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
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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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