Cloning And Functional Analysis Of The Promoters Of HbSUT3and HbSUT5in Hevea Brasiliensis

Sucrose transporters mediate transmembrane transportation of sucrose that rely on the cytoplasm electromotive force produced by protons. In the rubber tree, latex is synthesised with sucrose as primary raw materials in the laticifers. Due to lacking of plasmodesmata connections between laticifers and their neighboring cells, sucrose imports into the laticifers by a transmembrane pathway. The function of Sucrose transporter protein has been launched deep study, but its regulation mechanism is relatively scarce.Recent studies suggest that mutation of the non-coding sequences is the main driving force of evolution. The research of the promoters of sucrose transporters will contribute to a better interpretation of the evolution and the function assignment of the sucrose transporter family. What’s more, the assays of the sucrose transporter genes promoter sequences will improve our knowledge to the regulatory mechanism of sugar supply in the laticifers, which will provide possible ideas for increasing sucrose supply in laticifers through artificial manipulation.In this study, the protocol of the Adaptor-PCR for promoter cloning had been optimized. For the first time six sucrose transporter genes promoters in the rubber tree were cloned and their biological information analysis were conducted. Due to the more important roles of HbSUT3and HbSUT5, further analysis of the biological functions of the HbSUT3and HbSUT5gene promoters was performed in the transgenic Arabidopsis and the rubber tree simultaneously, using real time RT-PCR, GUS analysis and mRNA in situ hybridization methods, the results are as follows:1Promoter cloning library of the rubber tree were constructed by optimized adaptor-PCR method, six sucrose transporter genes promoter sequences were successfully amplified in the library, and the length of every genes promoter is more than1.5Kb.2Bioinformatics analysis showed a large variability in the promoter regions of the rubber tree sucrose transporter gene family. There were more stress-related and light-related response elements in Rubber tree sucrose transporter gene promoter regions. Their transcription start sites were located in a broad area beside the starting codon with the distance31bp-342bp, and the length of Rubber tree sucrose transporter gene5’UTR varied. GC content characteristic analysis showed that the six gene core promoter sequences could initiate transcription to some extent, and HbSUT2B core promoter possessed the strongest activity. 3Positive HbSUT3and HbSUT5promoter transgenic Arabidopsis plants were identified by PCR.GUS staining analysis showed HbSUT3and HbSUT5promoter had transcription initiation activity.4The tissue specificity of HbSUT3and HbSUT5promoter were analysed by mRNA in situ hybridization in rubber tree organizations and GUS reporter gene detection in transgenic Arabidopsis,the results showed they were identical in tissue localization basically.5The expression and regulation characteristics of the HbSUT3and HbSUT5promoters were also studied through gene expression analysis of total RNA in rubber tree latex using fluorescence quantitative PCR and GUS expression activity analysis by fluorescence quantitative in transgenic Arabidopsis,the results showed the expression pattern of both were opposed by the stimulations of ERE, SA,ABA, injuries and low temperature, the remaining processing is identical. The result also validated the potential cis-elements predicted by Promoter bioinformatics assayTake together, our results contribute substantially to further identification of upstream transcription factor related to sucrose transporter protein genes, the expression and regulation networks of sucrose transporter protein will be revealed.