Method for identifying transcription factor binding element in cotton fiber development period by DNA affinity protein sequencing

Abstract

The invention relates to a method for identifying a transcription factor binding element in a cotton fiber development period by DNA affinity protein sequencing. The method comprises the following steps of: 1) extracting cotton fiber genome DNA, randomly breaking into fragments of about 200bp, and constructing an Index-DNA library with a sequencing joint by using the broken DNA; 2) performing prokaryotic expression on a cotton transcription factor protein with a label, and performing immunoblotting to confirm the expressed target protein; 3) incubating the target protein and the Index-DNA library in vitro to fully combine the target protein and the Index-DNA library, and extracting Index-DNA specifically bound with the protein; and 4) performing amplification and sequencing, matching the obtained data to a cotton genome, and identifying and confirming the sequence of the obtained binding element after the obtained data corresponds to a DHS site in the cotton fiber development period. The method provided by the invention can quickly and accurately identify the downstream binding site of the cotton transcription factor without genetic transformation, and has the advantages of being fast, accurate, economical, labor-saving and the like.

Description

technical field [0001] The invention relates to a method for using DNA affinity protein sequencing to identify transcription factor binding elements during cotton fiber development, and belongs to the technical field of plant molecular biology. Background technique [0002] Transcription factors play an important role in the growth and development of animals and plants. Transcription factors regulate target genes by binding to cis-acting elements on the promoters of target genes. Studies have shown that a variety of cotton transcription factors are involved in cotton fiber development and stress resistance. For example, transcription factors such as GhWRKY34, GhWRKY6-like, GhATAF1, and GhABF2 can improve the salt tolerance of transgenic plants, and transcription factors such as GhMYB25, GhHOX3, and GhTCP14 can regulate cotton. Fiber development. However, it is difficult to identify the downstream binding element sites of cotton transcription factors, and it is difficult to ...

AI Technical Summary

Problems solved by technology

There are two main methods for chromatin immunoprecipitation: the first method is to directly obtain the nucleus of the required material, and perform chromatin immunoprecipitation followed by sequencing. There is often a family of transcription factors in organisms, and it is difficult to obtain a specific antibody for a certain member, which often causes a lot of background interference, resulting in a decrease in the accuracy of the results; the second method is to label the transcription factor with a known functional domain After constructing the fusion expression vector, the transgene is carried out, but the genetic transformation process of cotton takes 6-8 months, and the transgenic homozygous line must be screened in the follow-up before the chromatin immunoprecipitation experiment, which is time-consuming, laborious and costly

Existing studies on transcription factor regulatory sites during cotton fiber development mostly use transgenic methods, which are time-consuming, laborious, and expensive (Shan C M, et al., 2014, 5(5519):5519.), ordinary DAP The -seq method does not consider DNA methylation during specific developmental periods, does not analyze the open state of chromatin during specific developmental periods, and obtains more transcription factor binding sites (Ning Lihua et al., Science Bulletin, 2019, v.64(24 ):81-92.), increasing the difficulty of screening functional sites for subsequent analysis

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