Construction Of Rnai Plant Expression Vector In Barley And Establishing The Efficient Regeneration System For Barly Immature Embryo

Barley has great development Potential as feed crop and main raw material for beer production in China. The study of improving barley strains had fastened on barley resistibility and quality. Protein has a great influence on beer barley.In order to reduce the protein content and improve the quality of beer barley. In this study, as the material Gansu Beer Barley No.4 which has a high promotion area in Gansu province.Hordein gene was cloned conserved sequence, RNAi expression vector was constructed which for further improvements through the theoretical basis of barley quality. A pair of primers was designed according to the released DNA sequence of Hordein gene. B2PF5'-CAACCATTTCCACAGCAACCACCAT-3', B2PR5'-GAAAGATAGAGTAGACGATTGCACG-3',PCR was employed to clone a 349 bp fragment(GP4) from Gansu Beer Barley No.4. Based on the structure of vector pHANNIBAL and pART27, GP4 was insert with forward and reverse according to the enzyme position. The sequence analysis showed that the cloned DNA shared 92% identity with the data. A RNAi expression vector of the Hordein gene, named pART27-pHAN-GP4 was constructed and the RNAi expression vector pART27-pHAN-GP4 containing the Purpose gene.Immature embryos grow so fastly that have a great important in building a regeneration system. Immature embryos of Gansu Beer Barley No.4 was used as tissue culture material. The effects of different medium and exogenous hormones on callus induction of Gansu Beer Barley No.4 were studied. The result showed that Gansu Beer Barley No.4 in IM2 has a high callus induction,the rate is up to 71%.The quality of callus from IM1 and IM2 was better than that from IM3;In the same medium, it is obvious that dicamba can increase the callus induction rate.Different differentiation media has different effects on differentiation rate, and the differentiation of DM1 is better than DM2;The research proved that Gansu Beer Barley No.4 was identified with higher performance both in induction of embryonic callus and plant regeneration. To lay the foundation for genetic transformation.