Identification And Experimental Optimization Of RNA Polymerase Ⅱ Binding Map During Soybean Abscission Zone Development

Flower and pod abscission is one of the most important stages in plant growth and development.Because flower and pod abscission limits the cultivation of high-yield soybean varieties,the regulation mechanism of soybean flower and pod abscission is an urgent problem to be solved.Abscission Zone(AZ)is a structure composed of dense cells on the stem of tofu pudding.The development of abscission zone is a prerequisite for flower pod abscission and is regulated at multiple levels such as transcription.In order to study the gene expression pattern in soybean abscission zone development,two experimental methods,CUT&RUN(Cleavage Under Targets and Release Using Nuclease)and CUT&Tag(Cleavage Under Targets and Tagmentation),were used to detect the distribution of RNA polymerase Ⅱ(Pol Ⅱ)in the genome,and the differentially expressed genes in abscission zone and non abscission zone in different growth and development stages were obtained.In order to meet the research demand for the genomic distribution of soybean Pol Ⅱ in abscission zone,this study used Biochemical engineering methods to modify the Tn5 enzyme of CUT&Tag,improved the experimental efficiency of CUT&Tag,and Preliminary establishment of a distribution model for Pol Ⅱ in soybean abscission zone.The specific work carried out in this study is as follows:1.The pG/Tn5 expression vector was constructed by using the existing p A/Tn5 plasmid and p AG/MNase plasmid,expressed in Escherichia coli,and purified by affinity.The p A Tn5 and p G Tn5 enzymes with high purity were obtained,and their enzyme activities were verified.2.To investigate whether p A-Tn5 and p G-Tn5 in CUT&Tag will affect the experimental results due to different types of antibodies due to different affinity,this study selected different types of Pol Ⅱ antibodies and compared the CUT&Tag experimental results of different types of Pol Ⅱ antibodies.It was found that the affinity between antibodies and enzymes significantly affects the experimental results of CUT&Tag.3.CUT&RUN and CUT&Tag experiments were conducted on abscission zone and non abscission zone plant materials from soybean at different developmental stages,We found that compared to non abscission zone,the expression of transcription and translation related genes in abscission zone was more active,and a batch of genes specifically expressed in abscission zone were obtained.This paper optimized the experimental results of CUT&Tag and preliminarily explored the gene expression characteristics during the development of soybean abscission zone,providing a reference for reducing flower pod abscission and cultivating high-yield soybean varieties.