Pest Resistance Assessment Of An Engineered Gene CryNGc In GM Rice

Nearly half of the world's population depends on rice as their staple food.Rice is one of the most important food crops in China.However,pests are more serious in recent years with stem borer as the main pest that seriously affects the yield of rice and restricts stable development of agricultural.The traditional chemical pest control in rice production is of high cost,environmental pollution and the increase in pesticide tolerance and other negative impacts.Bacillus thuringiensis(Bt)is a gram-positive bacterium that produces the insecticidal crystal protein(ICP)of specific toxic to Lepidopteran insects.Bt as insecticide is a most popular microbial insecticide being extensively utilized as environment-friendly biological pesticides.With the development of plant genetic engineering,genes encoding these insecticidal crystal proteins were transferred to rice becoming a new strategy for rice borers control.Following cultivation of the genetically modified(GM)Bt plants,pest resistance occurs providing that exploration of new Bt proteins is of great interest to scientists.By means of genetic screening and molecular modification,our team selected the Cry1 Ab protein as a major donor protein that has resistance to Lepidoptera insects.They selected Cry1 Gc protein which was exchanged domains ? to Cry1 Ab protein's domains ?,and got a new hybrid protein,named CryNGc.Expecting hybrid protein that engineered by different pesticidal proteins can increase crop resistance to insects and expand the spectrum of insect-resistant.As part of the systems research,in order to prove whether CryNGc protein can be expressed successfully in crops and have resistance to Lepidoptera insects,the cryNGc gene encoding hybrid protein that engineered was tranformed to Jijing 88 of Jilin province predominant japonica in this study.By nucleic acid testing,protein quantification and insect-resistance identification of GM rice to get a new anti-stem borer GM rice material.The main results were as follows:1.Constructing the plant expression vector pTF101.1-ubi-NGc.With the method of agrobacterium-mediated genetic transformation,the cryNGc gene was transferred into Japonica rice Jijing 88.After Screening,15 resistant plants were obtained.2.PCR,Southern blot and RT-PCR detection of GM rice proved that the exogenous gene cryNGc had been integrated into the rice genome in the form of a single copy,and could transcript normally.3.With the method of ELISA,CryNGc protein could be detected in different organs at tillering and filling stage in the T1 transgenic line,and their CryNGc protein content were obviously higher than non-transgenic materials.The different organs in the same grow stage and the same organ in the different grow stages were different in CryNGc protein content.CryNGc protein content in leaves was highest.4.cryNGc gene expression analysis showed that exogenous gene cryNGc had been successfully expressed in T1 GM rice.The cryNGc transgenic rice' stems and leaves had no obvious symptoms of pests in the laboratory rice stem borer resistance identification.The mean adjusted mortality of borers was 96.16% and 95.12% in GM rice's leaves and stems.The T1 GM rice grew normally in the field rice stem borer resistance identification.There were no obvious symptoms of pests in the transgenic line.The GM rice's susceptible rate was 3.95%,significantly lower than non-transgenic rice.According to insect evaluation criteria,it can be concluded that cryNGc transgenic rice have high resistance to rice stem borer.In conclusion,the experimental results show that: the exogenous gene cryNGc had been integrated into the rice genome in the form of a single copy,and could transcript normally.Also it could be expressed successfully in GM rice,and had high resistance to rice stem borer.