Analysis On The Interactions Between The Peptide Fragments Of SBEIIb And SUS-SH1from The Endosperm Of Zea Mays L By Yeast Two-Hybrid System

Starch is not only the main source of food, but also the raw materials in industry. Recently, the value of starch in industry is becoming outstanding as the development of the economy and the consumption of the energy, such as:producing alcohol used in industry, food additives, plastic lunch boxes and paper. Corn starch is the best one for industry because of its good physical and chemical character. In addition, there is great cultivated area for corn in world, therefore, the cost of production is much low. So far, it is hard to culture the special corn which is used for industry with the traditional breeding method in a short time. So, culture the special corn with biotechnology is one of the main tasks in plant gene engineering.The metabolism of starch is much complex, the way in which starch is made in corn endosperm is the point that the biologically pay attention to, and the knowledge is also benefit for agriculture by providing the means to improve the quantity and quality of starch in cereal seeds. Starch branching enzymellb(SBEⅡb) and sucrose synthesis shrinkl(SUS-SH1) are the two main enzymes in the way of starch metabolism.It is reported that SBEIIb,SUS-SHland other enzymes in the way of starch metabolism are associate with each other in multisubunit complexes, which is about670KDa, but the function of the complexes is unknown. We have demonstrated that SBEⅡb and SUS-SH1interact with each other in yeast two-hybrid system, but the interactive system is unknown, the influence of the association of the two enzymes to the complexes is unknown, the function of the association of the two enzymes in the way of starch metabolism is unknown.We analysis of the interactions between the peptide Fragments of SBEⅡb and SUS-SH1with Yeast Two-Hybrid system in this paper, it will supply some evidence for deep understanding the way of starch metabolism and control system. The method and results as following:Determining the functional domain and non-functional domain of SBEⅡb based on the analysis of CDD and the definition of the NCBI for SBEⅡb; The coding sequence of the functional domains and non-functional domains were amplified, and then were cloned into pGBKT7, which are pGBKT7-SBEIIb-x(x=1,2,3,4,5,6). Afer being verified by sequencing, the bait vectors were transformed into the yeast cell AH109, and then were tested for the toxicity and the transcriptional activation. At the same time, the coding sequence of SUS-SHlwas amplified, and then was cloned into pGADT7, which is pGADT7-SUS-SH1.Six recombination plasmids (pGBKT7-SBEIIb-x(x=1,2,3,4,5,6)) pairs with recombination plasmid (pGADT7-SUS-SH1) respectively, and then were transformed into AH109competent cells, the transformed system were put on the selective medium SD/-Leu/-Trp, SD/-Leu/-Trp/-His and SD/-Leu/-Trp/-His/-Ade in order. Finally, detect the screening results with X-α-Gal.The cotransformed system of GBKT7-SBEⅡb-1, pGBKT7-SBEⅡb-3, pGBKT7-SBEⅡb-5, pGBKT7-SBEⅡb-6paired with pGADT7-SUS-SH1can grow on SD/-Leu/-Trp/-His, but not SD/-Leu/-Trp/-His/-Ade. The cotransformed system of pGBKT7-SBEIIb-2, pGBKT7-SBEIIb-4paired with pGADT7-SUS-SH1can grow on SD/-Leu/-Trp/-His and SD/-Leu/-Trp/-His/-Ade, but the detection results by X-a-Gal is not blue. It indicated that the binding ability of pGBKT7-SBEⅡb-1, pGBKT7-SBEIIb-3, pGBKT7-SBEⅡ-5, pGBKT7-SBEⅡb-6and pGADT7-SUS-SH1is weak or they are can not bind with each other actually; pGBKT7-SBEIIb-2, pGBKT7-SBEIIb-4and pGADT7-SUS-SH1can bind with each other, but the binding ability is weak. The results of this study need to be detected by other method.