Molecular Cloning And Functional Analysis On Sucrose Synthase Gene AhSuSy Of Peanut (Arachis Hypogaea L.)

Drought stress is the major environmental affecting factor in the growth and development of plant. Studies on the mechanism of plant gene expression and regulation have an important academic and realistic significant on clarifying the drought response of plant and drought-tolerant breeding. Sucrose synthase (SuSy), which is an exclusive necessary enzyme in the pathway of sucrose metabolism, plays an important role in plant growth, development and osmotic adjustment process. In this study, AhSuSy was isolated by homologous cloning combined with RACE and TAIL-PCR method from peanut. The AhSuSy expression pattern was characterized and its function was further researched. The results provided theoretical foundation for drought tolerance improvement of peanut. In order to reveal the mechanism of AhSuSy gene in peanut, the main results were as follows:1.Gene isolation and sequence blast analysis. In this study, AhSuSy (Accession number: JF346233) was isolated from peanut using AtSuSy as probe by homologous cloning and RACE. The full length was 2,790bp. It contained a 2,421 bp open reading frame, encoding a polypeptide of 806 amino acids. The estimated molecular weight of the gene was 92402.8Da and the isoelectric point of the putative protein was 6.31. The phylogenetic relationship also appeared that peanut SuSy protein was more similar to soybean protein than other SuSy proteins, and amino acid sequences alignment revealed that the homology was over 75% among peanut SuSy and other SuSy proteins.2.Gene cloning and sequence analysis of AhSuSy gene.In order to reveal the mechanism of SuSy gene in peanut, the TAIL-PCR technology of genome walking was utilized to obtain a genome sequence for the length of 6189bp, which included 13 exons, 12 introns and about an 825bp length partial promoter region. Sequence analyses showed that typical elements TATA-box, CAAT-box and some other regulate elements are existented in this region. For example cis-acting element involved in low temperature responsive element (LTR), GA responsive element (P-box), drought responsive of MYB binging site (MBS), anaerobic induction element (ARE) and light responsive element G-box and box4 were found in the sequence. It was supposed that the expression AhSuSy was regulated by low temperature, drought, anaerobic and hormone GA et al.3.The AhSuSy was ligated into pET-32a (+) and transformed into E.coli competent cell. The positive clone was selected and sequenced. After induced IPTG, the fusion protein relative molecular weight was 92kD, which was consistent with theoretical value.4.Analysis of AhSuSy expression patterns.The tissue specific expression analysis of peanut seedlings by using semi-quantitative RT-PCR assays indicated that AhSuSy was expressed in root, stem and leaf, and reached highest abundance in root. After treated with 10%PEG, peanut seedlings were used to determine AhSuSy transcript level, sucrose synthase enzyme activity and sucrose content. The result showed that all the there parameters were gradually increased during 4h-12h and then declined during 12h-24h, which indicated that AhSuSy may play an important role in plant adaptation to drought stress.