A method for rapid verification of promoter strength through protoplasts
A protoplast and promoter technology, applied in biochemical equipment and methods, material analysis by optical means, introduction of foreign genetic material using vectors, etc., can solve the problems of high cost, long cycle and large workload.
Active Publication Date: 2020-05-05
WUHAN BIORUN BIO TECH
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The routine method for analyzing plant promoter activity is to obtain stable transgenic lines using gus as a reporter gene through transgenic technology, and to judge the strength of the promoter according to the depth of staining by staining with gus. This type of technology has a long experimental cycle (4-6 months) month) can not achieve fine quantification, heavy workload and high cost
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[0013] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.
[0014] Such as figure 1 In order to construct the pBWA(V)HS-35scm-ccdbattpk1 vector, the vector is characterized by two gene expression units, one is the reference promoter + cmmkmarker (cell membrane localization signal + green fluorescent protein); the other is ccdb+attpkosgfp (vacuolar Membrane localization signal + green fluorescent protein). CCDB is the target promoter region to be replaced. This vector is easily constructed by con...
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The invention provides a method for quickly verifying intensity of a promoter with a protoplast. A same fluorescent protein is driven respectively by a target promoter and a reference promoter on a same carrier, so that the fluorescent protein is expressed in a same cell; however the fluorescent protein is located at different subcellular locations respectively and then is stimulated by a same laser beam; under the condition that all other parameters are consistent, the fluorescent brightness at the different subcellular locations can represent the intensity of the promoter; through comparison with the reference promoter, a multiple proportion relation between the target promoter and the reference promoter can be quantitatively analyzed. According to the method, a carrier, in which a sequence of a to-be-verified promoter is inserted, needs to be established one time only, then the protoplast is transformed by the established carrier, observation and photographing are performed under a laser confocal microscope, and the intensity of activity of the promoter is quickly evaluated in a manner of comparing the fluorescence intensity generated by the reference promoter and the fluorescence intensity generated by the to-be-verified promoter.
Description
Technical field [0001] The invention relates to a method for quickly verifying the strength of a promoter through protoplasts. Background technique [0002] With the rapid development of whole-genome sequencing technology, molecular biology, and bioinformatics, a large number of genes have been sequenced and cloned. In functional gene research, gene promoter research and analysis are the main components, so the activity of promoters Research is crucial. The conventional method to analyze the activity of plant promoters is to obtain a stable transgenic line using gus as the reporter gene through transgenic technology, and to determine the strength of the promoter according to the depth of staining by staining with gus. The experimental period of this type of technology is long (4-6 Month) cannot achieve precise quantification, the workload is large, and the cost is high. Summary of the invention [0003] The present invention proposes a method for quickly verifying the strength o...
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IPC IPC(8): C12N15/82G01N21/64
CPCC12N15/8202G01N21/6428
Inventor 李阳孙智光
Owner WUHAN BIORUN BIO TECH