Sclerotinia sclerotiorum induced oilseed rape promoter pBnGH, identification method and application

A promoter and sclerotinia technology, applied in the field of plant genetic engineering, can solve problems such as poisoning and excessive energy consumption of plants

Active Publication Date: 2019-04-19
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biggest disadvantage of this type of promoter is that it enables the foreign gene to be continuously and strongly expressed throughout the life cycle of the plant and in all tissues, causing excessive energy consumption or toxic effects on the plant.
However, the study of S. sclerotiorum inducible promoters in plants has not been reported

Method used

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  • Sclerotinia sclerotiorum induced oilseed rape promoter pBnGH, identification method and application
  • Sclerotinia sclerotiorum induced oilseed rape promoter pBnGH, identification method and application
  • Sclerotinia sclerotiorum induced oilseed rape promoter pBnGH, identification method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Changes in the expression of BnGH gene in Brassica napus disease-resistant materials and susceptible materials

[0021] The two materials used in this experiment are J964 (resistant material, R-line) and J902 (susceptible material, S-line), both of which are pure line materials of winter Brassica napus. obtained in the screening of resistant materials.

[0022] The pathogenic bacteria came from Sclerotinia SS-1 provided by Mr. Li Guoqing from the State Key Laboratory of Microbiology, Huazhong Agricultural University. The strain was stored in a 4°C refrigerator under dark light, and inoculated on potato medium (PDA, 25% potato extract, 2.5% glucose, 1.5% agar, pH adjusted to 5.86) before use, at 23°C, under dark light Used for inoculation after activation twice.

[0023]At the final flowering stage of rapeseed, the living stem inoculation method at the adult plant stage was used for inoculation. The specific method was as follows: use a 7mm puncher to punch ...

Embodiment 2

[0026] Example 2: Cloning of promoter pBnGH

[0027] According to the Brassica napus 'Darmor-bzh' reference genome sequence, the primers were designed to amplify the promoter sequence about 2000bp upstream of the start codon of the BnGH gene (BnaC01g21880D). The primer sequence is (BnGHpro-F:5'-CCAAAGGAAGACAAGGAGCA-3' (SEQ ID No. 2); BnGHpro-R: 5'-AGCAAGAAGAAGATACAGTGGGA-3' (SEQ ID No. 3)).

[0028] Genomic DNA of Brassica napus antisclerotinia strain J964 was extracted by CTAB method as a template, and PCR reaction was carried out. The reaction procedure was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 2 min, and 33 cycles of reaction Afterwards, fully extend at 72°C for 7min. After the end, the DNA was purified and recovered with the DNA recovery kit of Kangwei Company, and the purified DNA fragment was connected to the pEASY-Blunt simple vector (full type gold, China), incubated at 25°C for...

Embodiment 3

[0031] Example 3: Relative expression of BnGH gene in each tissue of Brassica napus

[0032] The test material J9712 was planted in the experimental field of Yangzhou University, Jiangsu. The cotyledon, seedling stage (1 month old) leaf, seedling stage (1 month old) root, stem, sedge stem leaf, apical meristem (SAM), flower bud, unpollinated ovary, 14d seed, 14d silique , 24d seeds, 24d silique peels, 34d seeds, 34d silique peels, 50d seeds and 50d silique peels were collected. All tissue samples were obtained from multiple plants and mixed as one sample, and three biological replicates were set. After sampling Immediately placed in liquid nitrogen, and then stored in a -80°C refrigerator.

[0033] RNA was extracted using a plant total RNA extraction kit (Quanshijin, China), and the experimental procedure was referred to the instruction manual of the kit. Genomic DNA contamination was removed with RNase-free DNase I (Thermo Scientific, USA). The same method as in Example 1 ...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a sclerotinia sclerotiorum induced oilseed rape promoter pBnGH, an identification method and application. Separation and cloning are performed on Brassica naps, and a sclerotinia sclerotiorum induced promoter is identified. The nucleotide sequence of the promoter is shown in SEQ ID No.1. An experiment proves that a gene controlled by a promoter can generate a specific response reaction on sclerotinia sclerotiorum infection, the expression quantity in brassica naps and arabidopsis leaves, stalk, flower buds, pods and seeds is low, and the promoter only has certain expression in roots. In addition, the promoter is not induced by hormone treatment and most abiotic stress, and only has the certain response reaction for high temperature. By means of the pBnGH promoter, expression of resistance genes is controlled, expression of the resistance genes is only violently induced during sclerotinia sclerotiorum invasion, and the resistance of Brassica napus in sclerotinia sclerotiorum is enhanced.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a rapeseed promoter pBnGH induced by sclerotinia sclerotiorum, an identification method and application. The promoter can be used to improve the resistance of plants to sclerotinia through plant genetic engineering technology. Background technique [0002] A promoter is a DNA sequence that RNA polymerase specifically recognizes, binds, and initiates gene transcription. It acts as a "switch" for gene transcription and regulates the transcription time and expression level of genes. Promoters can be divided into constitutive promoters, tissue-specific promoters and inducible promoters according to their functions and modes of action. Plant genetic engineering introduces exogenous genes into recipient plant cells and integrates them into recipient chromosomes, so that recipient plants can obtain corresponding genetic phenotypes. Traditional genetic engi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/10A01H5/00C12Q1/6895
CPCC07K14/415C12N15/8282C12Q1/6895C12Q2600/158
Inventor 吴健王幼平林俐孙勤富刘东晓方玉洁蒋金金吴德伟
Owner YANGZHOU UNIV
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