Anti-Sense Genes Construction Of Key Enzymes In Starch Synthesis And Transformation In Wheat (Triticum Aestivum)

Starch,being the major form of storage carbohydrate in many crops,has many applications in food processing and other industries.Therefore,improvement of starch quality and quantity in major crops is extremely important. AGP,GBSS and SBE are key enzymes in the starch biosynthesis pathway and have a major influence on amylose and amylopectin content and ratio and thus on starch quality.The ultimate goal of this study is to be able to manipulate the starch synthesis pathway,such that wheat starch with targeted starch content or amylose/amylopectin compositions can be produced at will.Our initial strategy is to reduce the expression of key genes in wheat endosperm starch biosynthesis pathway by antisense inhibition.A series of antisense constructionss of different lengths have been produced in order to determine the length of the constructions which gives optimal down-regulation.Full-length of antisense AGP/GBSS/SBE coding gene replaced ipt gene of pCAMBIAl301-MAR-LI and sequence deletions were performed by using appropriate enzyme digestion and ligation.Al1 these constructions have MAR sequence flanking the foreign gene,which should ensure better integretion and more stable expression;Use of wheat LMW Glu 1D1 promoter sequence should result in strong ,timely and endosperm-specific expression of foreign genes.Wheat (Cultivar Zhengmai 9023)young embryos(12-14 days after anthesis)have been transformed by particle bombardment using the above constructs. We have got Hyg-resistant pantlets through tissue culture and Hyg selective(20mg/l).We have transformed 3600 wheat embryos and got 98 regenerated plantlets.We have confirmed these regenerated plantlets by PCR analysis and got 22 trangeneic plantlets(thetransgenic efficiency is 0.61%),which indicates foreign genes have been successfully integrated into wheat genome.