Construction Of RNA Interference Vectors Of Pin Genes Determining Grain Hardness And Genetic Transformation In Wheat

Wheat grain hardness is one of the most important traits that determines milling quality, and strongly affects ?our yield, starch damage and water absorption of dough. Grain hardness is mainly controlled by puroindoline a and b (PinA and PinB) genes located on the short arm of wheat chromosome 5D. Lacking or mutations of PinA and PinB result in harder endosperm. The functional research and transgenic application of Pin genes are of great importance to wheat quality improvement.In the present study, RNA interference was used to trigger efficient post-transcriptional gene silencing of Pin genes in order to gain reduced Pin protein expression level and hard kernel-texture in common hexaploid wheat. These would lay the foundation for futher study on puroindolines'biological function and their interaction mechanism in determing grain hardness. On the other hand, transgenic wheat lines obtained by RNA interference of Pin genes will provide hard-kernal germplasms for breeding lines with different end-use qualities.A PinB RNAi vector was constructed, which expresses a hairpin RNA structure containing specific PinB gene fragment. This vector, together with a PinA RNAi vector previously constructed by our laboratory, was used in three combinations (PinA RNAi vector/PinB RNAi vector/PinA RNAi vector+PinB RNAi vector) and was transformed into soft hexaploid wheat L88-31 via particle bombardment. Through tissue culture, a large number of regenerated wheat seedlings were obtained. Several positive transgenic lines were screened by PCR amplifications. The results of the present research thus provided with bases for further studying on the function of Pin genes and breeding new wheat varieties with high milling quality.