用于农杆菌转化的雪花莲外源凝集素(GNA)基因植物中间表达载体的构建

雪花莲外源凝集素(GNA)是一类能特异地和甘露糖残基结合的蛋白质成分,对人畜毒性极低,但对同翅目昆虫有强烈的抗性.为利用雪花莲外源凝集素对同翅目害虫的抗性,将雪花莲外源凝集素基因置于Ubi启动子和Tnos终止子的控制之下,插入pCAMBIA3300的SmaI位点,构建了可用于农杆菌转化的植物中间表达载体-pCUGAN-Bar.将pCUGAN-Bar电击转入农杆菌,得到了带有pCUGAN-Bar的农杆菌菌株,可用于小麦和水稻的转化工作.

Construction of a plant medial expression vector containing snowdrop lectin (GNA) gene for Agrobacterium tumefaciens mediated transformation

Many insect resistant genes with different pest resistant ability,such as Bt ( Bacillus thuringiensis) toxin genes,proteinase inhibitor genes and plant agglutinin genes,have been discovered and used in plant anti insect genetic engineering.Plant lectins are a kind of proteins that bind reversibly to specific mannose and can protect plants against insect and pathogenic bacteria.Lectins from snowdrop( Galanthus nivalis. GNA)have very low toxicity to higher animals,but have an effective resistant ability against diptera insects,such as wheat aphid and rice brown planthopper which are the most damaging pests in terms of crop losses.Studies revealed that GNA can binds to specific mannose of glycoprotein.Mannose is an abundant cell surface sugar present in glycoproteins that are implicated in a wide range of important receptor mediated cellular processes.The interaction between lectin and glycoprotein blocked the normal function of glycoprotein and futher impeded the biololgical function of cells.Although little is known about the insect resistant mechanism of letin,new unique biological properties have been observed.Those included:inhibitory activity against retrovirues and the ability to block Escherichia coli proliferation in the small intestines of rats.Recent studies discovered the functional form of GNA is a tetramer of identical subunits.The mature subunit is composed of 105 amino acids,which is generated from a prepeptide of 157 amino acids by post translational excision.For the purpose of the GNA application in plant anti insect genetic engineering,we constructed a plant medial expression vector,pCUGAN Bar,in which the GNA was under the control of Ubi promoter and Tnos terminator.Ubi promoter is the known most effective promoter of monocotyledonous plants and combined with the Tnos terminator it will ensure the effective expression of GNA in transgenic plant.Finally,the plasmids of pCUGNA Bar was electrically pulsed into Agrobacterium tumefaciens LBA4404 and the resulted Agrobacterium tumefaciens can be used for the genetic transformation of wheat and rice.