Family Gene Characterization Of Ethylene Synthesis Enzyme ACO In Hevea Brasiliensis

Natural rubber is an important industrial raw material.Studies have shown that ethylene can promote the production and degumming of rubber tree latex tubes,and the application of ethephon on the bark can significantly increase the production of latex.In the preliminary research of the research group,the Hevea genome and transcriptome data were obtained,and 16 HbACO gene family members were predicted.Based on these data,we performed a functional analysis of the HbACOs gene family and obtained the following results:1.8 HbACOs family genes were screened out through bioinformatics analysis and successfully cloned,named HbACO1,HbACO2,HbACO3,HbACO5,HbACO7,HbACO10 and HbACO11.These 8 genes all have 2OG-Fe II＿Oxy and DIOX＿N domains unique to ACO enzyme.2.The expression patterns of HbACOs family genes in different tissues and under the stimulation of ethephon were studied,combined with transcriptome data and q RT-PCR data,the HbACO genes that were highly expressed in bark were screened out,namely HbACO2,HbACO3,HbACO7 and HbACO10.3.During the rubber tapping period of 2019 and 2020,samples of bark and latex were collected in different months of the year,the physiological parameters of latex and bark were measured,the bark RNA of different months was extracted,and the highly expressed genes were tapped during the rubber season.Analysis of changes in expression patterns between different months.4.The correlation analysis between the physiological parameters of rubber latex and bark in different months showed that the ethylene release from the bark and the latex production are likely to be negatively correlated,and the dry rubber content and the total solid content are positively correlated.5.The correlation analysis between the physiological parameters of rubber latex and bark and the expression of HbACOs gene in different months showed that the expression of HbACO2,HbACO3,and HbACO7 was negatively correlated with the ethylene release of the bark,and the expression of HbACO10 was negatively correlated with that of the tree.The release of skin ethylene is positively correlated.6.Prokaryotic expression vectors of HbACO2,HbACO7 and HbACO10 genes were successfully constructed,and their induced expression in E.coli BL21 was realized.The purified HbACO2,HbACO7 and HbACO10 recombinant proteins have obvious ACO enzyme catalytic activity and successfully catalyze the synthesis of ethylene in vitro,and the activity of HbACO10 fusion protease is about nine times that of HbACO2 and HbACO7 fusion protease.In this study,the HbACOs gene highly expressed in the bark was screened,the prokaryotic expression vector was constructed and the recombinant protein with ACO enzyme activity was successfully purified.The study of HbACOs gene function and its regulation mechanism in ethylene biosynthesis pathway has important guiding significance for understanding the physiological effects and regulation mechanism of ethylene on plants,and the molecular mechanism of ethylene response to external stimuli.The results of the study have laid a good foundation for understanding the molecular mechanism of ethylene stimulating rubber tree production,revealing the correlation between the differences in rubber production effects of different rubber tree species and gene expression,and providing new ideas for the study of ethylene stimulation mechanism,which has important theoretical significance.It also has potential application value.