渗透胁迫相关基因高通量筛选技术体系的建立

Construction of the High Throughput Technology for Screening Osmotic Stress Relevant Genes

随着功能基因组学的发展,在基因组水平上获得了大量渗透胁迫相关基因。农杆菌介导的超量表达是筛选和鉴定这些基因功能最为常用的技术。以植物表达载体pBI121为基础,在不改变其氨基酸序列的基础上通过定点突变消除了其上与质粒复制和稳定性有关的trfA基因(X00713)内部的SfiI酶切位点,并在表达单元CaMV35s启动子和NOS终止子之间引入了SfiIA和SfiIB位点,改造成通用植物表达载体卡盒pBHT-5。该载体卡盒可直接用于连接Clontech SMARTTM技术构建的cDNA文库,提高了大规模构建cDNA文库基因植物表达载体的工作效率。为高通量的筛选与鉴定基因功能,从大量的渗透胁迫相关基因中筛选出具有独立知识产权的、对植物抗渗透胁迫起重要作用的新基因奠定了坚实的基础。

With the development of functional genomics, high throughput analysis of genes＇ function has been the mainstream of research, and exogenous gene＇s over expression via Agrobacterium-mediated transformation is the most commonly used method in gene functional analysis. The versatile plant expression vector cassette named pBHT-5 was constructed by the method of site-specific mutagenesis based on pBI121. First of all, the restriction enzyme SfiI recognition site in trfA gene （X00713） which was relevant to plasmid replication and stability was replaced without changing its amino acid composition. And then the SfiIA, SfiIB sites were added between promoter CaMV35s and terminator NOS. The versatile plant expression vector used to construct plant expression vector containing the full- length genes cloned technology, which will raise the efficiency of vector construction. The result will provide te can be directly ontech SMARTTM basis of new genes＇ high throughput screening and functional analysis, then get the new genes functioning in plant osmotic stress resistance.