The Cloning Of SuSy1and SuSy4Gene In Cassava And Determining The Core Promoter Of SuSy1

Sucrose synthase is one of the most important enzyme in starch synthesis. It has physiological functions in sucrose metabolism, influencing sink strength, the fiber, cell wall synthesis and also in regulation of starch synthesis and so on. Sucrose synthase can express in storage organs in most plants. The mechanism of sucrose synthase in starch synthesis is by reversibly catalyzing sucrose and UDP into UDPG and fructose, so as to provide the substrate for starch synthesis.Research on genes and promoters involving in cassava starch synthesis is useful in understanding the molecular mechanism of cassava starch synthesis. The tobacco transgenic technology is relatively mature, and has high efficiency and short cycle. Therefore, in this study we use Nicotiana benthamiana to conduct research on cassava SuSy gene and promoter. The results are as follows:1、Basing on six SuSy sequences from Arabidopsis, Six SuSy subtypes were identified by searching the database (http://www.phytozome.net/IEsorry.Php?refer=/) in cassava. Phylogenetic analysis of six cassava SuSy subtypes was conducted and result showed that six SuSy isoforms can be broadly divided into three categories with different functions. SuSyl and SuSy4belonged to SuSyl family; SuSy2and SuSy3belonged to dicotyledonous SuSyA family; SuSy5and SuSy6belonged to NG (New Group) family.2、Expression analysis of six different subtypes in cassava functional leaves and roots from various developmental periods reviewed that SuSyl, SuSy3and SuSy4were expressed in roots from all periods and the expression levels of SuSyl, SuSy4were basically the same, followed by the expression level of SuSy3. However, SuSy2, SuSy5and SuSy6could hardly been detected in roots from different developmental periods.3、The exon-intro structures of six sucrose synthase isoforms were studied and the result showed that the six SuSy isoforms could be broadly classified into three categories: SuSy1/SuSy4、SuSy2/SuSy3and SuSy5/SuSy6which evolved from three genes respectively.4、The coding sequences of SuSyl and SuSy4were obtained through PCR amplification based on cDNA of KU50and W14. Both the sequences were2421bp and bioinformatics analysis showed that they had similar functional domains and three dimensional structures. And we obtained about2K upstream sequences from SuSy1ATG based on DNA from KU50and W14. 5、The prokaryotic expression vectors of W14SuSy4, KU50SuSyl and KU50SuSy4were constructed and transformed into BL21competent for expression analysis. All three vectors could express proteins about90KD.6、The over-expression vectors of W14SuSy4, W14SuSyl and KU50SuSy4were constructed and transformed into tobacco.7^A series of5’promoter deletion vectors of KU50and W14SuSy1were constructed and transformed into tobacco. GUS staining analysis of a series of W14transgenic seedlings showed that a phloem-specific DNA sequence existed between-1-(-299) while an enhancing expression DNA sequence was found at-299-613.