Reasearch On Cell Death And Expression Of Anti-apoptotic Genes Of Malus Hupehensis New Roots

Malus hupehensis(Pamp)Rehd.var pinyiensis Jiang is a parental stock of apple which isonly found in China. In this study, combined with the Apple Genome Databasehttp://genomics.research.iasma.it/, the MhBI-1gene was cloned and the character and functionof MhBI-1and the protein that it encoded was analyzed. The sequences of MdBI-1s were alsogot from the Apple Genome Database and their expression patterns were investigated indifferent varieties, organizations and growth periods and under42℃treatment. The senseand anti-sense expression vectors of MhBI-1were successfully constructed. The expressionpatterns of anti-apoptotic genes: MhBI-1, MhBAG and MhHSP70of Malus hupehensis newroots (absorbing roots and extensive roots) were studied under normal growth,2,4-D andtreatment of alternation of wetting and drying.1) The full CDS of MhBI-1gene was cloned which had747bp long and encoded248amino acids. The accession number of MhBI-1in GeneBank is KC456613.2) The MdBI-1s family of Malus domastic was got and the phylogenetic tree of thefamily was constructed. The result of the semi-quantitative RT-PCR showed that theexpression patterns of the MdBI-1s showed a similarity. The expression levels of MdBI-1s instem was found to be higher than that of root and leaf and Malus sieversii got a higherexpression levels than Malus baccata, Malus hupehensis and Malus micromalus. In seedlingstage of Malus hupehensis roots, as the growth, MdBI-1s present downward trend after risingfirst, reached its peak at six-leaf stage. At42℃, MdBI-1s expressed with the intensified ofstress level and declined after rising first which reached the peak at2h after the treatment.3) The pET-30a-MhBI-1was constructed and expressed in E. coli and the protein ofMhBI-1encoded is about25kD.4) The pBI121-MhBI-1vector was successful constructed in this study and transformedArabidopsis thaliana col-0using the floral dip method via agrobacterium mediated and the T0seeds had been harvested.5）Under normal condition, the changes of cell death rate, root activity, ATP content andcaspase3/7-like activity of absorpting root presented a degree of volatility.The absorbing rootsshowed higher level of changing frequency than the extensive roots. And the expression ofMhHSP70, MhBI-1and MhBAG in absorbing roots showed the same changing trend. All the changing trends above reflected the character of absorbing roots which have short life cycleand high level of cell metabolic activity. While changes of extensive roots were relativelystable, showed "terraced changing trends", were inhibited after the actived phase. In the29days of normal condition, the MhHSP70, MhBI-1of extensive roots were in suppressed statehowever the MhBAG was induced and reached its peak at17d, which may related to thegrowth state of extensive roots.6) After the2,4-D and wetting and drying alternation treatment, the new roots(extensiveroots and absorbing roots) showed a increasing trend of cell death rate and caspase3/7-likeactivity and a decreasing trend of the ATP content and root activity. The anti-apoptotic geneswere induced by the two treatments. High expression levels of antiapoptotic genes werehelpful to reduce cell death rate. When at expression peak, the increasing ratio ofantiapoptotic genes of extensive roots were significantly higher than absorbing roots,suggested that the extensive roots had better ability to resist stress. At the early time of2,4-Dtreatment, the amplitude of cell death rate increased and root activity decreased of absorbingroots were higher than the extensive roots and at the treatment of alternation of wetting anddrying, after watered, the change of cell death rate of extensive roots lagged behind absorbingroots which showed that the absorbing roots were more sensitive than the extensive roots to2,4-D and the treatment of alternation of wetting and drying.