The Genetic Transformation Of Chinese Dwarf Cherry With RolC Gene Detection And Research On Function Expression

Chinese dwarf cherry (Cerasus humilis (Bge) Sok.), is a small shrub fruit trees which is widely distributed in northern China, the study is mainly about the molecular detection of transtormated rolC plant which named T1、T2、T3、T4、T5、T6. The plant is obtained by Agrobacterium-mediated and GUS staining positive. Using RT-PCR to detect exogenous rolC gene expression at the transcriptional level; the rolC gene by absolute real-time fluorescence quantitative PCR detection will got the copy number. Meanwhile, study on biological characteristics of rolC transgenic plants. The study results are follows:1.The GUS staining positive Chinese dwarf cherry and CK leaves as material. The rolC gene transcription By RT-PCR expression found:in all the6plant of rolC gene transformation, T1, T2, T4at the transcriptional level expressed, expanding a gain band with the the rolC same size strips, T3, T5, T6is no amplification of the corresponding strip. In order to detect the stability of the genetic expression of the rolC gene transformation.using asexual reproduction subculture seedlings by tissue culture and chilling stress subculture seedlings RT-PCR expansion. The results show that the rolC gene transformation of Chinese dwarf cherry asexual reproduction under low temperature stress, exogenous rolC gene stable inheritance and expression at the transcriptional level.2. The rolC gene quantitative PCR determination of exogenous absolute copy experimentally constructed plasmid standard curve, standard curve regression equation is Y=-3.068lgX+41. The correlation coefficient R2=0.986, dissolved unimodal curve detection results showed that the transgenic plants obtained by Agrobacterium-mediated transformation method, the type containing an exogenous gene copy number, the starting copy number of the gene in strain T3rolC up to3.38×106, followed by T2copy number is1.87x106, T5and T6in the copy number of the relatively low.3. Study on the transformation rolC plant and CK stem segments multiplication and rooting characteristics in different types and concentrations of medium, found the lines and the control of genetically rolC gene on the biological characteristics of the significant changed. T2and T4tissue culture seedlings cultured root. When access to various strains in the same rooting medium, T1, T2, T3, T4rooting rate very significantly higher than CK rooting rate, theT5rooting rate was significantly than CK, T6was not significant with CK. In root length, T1, T2, T4root length is extremely significant than CK longer;T3、T5significant than that of CK growth; T6and CK were no significant differences. Different hormone concentrations in differentiation medium, genetically rolC lines and CK showed a trend of increased and then decreased with the increase of the concentration of6-BA.when6-BA0.3mg/L, the multiplication coefficient are the highest; When6-BA greater than0.3mg/L, CK of proliferation rate was significantly higher than the transfomation of rolC strains. When the rooted plants after25days transplanting, CK、T5growth in plant height was significantly higher than other strains, after transplanting four months the CK plant height increased significantly in rolC transformate lines and the plant height of CK was very significantly than T1、T2、T4,and significant thanT3、T5、T6. CK section rooms significantly more than the transformation of rolC lines, the section length was not significant with T5, significant than other strains.