Study On Agrobacterium-mediated Genetic Transformation Of Indica Rice With SBE Gene

Starch distributes among different crops universally and it can be chemically fractionated into two types of glucan polymer: amylase and amylopectin. The starch structure can be changed by varying the ratio between the two forms, and furthermore, the quality and economic value of crops can be influenced. Initiating, elongating and branching of chain are three key steps in the starch biosynthesis, and starch branching enzyme(SBE) is known as the key enzyme in amylopectin chain length and branch points. Thus, the quality of crop starch can be improved by transforming the sbe gene into crop through genetic engineering.In this study, three expressing plasmids of maize SBE gene (sbeâ… , sbeâ…¡a, sbeâ…¡b) were constructed and introduced into the embryogenic calli of indica rice variety Kangwenqingzhan by an Arobacterium-mediated transformation system. The results are as follows:(1) The regeneration system for Kangwenqingzhan was established. N_1.5S, which contained 5.0mg/L Na₂S₂O₃ and 1.5mg/L 2,4-D was screened from five candidate calli-inducing media ((1/2)N_1.5S; (1/2)NS; N_1.5S; NS; NB), and was best for the calli induction and subculture of the variety. Then the NNK₂ with 5 ml/L KT and 0.5 ml/L NAA was screened as the optimal differentiation medium according to the experiment on different concentrations of KT.(2) Three expressing plasmids of maize SBE gene were constructed. Target genes pLf8/ PA2-4/ pMAâ…¡, which were cloned in pBluescript plasmid, were previously subcloned into pBBR1MCS plasmid and then the obtained target genes pLf8/ PA2-4/ pMAâ…¡with Kpnâ… /BamHâ… restriction enzyme sites were inserted into the corresponding positions of the expression vector pGA1621 and the plant expression vectors containing target SBE genes were established. The recombinant plasmids were transformed into Agrobacterium tumefaciens by freezing-thaw method and the target strains were further confirmed by restriction enzyme digestion.(3) Transformation system was optimized. By corresponding experiment, OD₆₀₀ = 1.1 was thought to be the best concentration of co-culture, 40mg/L of hydromycin for screening, and 250 mg/L of Timentin as bacteriostat. With the high frequency Agrobacteriums-mediated rice genetic transformation system and the Agrobacterium tumefaciens clones of pLf8/PA2-4/pMAâ…¡, the resistant calli were obtained and GUS activity and PCR detection indicated that in the calli of pLf8/PA2-4/pMAâ…¡, 4/2/2 positive resistant calli were acquired and the transformation efficiency were 1.25%, 1.10%, 1.04%, respectively.