Application of Arabidopis thaliana AtGDSL gene in prevention of sclerotinia rot and promotion of seed germination of rape

An Arabidopsis, gene technology, applied in the field of plant genetic engineering and biology, can solve very few problems, and achieve the effects of low cost, simple operation and fast speed

Inactive Publication Date: 2016-01-20
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] But for Arabidopsis AtGDSL The role of the current relevant literature and patent reports are very few

Method used

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  • Application of Arabidopis thaliana AtGDSL gene in prevention of sclerotinia rot and promotion of seed germination of rape
  • Application of Arabidopis thaliana AtGDSL gene in prevention of sclerotinia rot and promotion of seed germination of rape
  • Application of Arabidopis thaliana AtGDSL gene in prevention of sclerotinia rot and promotion of seed germination of rape

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: AtGDSL Gene acquisition

[0037] 1. Seedling cultivation

[0038] The wild-type Arabidopsis was used as the experimental material, and the growth conditions were as follows: temperature 20±2°C; humidity 60-90%; daily photoperiod of 8 hours of light and 16 hours of darkness; light intensity of 44 μmolm –2s–1 .

[0039] 2. RNA extraction and cDNA first-strand synthesis

[0040] RNA extraction: Use UNIQ-10 column Trizol kit (purchased from Shanghai Yingjun Biotechnology Co., Ltd.), take a small amount of sample, freeze it with liquid nitrogen in a mortar, grind it into powder, add 1mL TRNzol-A reagent (day Root Biochemical Technology Co., Ltd.) in a 2mLEP tube, after fully shaking, centrifuge at 4°C, 12000rpm for 10min; then take about 800μL of the supernatant, add 300μL of chloroform (pre-cooling is easy for separation), shake vigorously for 15s, and place at room temperature for 3min; After centrifugation at 12,000 rpm at 4°C for 10 min, transfer abo...

Embodiment 2

[0054] Embodiment 2: construct overexpression AtGDSL Transgenic Rapeseed Plants

[0055] 1. AtGDSL Overexpression vector construction

[0056] Add the CaMV35S strong promoter to the upstream EcoRI / KpnI restriction site of the vector pCAMBIA1300 (Beijing Dingguo Changsheng Biotechnology Co., Ltd.), and add the CaMVNos terminator to the downstream BamHI / HindIII restriction site to transform it into Recombinant vector pCAMBIA1300-35S-Nos. The specific process is as follows:

[0057] The specific process is as follows: according to the CaMV35S sequence (GeneID: AJ007626) published on NCBI, use PrimerPremier5.0 software to design primers: upstream primer: 5′- GAATTC TTAATTAAGAGCTCGCATGCC-3' (SEQ ID NO.5) contains EcoRI restriction site (underlined part), downstream primer: 5'- GGTACC GTCCCCGTGTTTCTCCAA-3' (SEQ ID NO.6) contains the KpnI restriction site (underlined part), and the CaMV35S fragment was amplified from the pEGAD vector (purchased from Bao Biotechnology (Dali...

Embodiment 3

[0082] Example 3: Overexpression AtGDSL Response of Rapeseed Rapeseed Plants to Sclerotinia Infection

[0083] Sclerotia sclerotia were cultured in PDA medium. PDA medium (1L) formula: 200g potato, 10-20g sucrose, 17-20g agar powder, 1000g double distilled water; cut the peeled potato into small pieces, add 800mL water, boil for 0.5h, filter the residue with gauze, add water Make up 1000mL, then add sugar and agar, heat to melt the agar completely, aliquot while hot, and autoclave for later use.

[0084] Take the four-leaf-one-heart stage of the above-mentioned PCR test positive AtGDSL The fourth true leaf of positive plants was inoculated in vitro. Cut the leaves and place them in a white porcelain plate. The leaves of each transgenic plant and the non-transgenic control are placed side by side in a plate, and a layer of wet gauze is laid under the leaves. Take a freshly prepared φ5mm mycelium block, with the mycelium side facing down. , inoculated at the position sligh...

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Abstract

The invention discloses a gene for resisting sclerotinia rot of cabbage type rape and promoting rape seed germination and application thereof and specifically relates to an AtGDSL gene capable of improving resistance of plants to sclerotinia sclerotiorum biotic stress and promoting rape seed germination. Verification is carried out on the function of the AtGDSL gene in improvement of resistance of plants to sclerotinia sclerotiorum biotic stress and promotion of rape seed germination, and application of the gene is also verified. According to the invention, the exogenous Arabidopis thaliana AtGDSL gene is introduced into a rape plant through a genetic engineering technological means, and after excess expression of the exogenous Arabidopis thaliana AtGDSL gene in the cabbage type rape, the rape plant has substantially improved sclerotinia rot resistance and seed germination capability; and the AtGDSL gene exerts obvious resistance effect on sclerotinia sclerotiorum, can be applied to stress resistance breeding of plants, improves stress resistance of the plants and improves the speed of seed germination, thereby providing excellent breeding materials for large-scale mechanical production of rape.

Description

technical field [0001] The present invention relates to Arabidopsis AtGDSL The application of the gene in anti-sclerotinia and promoting seed germination of rape belongs to the field of plant genetic engineering and biotechnology. Background technique [0002] Rapeseed (Brassicanapus) is the largest oil crop in my country. 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. The rapeseed oil it produces accounts for the entire supply of edible vegetable oil in my country 40% (Shen Jinxiong et al., 2007). In addition, rapeseed oil is also used to produce biodiesel, which is an important raw material crop to solve the global energy shortage. Therefore, rape is not only the main source of edible oil, but also an important energy crop, which has a very important strategic position. However, due to the limited arable land area in my country, it is impossible to increase the tota...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/20C12N15/84A01H5/00
Inventor 谭小力郭小娟王政张志燕闫素珍陈克平
Owner JIANGSU UNIV
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