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Intermediate factor BnMED16 gene to regulate Sclerotinia stem rot resistance in Brassica napus and application thereof

A technology for sclerotinia sclerotiorum and genetics of rapeseed, applied in application, genetic engineering, plant genetic improvement and other directions to achieve the effect of improving resistance

Inactive Publication Date: 2019-10-25
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Rapeseed rape and Arabidopsis thaliana belong to the Brassicaceae Brassica plant, and the genomes of the two have a high homology. Sequence comparison analysis shows that the nucleotide sequence similarity of rapeseed and Arabidopsis MED16 gene is 91%, and the protein sequence is similar The degree is 90%, and Sillito et al. found that Arabidopsis genes usually exhibit similar functions in rapeseed (Sillito D, Parkin IA, Mayerhofer R, et al. Arabidopsis thaliana: a source of candidate disease-resistance genes for Brassica napus. Genome.2000,43(3):452-60); In addition, MED16 protein is highly conserved in yeast, human and plant (Deepthi LW, Shane AR, Heather K, et al.OsSFR6 is a functional rice orthologue of SENSITIVE TO FREEZING-6 and canact as a regulator of COR gene expression, osmotic stress and freezing tolerance in Arabidopsis. New Phytologist, 2011, 191:984-995); therefore, to further explore the molecular mechanism of rapeseed BnMED16 regulating sclerotinia resistance, It will provide theoretical guidance and new materials for anti-sclerotinia breeding of rapeseed, but there are no relevant literature and patent literature reports on the role of BnMED16 in rapeseed

Method used

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  • Intermediate factor BnMED16 gene to regulate Sclerotinia stem rot resistance in Brassica napus and application thereof
  • Intermediate factor BnMED16 gene to regulate Sclerotinia stem rot resistance in Brassica napus and application thereof
  • Intermediate factor BnMED16 gene to regulate Sclerotinia stem rot resistance in Brassica napus and application thereof

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Experimental program
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Embodiment 1

[0030] Embodiment 1: BnMED16 gene isolation clone

[0031](1) Isolation and cloning of BnMED16 gene

[0032] The Brassica napus westar variety (provided by the Rapeseed Research Laboratory of Huazhong Agricultural University) was used as the experimental material. Super Total RNA Extraction Kit (purchased from Promega, U.S.) was used to extract the total RNA from leaves of Brassica napus westar variety. After the RNA extraction was completed, it was treated with DNaseI (purchased from Promega’s kit), and the RNA integrity passed 1.2% (w / v) Agarose gel (EtBr) electrophoresis detection (5V / cm). The determination of nucleic acid concentration was carried out on an IMPLENNanoPhotometer-N50 series ultra-micro ultraviolet spectrophotometer (Germany). The RNA 260 / 280 ratio is between 1.9 and 2.1, the 260 / 230 ratio is greater than 2.0, and the RNA with a concentration greater than 500ng / μL is used for the next analysis. cDNA was synthesized using IIQ RT SuperMix for qRNA (+gDNA...

Embodiment 2

[0036] Embodiment 2: the acquisition of overexpression BnMED16 transgenic rape plant

[0037] (1) Construction of plant overexpression vector

[0038] According to the CaMV 35S sequence (Gene ID: AJ007626) information published on NCBI, use Primer Premier5.0 software to design the following primers:

[0039] 5′-GAATTCTTAATTAAGAGCTCGCATGCC-3′ and

[0040] 5'-GGTACCGTCCCCCGTGTTTCTCCAA-3', the Double CaMV 35S fragment was amplified from the pCAMBIA1300s vector (purchased in Beijing Dingguo Changsheng Biotechnology Co., Ltd.) by PCR method, and then connected to the pCAMBIA2301 vector (Mr. Wang Jing, Huazhong Agricultural University Kindly donated) EcoRI / HidIII restriction site, and finally the final vector pCAMBIA2301-1300s (11634bp) was obtained.

[0041] EcoRI and SalI restriction sites and corresponding protective bases were added to the two ends of the BnMED16TA cloning amplification primers respectively, and the primers were named OE-BnMED16-F (5'CGGAATTCTTGCTCTCTCGCTCGACA...

Embodiment 3

[0074] Example 3: Evaluation of BnMED16 Overexpression Transgenic Rapeseed Against Sclerotinia

[0075] (1) Immune response of BnMED16 overexpression strain to S. sclerotiorum

[0076] The in vitro inoculation treatment of Sclerotinia sclerotiorum (sclerotias clerotiorum, the pathogen of Sclerotinia sclerotiorum was preserved by the Laboratory of Life Science College of Huazhong Agricultural University and activated by the laboratory) was carried out on the second leaf of rapeseed seedlings (at the stage of four leaves and one heart). For the second leaf of the positive line with significantly increased expression in Example 2, the leaves were cut and placed in a transparent square box. The leaves of each transgenic plant were placed side by side with the non-transgenic control in a box, and several layers of filter paper were laid under the leaves. sheets and damp gauze, freshly prepared Mycelium block, with the side of mycelium facing down, was inoculated at the position s...

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Abstract

The invention belongs to the field of plant genetic engineering and particularly relates to an intermediate factor BnMED16 gene to regulate Sclerotinia stem rot resistance in Brassica napus and application thereof. A gene BnMEDIATOR16(BnMED16 to control Sclerotinia stem rot in Brassica napus is acquired from Brassica napus westar by isolating and cloning; a nucleotide sequence of the gene is shownas SEQ ID NO: 1; a protein sequence encoded by the gene is shown as SEQ ID NO: 2. Functional verification is performed on the gene BnMED16 herein. By increasing the expression quantity of BnMED1 in Brassica napus through genetic engineering techniques, it is possible to activate a disease-resistant signal pathway of jasmonic acid and to increase accumulation of active oxygen and expression of disease resistance-related genes; lesion area of leaf Sclerotinia stem rot of transgenic oilseed rape is decreased by 81.20%; the oilseed rape is more resistant to Sclerotinia stem rot. The gene herein has an important application prospect in enhancing the resistance of Brassica napus to Sclerotinia stem rot.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and specifically relates to an intermediary factor BnMED16 gene for regulating Sclerotinia sclerotiorum resistance of Brassica napus and its application. The gene of a Mediator Complex member of Brassica napus was isolated and cloned, and the function verification and preliminary transformation application of the gene were carried out. According to the sequence of the mediator complex subunit AtMED16 reported in the model plant Arabidopsis thaliana with the most significant resistance to sclerotinia, sequence alignment is performed in Brassica napus with high homology, and the screened The sequence of BnMED16 with the highest homology was obtained, the gene was cloned and overexpressed in Brassica napus Westar, and inoculated with Sclerotinia sclerotiorum to quickly identify its function in Sclerotinia sclerotiorum resistance. Background technique [0002] Rapeseed is the most important ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29A01H5/12A01H6/20
CPCC07K14/415C12N15/8282
Inventor 杜雪竹胡慧贞汤依唯潘宣丞
Owner HUBEI UNIV
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