Construction And Transformation Of Antisense Expression Vector Harboring Puroindoines Genes And Genetic Characterization Of Transgenic Wheats With Over-expression Vector

Grain hardness is an important factor in determining wheat end-use quality, and is one ofthe most important characters in wheat quality improvement. Two major genes controllinggrain hardness,Pina and Pinb, was located at the short arm of chromosome 5DS. Theobjective of this study is to further understand the function of puroindoline genes throughtransgenic plants and provide information for understanding the formation of kernel textureand improving grain hardness throught genetic engineering.Antisense expression vector pAHantipA and pAHantipB,harboring the reverse sequenceof Pina and Pinb, respectively, were constructed using pAHC25, as basic plasmids, whichharbors the ubiquitin promoter and selection gene Bar. It can be used in wheat genetictransformation. Nine transgenic plants with antisense Pina and five transgenic plants withantisense Pinb were obtained via pollen-tube pathway with Jing411 as receiptor. Bastaresistance test and PCR analysis indicated that the transgenic plants with foreign Pina andPinb gene were obtained with the efficiency of 1.4% and 0.9%, respectively. Southern dotblot analysis further confirmed the result.At the same time, the gramineous high efficient expression vector pUBPa and pUBPbharboring Pina and Pinb gene were transferred into wheat variety Zhongyou 9507(Pinb-Dlb)via pollen-tube pathway. Basta resistance test, PCR and Southern blotting analysis indicatedthat the transgenic plants with foreign Pina and Pinb gene were obtained with the efficiencyof 2.2% and 2.0%, respectively. SDS-PAGE analysis showed that different friabilin level wasobserved in different transgenic lines.