Cloning, Expression And Vector Construction Of A Novel Gene MhCDPK And Etablishment Of Regeneration Systerm Of Malus Hupehensis Rehd

Calcium dependent protein kinases (CDPKs) are a kind of serine/threonine protein kinases that are present in plants. CDPKs play an important modulating roles in the plant calcium signal transduction as well as growth and development. Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang (Chinese name is PingYi TianCha, PYTC) is one kind of excellent rootstocks of apple species. In this thesis, a series of studies based on the materials of PYTC have been conducted on the gene isolation, sequence comparison, expression analysis in stress and the establishment of regeneration system. The main results are as follows:1. According to the homologous sequences from other plants, degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from PYTC. By RT-PCR, we isolated a CDPK gene and named MhCDPK, with the accession number of GeneBank EF519915. The MhCDPK cDNA is 1999bp long with an open reading frame of 1701bp encoding for a protein of 566 amino acids. The predicted MW and PI were 62.9kD and 5.26. The predicted MhCDPK protein contained all the structural characteristics of reported CDPKs and there were four domains of a variable domain (1-108), a kinase domain (109-366), an autoinhibitory domain (367-402) and a calmodulin-like domain (403-566) from N- to C- terminal end. Sequence comparison showed that MhCDPK had a high homolog of other plants.2. The real time fluorescent quantitative PCR showed that the MhCDPK gene expression increased under the high salinity, drought and heavy metal stress.3. In order to study the function of endogenous MhCDPK in plants, construction vectors pBI121-MhCDPK containing full-length cDNA of MhCDPK gene in sense and pBI121-antiMhCDPK containing 3'segment in antisence orientation driven by the constitutive cauliflower mosaic virus 35S promoter were assembled and introduced into tobacco, and we gained transgenic plants in antisence. In order to study the subcellular localization of MhCDPK protein, we also constructed the vector of pBI121-gMhCDPK-GFP, which then was introduced into onion inner epidermis, the result showed that MhCDPK protein localized in the cytoplasm.4. The stems of seedlings were used for suitable explants. The medium MS+0.5mg·L-1BA+0.2mg·L-1ZT+0.1mg·L-1NAA was more suitable for plantlet propagation with proliferation coefficient of 15.6. The medium 1/2MS+0.5mg·L-1IBA +0.3 mg·L-1NAA was more suitable for rooting, and rooting rate reached 85.6% and mean number of roots was 15.5.5. 6-BA, ZT, NAA and dark treatments could increase adventitious shoot differentiation. The best result was achieved in the medium MS+6.0 mg·L-1 BA +0.5 mg·L-1 NAA + 0.2 mg·L-1 ZT+ 6.0 mg·L-1 SNP, in which the percentage of adventitious shoot regeneration were 67.8% after dark treatment for 15d.6. The proliferation of PYTC was promoted significantly by applying 20μmol·L-1 SNP. The differentiation and regeneration of adventitious shoots from cotyledons or leaves increased significantly when 20~30μmol·L-1 SNP was supplied to the regeneration medium. Adventitious shoots regeneration frequency from cotyledons was higher than that from leaves at the presence of SNP. The best result for rooting was achieved in the half-strength MS medium containing 75μmol·L-1 SNP.