Transformation Of Lipid Trenafer Protrin-Like Antimicrobial Protein LTP Gene From Leonurus Japonicus Houtt Into Wheat

Wheat is one of the important food crop.The requirement for wheat is rising with the development of economy and society and the growth of world population.Wheat disease is one of the important factors that impact the quality and quantity of wheat. And 10% to 50%production reduce are cause by disease each year.Therefore,to cultivate the wheat with high disease resistance is very important. Transgenic breeding had became the important method for wheat resistant breeding because it not limited by the distance of genetic relationship,so that expanded the range of available exogenous resistance genes and can effectively shorten the breeding cycle.LTP gene is provided by professor Pei Yongyan in Southwest University and come from the seeds of Motherwort (Leonurus japonicus Houtt). It belong to lipid transfer protein-like antimicrobial protein gene,its encoded protein with broad-spectrum antimicrobial effect in vitro and over-express LTP gene can improve plant disease resistance.At the same time, by transferred the LTP gene into tobacco and tomato, the resistance for brown spot and bacterial wilt of the obtained transgenic tobacco significantly increased and the resistance for Verticillium wilt and early blight of the obtained transgenic tomato also obviously improved. This experiment is to further optimize the genetic transformation system of wheat and transfer the LTP gene into wheat.So that to obtain the wheat whose resistance steadily increased.1 screen the transformation conditions Find out that 100 mg/L Timentin is the optimal concentration for inhibiting the Agrobacterium by analyzing the effect of Agrobacterium inhibitor (cefotaxime and Timentin) on wheat differentiation, at that time the differentiation rate is 57%. Find out the optimal concentration for PPT screen is 3 mg/L in subculture, 2 mg/L in differentiation, 5 mg/L in rooting and 10 mg/L in painting the leaves though screening the optimal PPT concentration in subculture, differentiation, rooting and painting the leaves.2. LTP gene transform wheat In order to transfer the LTP gene into wheat,expression vector pAHC25-LTP, pBI121-LTP and pCAMBIA1302-LTP were successful constructed and Northwest 1376, Xiaoyan 22 and Kelong 199 were used as receptor materials, pollen tube pathway,Agrobacterium-mediated method and particle bombardment were used for transformation. In pollen tube pathway,1670 seeds and 31 resistant plants were obtained by using pAHC25-LTP plasmid to transfer 3000 wheat flowers,which setting rate is 55.67% and resitant rate is 1.856%;1731 seeds and 43 resitant plants were obtained by using plasmid pCAMBIA1302-LTP to transfer 3000 wheat flowers,which setting rate is 57.7% and resistant rate is 1.484%.In particle bombardment transformation,12869 caulls were transferred and 203 PCR positive plants were obtained, transformation rate is 1.87%. Initially proved that LTP gene have been transferred into wheat.Find out the resistance for wheat stripe rust of the T1 genreation of the transgenic plants improved by inoculated wheat stripe rust on them,And further experiments are still in progress.