Screening And Analysis Of Transcription Factors Regulating SRPP Gene Of Eucommia Ulmoides

Eucommia ulmoides is a unique economic tree species in our country,which can produce valuable Eu-rubber.The structure of Eu-rubber is trans-polyisoprene,and its synthesis pathways are seldom studied,especially the small rubber granule protein(SRPP)family of proteins,which are crucial for the biogenesis and stability of rubber particles.Therefore,in this study,the detailed sequence of SRPP gene promoter was obtained,analyzed the cis-acting elements of SRPP gene promoter,and further analyzed the expression pattern of SRPP gene;By screening the transcription factors interacting with the promoter region of SRPP gene,bioinformatics method was used to analyze the function of transcription factors and the mode of transcription factors regulating SRPP gene expression.1.Clone and analysis of SRPP gene promoter sequenceThrough PCR amplification experiment,the promoter sequence of SRPP6 and SRPP 7 gene was obtained,and the length of them was 1994 bp and 2011 bp,respectively.The comparison rate with the genome was 99.45%.The 1895 bp promoter sequence of SRPP1 gene in the 5 'direction was obtained by gene walking experiment;SRPP1,SRPP6 and SRPP7 gene promoters have cis-acting elements related to plant hormone regulation,environmental stress,light regulation,and growth regulation.There are a large number of cis-acting elements related to light regulation in the promoter of SRPP6 gene.2.Analysis of SRPP gene expression patternThe results of the expression patterns of the same gene in different tissue showed that the expression patterns of SRPP1,SRPP2,SRPP3 and SRPP5 genes were similar,and the expression levels decreased in the bark,leaf,seed coat,and root in turn;the expression levels of SRPP6 and SRPP7 were the highest in the seed coat,the expression of levels in bark and leaves were followed,and the expression levels of root were the lowest;While the overall expression level of SRPP4 was relatively low,and the difference between the expression of roots and other tissue was the smallest.The expression results of different SRPP genes in the same tissue showed that the expression levels of SRPP1 and SRPP7 gene were higher in leaves,the expression levels of SRPP1,SRPP6 and SRPP7 gene were higher in fruits,and the expression levels of SRPP1,SRPP 2,SRPP 5 and SRPP 7 were higher in bark.The results of external application of plant hormones showed that the expression levels of SRPP1,SRPP6 and SRPP7 genes all showed a downward trend after spraying ABA,GA3 and methyl jasmonate.After spraying ethephon,the expression levels of SRPP1,SRPP6 and SRPP7 genes showed a trend of increasing first and then decreasing,indicating that external application of ethylene may contribute to the synthesis of Eurubber.The promoter activity of SRPP1,SRPP6 and SRPP7 gene weakened in root,stem,and leaf in turn,which was not completely consistent with the expression trend of SRPP1,SRPP6 and SRPP7 in the stem,leaf,and root of Eucommia ulmoides.The trend was same as the trend in stem and leaf,the main difference was existing in the root.3.Screening and analysis of transcription factors interacting with SRPP geneIn this study,the bait expression vectors pHIS2-SRPP1,pHIS2-SRPP6,and pHIS2-SRPP7 were successfully constructed.SRPP1 yeast one-hybrid obtained 14 positive clones that passed the His reporter gene detection,and SRPP6 yeast one-hybrid obtained 16 positive clones that passed the His reporter gene detection.As for the positive clones,SRPP7 yeast one-hybridization obtained 10 positive clones that passed the His reporter gene test.Through sequencing and bioinformatics analysis,it was found that there were no transcription factors in these positive clones.