Optimization Of Genetic Transformation System And Dwarfing Functional Analysis Of GA2ox8 Gene And In Pyrus

Pear,woody deciduous trees of the genus Pyrus of the family Rosaceae,which is one of important fruits in China.High-density pear orchards on dwarfing rootstocks have become common,this has allowed pear growers to achieve higher production,lower cost and easier to mechanization than previously.Research on breeding and mechanism of dwarf rootstock lays the foundation for dwarf tree cultivation of pear.Genetic engineering is widely used to selective breeding,and it has advanced the understanding of many theoretical foundation.According to the basis of previous studies,the method to construct the system of pear leaves and callus were designed.And pear was used as material to study the function of GA2ox8 gene by transgenic technology.The main results are as follows:1.Study on genetic transformation system of pear leaves and callus.To improve the transient efficiency of pear leaves mediated by Agrobacterium tumefaciens,the effects of inoculation producers,carbon source of infection solution and p H value on the transfection efficiency of leaves were studied.Compare to ‘Conference' and ‘OHF 333',the number of blue point was the most under the condition of injury by the head of pipette tips in ‘Shanli'.The transient effieciency of pear leaves showed optimal GUS activity at p H 5.5 and an upward trend when p H was determined ranging from 4.5 to 5.5.However,different combinations of glucose and sucrose in the infection solution had no significant effect.In addition,‘OHF 333' pear leaves were unable to grow normally on regeneration media containing 80 mg/L neomycin or 5 mg/L kanamycin.In order to establish the genetic transformation system of ‘Starkrimson' callus,the optimum concentration of cytoknin(6-BA)and auxin(2,4-D)in the culture medium were 0.5 mg/L and 1.0 mg/L or 1.5 mg/L,respectively.And we also found that it negatively promote callus cell proliferation when the kanamycin and hygromycin were 25 mg/L and 2.5 mg/L in the medium,respectively.2.Functional analysis of PbGA2ox8 gene.The sequence of GA2ox8 gene in pear was found by searching related databases of NCBI.We isolated the PbGA2ox8 gene and the overexpression vector was successfully constructed.Amino acid analysis showed that PbGA2ox8 protein had DIOX?N and 2OG-Fell?Oxy domain,and it is similar with the GA2 ox in Arabidopsis and Rice.Phylogenetic analysis indicated that the selected GA2ox8 show high homology with Arabidopsis thaliana and Oryza sativa,especially it divided into the same branch with At GA2ox8.The subcellular location of tobacco and onion showed that the protein encoded by this gene was located in the nucleus and cytoplasm.In contrast with the wild type,the transgenic plant with overexpression of PbGA2ox8 gene exhibited more short stature,extremely short internode,and reduced leaves length and width.