Construction Of Wheat Transformation Vector Of Gene Determining Grain Hardness For Rna Interference

The hardness, or texture of wheat grains is a primary determinant of their milling quality and end-use quality, thus their international commercial value. Because Puroindoline a and b (PinA and PinB) , locating on wheat chromosome 5DS, represent the molecular-genetic basis of grain hardness, their functional research and transgenic application are of great importance to wheat quality improvement. Because of the existence of wildtype functional PinA and PinB in common wheat (Triticum aestivum L., AABBDD, 6n = 42), grain texture is soft. Many studies suggest individual yet co-operative modes of action of the PIN proteins in determining kernel texture. Moreover, recently, direct physical interaction between PINA and PINB has been reported. Generation of transgenic wheats with the silencing of either PinA or PinB by RNA interference is of much value to elucidating their biological functions, and to making useful germplasm with hard kernel texture in common wheat.In this study, an RNAi construct for PinA, which expresses a hairpin RNA structure, has been made. We cloned a 166-bp segment in the sense orientation between restriction sites Bam Hâ… -Speâ… and in antisense orientation between restriction sites Xbaâ… -Sacâ…¡, with a intron as a linker. The RNAi construct was made in the wheat transformation vector pAHC25, in which the expression of the transgene is driven by Ubi-1 promoter. This provides a basis for working out the mechanism of the co-operative and interdependent roles of puroindolines.