Genetic Transformation Of Artificial Mutants Of Milling-Quality Related Gene Pina In Wheat (Triticum Aestivum L.)

PINA and PINB are very important quality proteins and play key roles in determining the wheat grain texture hardness, and also were reported to have potential antimicrobial activities. PINA contains a unique tryptophan-rich domain, exhibiting activity against gram-positive and gram-negative bacteria in vitro. Previously, several mutations of PINA, which contain different number of copies of Trp-rich domain, were constructed in our lab. In the present research, in order to investigate the effect of PINA mutants on wheat grain hardness and antimicrobial activity, expression vectors containing these mutant genes driven by endosperm specific promoter 1DX5, were constructed and transformed into durum wheat Ofanto embryo via particle bombardment. The main results are as follows:(1) pLRPT, which was constructed on the basis of pBluescript, containing an endosperm peomoter 1DX5 promoter , was used. Pina mutants ABBC and ABBBC were inserted into pLRPT under the control of 1DX5 promoter. pLRPT-ABBC and pLRPT-ABBBC were transformed to durum wheat Ofanto Embryo by Particle bombardment.(2) The wheat tissue culture condition were optimized , and a large number of regenerated wheats seedlings were obtained by using this system.(3) Two pairs of primers designed based on pina sequence, were used to identify the transgenic seedlings, and a total of 13 positive regenerated seedings were indentified.(4) According to the solubility of PINA in TX-114, PINA proteins were extracted by phase separation method. The expression of mutant proteins were confirmed by SDS-PAGE.The results thus obtained provided with the basis for series of further experiments on the effect of these mutants on grain hardness and their antimicrobial activity.