used

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  • A gene for controlling Sclerotinia sclerotiorum and its application
  • A gene for controlling Sclerotinia sclerotiorum and its application
  • A gene for controlling Sclerotinia sclerotiorum and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: the isolated clone of BnWRKY33 gene

[0029] (1) Isolation and cloning of BnWRKY33 gene

[0030] The variety Zhongshuang No. 9 of Brassica napus was used as the experimental material, and the growth conditions were: temperature 20±2°C; humidity 60-90%; daily photoperiod of 8 hours of light and 16 hours of darkness; light intensity of 44 μmolm –2 the s –1 .

[0031] Extraction of total RNA from rapeseed leaves and synthesis of cDNA were carried out using UNIQ-10 column Trizol kit (purchased from Shanghai Yingjun Biotechnology Co., Ltd.), and DNase I (Bao Bioengineering (Dalian) Co., Ltd.) was used to remove a small amount of mixed total RNA. DNA, and finally the integrity of total RNA was checked by 1% agarose gel electrophoresis. The cDNA was synthesized according to the instructions of the MMLV Reverse Transcriptase Kit (Promega (Beijing) Biotechnology Co., Ltd.), and 50 μmol / LOligo(dT)18 (ThermoFisher Scientific) was used as the primer. Then use prim...

Embodiment 2

[0034] Example 2: The response of BnWRKY33 to Sclerotinia

[0035] (1) Treatment of Sclerotinia

[0036] The third leaf of rapeseed seedlings (at the four-leaf one-heart stage) was inoculated with Sclerotinia sclerotiorum, the pathogen of Sclerotinia sclerotiorum, and the plants were incubated at 23°C for 4-5 days at a constant temperature before treatment, and then the leaves were kept in a dark environment for 24 hours. Then inoculate with mycelium blocks of Sclerotinia sclerotiorum, and control with sterile medium blocks. Samples were taken at 0h, 12h, 24h, 36h, and 48h after inoculation.

[0037] (2) Extraction of Brassica napus RNA

[0038] Total RNA was extracted using plant Trizol (Shanghai Handsome Biotechnology Co., Ltd.) reagent.

[0039] (3) Real-time quantitative PCR

[0040] Fluorescent quantitative PCR uses SYBR?GreenRealtimePCRMasterMix–Plus–kit (Bao Biological Engineering (Dalian) Co., Ltd.), using B.napusUBC21 (ubiquitin-conjugatingenzyme21) as the interna...

Embodiment 3

[0043] Embodiment 3: the acquisition of overexpression BnWRKY33 transgenic rape plant

[0044] (1) Plant expression vector construction

[0045]Design primers 5'-gaattcTTAATTAAGAGCTCGCATGCC-3' and 5'-ggtaccGTCCCCGTGTTCTTCCAA-3', and use PCR to amplify the CaMV35S fragment from the pEGAD vector (purchased from Bao Biological Engineering (Dalian) Co., Ltd.), and then connect it to pCAMBIA1300 The EcoRI / KpnI restriction site of the vector (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.); simultaneously design primers 5′-ggatccGAATTTCCCCGATCGTTCAA′-3′ and 5′-aagcttGATCTAGTAACATAGATGACACCGC-3′, and amplify from the pEGAD vector by PCR The CaMVNos fragment was amplified and connected to the BamHI / HindIII restriction site of the pCAMBIA1300 vector to obtain the pCAMBIA1300-35S-Nos vector. The endonucleases EcoRI, KpnI, BamHI and HindIII used were all purchased from Treasure Bioengineering (Dalian) Co., Ltd., and the enzyme digestion conditions were 37°C for 12h. ...

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Abstract

The invention discloses a gene for controlling Sclerotinia sclerotiorum (Lib.) de Bary of Brassica napus L. and a use thereof and especially relates to isolation and cloning, functional verification and application of a gene BnWRKY33 for controlling Sclerotinia sclerotiorum (Lib.) de Bary of Brassica napus L. The gene BnWRKY33 has the function of controlling Sclerotinia sclerotiorum (Lib.) de Bary of crops. In Sclerotinia sclerotiorum (Lib.) de Bary treatment on Brassica napus L., a BnWRKY33 expression level is substantially improved. Through the gene engineering technology, a BnWRKY33 expression level is improved and Sclerotinia sclerotiorum (Lib.) de Bary of Brassica napus L. can be controlled and thus the gene provides an important application prospect for improving Sclerotinia sclerotiorum (Lib.) de Bary resistance and yields of Brassica napus L. and other crops.

Description

technical field [0001] The invention relates to a gene for controlling Sclerotinia sclerotiorum and its application, in particular to a gene for controlling Sclerotinia sclerotiorum of Brassica napus and a carrier containing the gene or its homologous gene, and relates to the use of the gene or its similar function The invention relates to the application of biological regulation of plant anti-sclerotinia in agricultural production, which belongs to the field of plant genetic engineering. Background technique [0002] As an important oil crop, rape plays an important role in China's oil production. The annual planting area of ​​rapeseed in my country is 7 million hectares (100 million mu), accounting for 1 / 3 of the world, ranking first in the world, and the rapeseed oil it produces accounts for 40% of my country's entire edible vegetable oil supply (Shen Jinxiong et al., Chinese rapeseed production and Potential of Genetic Improvement and Prospect of Rapeseed Biodiesel [J], ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00
Inventor 王政方和娣谭小力陈克平张志燕陈宇李冠英顾守来
Owner JIANGSU UNIV
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