Using Rnai Technology To Inhibit The Synthesis Of Grain Ppo Improved Wheat Flour Whiteness

Flour whitness is one of the most important characters in weighing flour quality,which is controlled by multiple genes and environmental effects.Among them,polyphenol oxidase (PPO),which was encoded by ppo ploygene families,is the key effector for noodles(paste) browning.It is hard to produce useful cryptic mutant for breeding by traditional breeding mathods,such as natural and manual mutation.In order to overcome the restriction of traditional breeding,genetic engineering technology has been applied in improving and breeding importing genes to gain new character or knock out unfavorable genes.Post transcriptional gene silencing(PTGS) were widely used to down-regulate expression of unfavorable genes in the plant metabolic process.As a very efficient method of PTGS,plant RNA interference(RNAi) technology has been widely used to silence unfavorable gene in recent years.In our study,the RNAi vector of kernel PPO gene was constructed,which was driven by endosperm specific expression promoter of high molecular weight glutenin gene 1Dx5,and named as pBAC47P+ppoIR.The constructed RNAi vector and selection vector pBAC35Sbar were used to co-transform immature embryos and immature calluses of common wheat cultivars- Jinghua No.1 and Zhongyou9507 with biolistic microprojectile bombardment (PDS-1000/He).Following screen of PPT resistance on selection medium and differentiation culture,27 transformed plantlets were regenerated.PCR and Southern Blot analysis of transgenic plants proved that the foreign target gene was integrated into wheat genome of 12 lines of T₁ generation.Semi-quantitative RT-PCR and Northern blot analysis indicated that ppo gene was well down-regulated in offspring of different lines.Isozyme electrophoresis and enzyme activity analysis of transgenic wheat T₂ generation kernel showed that PPO activity was remarkably reduced in 6 lines.Our research indicated that the expression of endogenesis ppo gene in kernel can be well controlled by RNA interference of ppo multiple genes.