The Establishment Of Cotton Cotyledon Transient Expression System And The Activity Analysis Of Artificially Synthesized Inducible Promoter

Plant transient expression system is when exogenous genes are transfected into plants and produce transient effects in host cells during a short time,instead of integrating into host cell chromosomal DNA to steady expression.Transient expression system has an important role in research cellular localization,protein interaction,promoter activity and so on.In addition,transient expression system is easy to operate without special equipment,decreases risk of exogenous genetic drift,and effectively avoids gene silencing,etc.As a result,the transient expression system is widely used in transgenic engineering.Promoter is a DNA sequence with the transcription initiation complex and multiple transcription factors interacting,in the ribonucleic acid(RNA)synthesis,promoter controls gene expression(transcription)of the start time and the degree of expression.Promoters can be classified into three types:constitutive promoters,tissue-specific promoters and inducible promoters,which depend on the pattern of transcription.Constitutive promoters drive genes expressed in each tissue of plants;tissue-specific promoters drive genes expressed in a tissue-specific site only;inducible promoters under various environmental stress conditions can induce gene expression numerously,but normally not expressed or the gene expression level is very low.Here,we established a transient expression system in cotton cotyledons with eGFP as reporter gene.Based on the identification of D-7 247 bp region,we constructed DREB1,added 28 bp SynJ(a synthetic enhancer sequence)promoter and series of D-7 artificial promoters fused with eGFP,and verified their promoter activity using this transient expression system,results was consistent with the results of transgenic Arabidopsis,so the system can be well applied to the study of promoter activity.The results of our experiment are as follows:1.The best optimum conditions of the system with cotyledon growth time,agrobacterium concentration,injection volume and co-culture time were studied and determined.The results showed that the highest efficiency of exogenous gene expression were obtained by using cotton cotyledons germinated for 6 days injected 50?L Agrobacterium LB4404 including heterologous gene with OD600 equal to 0.5 and co-cultured for 3 days.2.We constructed DREB1,added 28 bp SynJ promoters and series of D-7 artificial promoters fused with eGFP,named pDREBl-eGFP,pDrought 1,pDrought 3,pDrought 5,pDrought 7,p28nt-eGFP and verified their promoter activity using this transient expression system,results showed that pDREB1-eGFP,pDrought 1,pDrought 3,pDrought 5,pDrought 7 respond to drought,which may be inducible promoters,and p28nt-eGFP as a strong constitutive promoter.3.We transfered pDREBl-eGFP,pDrought 1,pDrought 3,pDrought 5,pDrought 7 into Arabidopsis thaliana mediated by Agrobacterium GV3101.We detected the expression of eGFP and analyzed the promoters' activity.The results showed that each promoter we constructed above has a response to drought.4.The result of transient expression with cotton cotyledon was consistent with the result of transgenic Arabidopsis.It showed the transient expression system can be well applied to the study of promoter activity.However,the activity of artificially synthesized promoters from D-7 was still weak.Therefore,adding additional flanking sequence in upstream of promoter region to reform D-7 promoter into a strong inducible promoter is our next project.