Transformation of HVA1 Gene Into Malus hupenensis var. `Pingyitiancha'

Abstract

Malus hupenensis var. `Pingyitiancha' is an important apple stock with many good characteristics, including waterloggig resistance, cold resistance, salt resistance and so on. The three group gene-HVA1 come from barley was transformed into `Pingyitiancha' mediated by Agrobacterium tumefaciens and transformed regeneration plants were obtained in this research. The HAV1 gene cloned from plasmid containing it (offered by Dr. Guo Weidong) by PCR with high fidelity pfu Taq DNA polymerase. It was ligated between BamH 1 and Sac 1 site in PUC118 vector, and identified by electrophoresis after digested with BamH 1 and Sac 1. Through nuclear sequence detecting, it is confirmed that the HAV1 gene cloned in this research is 703bp.This fragment was ligated with 11kb fragment from pB121 plasmid and constructed pBHA vetor. The pBHA vector was introduced in A.tum LBA4404 by triparental mating and the binary vector was obtained. It is cinfirmed that HVA1 gene had been insert in T-DNA by in situ hybirdization. Using `Pingyitiancha' shoot apex, mediated by A. tum. System, the HAV 1 gene was transformed into the plant. Kam resistance regeneration plants were obtained, 6 of them were confirmed as transformation plants by PCR and dot blot.