The Construction Of Low-temperature Induced Site-specific Recombinant Expressiong Vector And Wheat Transformation

Selectable marker genes play a vital role in plant genetic transformation,using for selection of positive transformants.In the later stage of transformation,it has no use,adversely,marker gene in transgenic crop would increase security risks.The completely effective way to solve this problem is marker gene deletion.The induced site-specific recombinant system is the most effective choice which has great application prospect.This study constructed a low-temperature induced plant expression vector pXL5523-fwcs-RDA using Par/MRS site-specific recombinant system,then used gene gun and agrobacterium transformate wheat(M19)immature embryo callus and mature embryo callus respectively.Finally the transgenic plants were detected for target gene PYL5 related properties.The total results obtained are as follows:(1)Construction of low-temperature induced plant expression vector using ParA/MRS system and wheat transformationThis study constructed low-temperature induced plant expression vector pXL5523-fwcs-RDA containing drought-resistant gene PYL5,in which the recombinase ParA gene was induced by cold-induced promoter wcs120 derived from wheat genome.Then using gene gun and agrobacterium to transform wheat(M19)callus,obtaining 6 and 7 positive transgenic plants respectively,after PCR analysis,3 of 6 plants from gene gun transformation have finished gene deletion.This study laid a foundation for further research on marker-free transgenic plant through ParA/MRS system.(2)Drough-resistance analysis of positive transgenic plantsThe analysis of drought-resistance properties showed that,compared with non-transgenic wheat,transgenic plants containing PYL5 gene showed higher drought tolerance:after drought treatment,transgenic wheat wilted slighter than control,and showed faster recovery after re-water;during the treatment,the decrease degree of chlorophyll and relative water content are all lower than control,adversely,the accumulation of soluble sugar is higher than control.Besides,under high moisture condition,transgenic plants showed growth retardation,resulted in lower shoot and root extention speed.This study preliminarily proved that transgenic plant with PYL5 gene have high drought tolerance,and could lay the foundation for the research of wheat drough resistance.