Studies On Transformating HVAI Gene Into'Pingyitiancha'(Malus Hupenensis Borkh)

It is well known that water deficiency has become a critical ecological problem progressly. Compared with developed country, it is worse in china. Fruit production in our country will face with the problem of water deficiency as well and the focus should be put on cultivar with drought resistant in fruit breeding. 'Pingyitiancha'(Malus hupenensis Borkh) is an important apple storck with many good characteristics, including waterloggig resistance, cold resistance, salt resistance and so on. However it is poor in drought resistance, which has become a key factor limited its application. The three group gene-HVA1 come from barley was transformed in to 'pingyitiancha'mediated by Agrobacterium .tumefaciens and transformed regeneration plants were obtained in this research. Section 1: The HVAI gene cloned from plasmid containing it by PCR with high fidelity pfti Taq. It was ligated between BamHI site and Sad site in PUC 118 vector. Identified by electrophoresis , digested with BamHI and Sad and PCR, it is confirmed that HVA1 gene had been ligated into PUC1 18 vector. Through nuclear sequence detecting, it is confirmed that the HVA 1 gene cloned in this research is 703bp,its nuclear sequence and the amino acid sequence encoded by it is identical to the sequence reported by homology with PCGEN7E program. PBHA vector obtained by ligation the 703bp fragment frOm recombinant plasmid digested with BamHI and Sad and 11kb fragment from pBIl2l plasmid digested with BanIHl and Sad. It was confirmed that the pBHA vector had constructed successfully by identified by PCR and digested with BamHL and Sad.. Section II :The pBHA vector was introduced into A.tum. LBA4404 by triparental mating and the binary vector was obtained. It is confirmed that HVA I gene had been inserted into T-DNA by in situ hybridization The effects of variety concentration of Kam .. Cef and Carb on differentiation of 'pinyitiancha'shoot apex were studied. The effects of variety preculture time, activate medium of A.tum, concentration of Ca, inoculation time, treated with AgNO3 and PVP or not, delay selection time on transformation frequency were studied as well. The results demonstrated that using 20Omg.LCef, 8 mg.L1Kam. GCJ8 activate medium, preculture 2 days, 5 X Ca concentration in preculture medium and coculture medium, inoculation I mineture,4mg li1 AgN0 and 0.7% P\P, delay selection 5 days will obtain the highest transformation frequenc Using 'pingyitiancha'shoot apex, mediated by A.tum as above system, the HVAIgene, was transformed into pingyitiancha and obtained Kam resistance rerzeneration plants, it is confirmed that 6 plants of them is transformation plants by identified by PCR and dot blot...