Combining Ability And Transient Expression Analysis Of Trangenic Bt Gene In Rice

In recent years,rice production has been facing the hazards of pests and diseases,including Chilo suppressalis,Chilo suppressalis most seriously,mainly due to the current in rice has not been found resistant to stem borer varieties,anti-stem borer gene mining and positioning has been slow.At present,the main strategy is to transfer the Bt gene into rice through transgenic technology to obtain a transgenic line with insect resistance.Further understanding of the expression pattern of Bt gene in transgenic lines and evaluation of its resistance to stem borers is beneficial to understand its pest resistance characteristics and provide a reference for guiding the application of rice production.However,there are always many worries about genetically modified technology,academia and society.Whether gene insertion can achieve the safety problem caused by other unanticipated effects while gaining the target trait is one of the main problems.So far,the unanticipated effects of GMOs remain at the phenotypic level,and seldom involve molecular level analysis.Therefore,it is necessary to carry out studies on the unanticipated effects of GMOs at the molecular level.1?35 combinations were made by NCII incomplete diallel cross,and the parents with strong combining ability and excellent hybridization combinations were obtained by analyzing the combining ability.GCA analysis showed that the GCA effect of Hong A,Quan 9311A,Changhui 121T and Changhui T025T was high.The analysis of the SCA effect of hybrid combinations showed that the average GCA value of Quan 93A/Changhui T025T Nine agronomic traits had the best comprehensive SCA effect value;the highest SCA effect value per plant was obtained with 67A/R205T;the lowest plant height SCA effect value was found in Quan 9313A/Changhui 606T;the highest SCA effect was 843A/ChangHong 891T;SCA effect value of empty grain number 843A/Changhui 121T was the lowest,seed setting rate SCA was the highest.Tsuen 9311A/Chang Hui T025T,67A/R205T,Hong A/Chang Hui 121T,843A/Changhui 121T as a good hybrid combination.2?The expression patterns of cry1C and cry2A in cry1C and F1 were studied by Real-time fluorescent PCR.The gene expression of cry1C and cry2A in new insect-resistant restorer line and F₁ was as follows:The expression levels of cry1C and cry2A in leaves and stems increased gradually at tillering stage and young panicle differentiation stage,reached the peak at heading stage,remained high at filling stage and rapidly decreased at maturity stage.The expression levels of cry1C and cry2A gradually increased from the young panicle differentiation stage until the peak reached the heading stage and then decreased rapidly after the filling stage,indicating that the expression of cry1C and cry2A tended to go up and down after the whole growth cycle,and the expression process The expression of cry1C and cry2A in tillering stage to mature stage was in leaf tissue>stem tissue>ear tissue>endosperm tissue,indicating that cry1C and cry2A genes were mainly expressed in the main harms of rice leaf roller and rice stem borer;cry1C and cry2A expression levels in parental restorer lines were much higher than those in F1 hybrids,indicating that some forms of dose-effect may exist for cry1C and cry2A;cry1C,cry2A was lower than that of monovalent pest-resistant rice,and the expression pattern was complicated,indicating that cry1C and cry2A may have complex and diverse interaction effects.There were some differences in the expression levels of cry1C and cry2A between different varieties,indicating that the expression of insect-Rice genetic background is closely related.3?Elisa detected the spatiotemporal expression of Bt protein in new insect-resistant restorer lines and F₁,and calculated the lethal rate of stem borers,new insect-resistant restorers and F₁ in new insect-resistant restorers and F₁ The expression of Bt protein and the mortality of stem borers were as follows:The expression of Bt protein was in the range of 1.82ug/g⁴.95ug/g for the expression of cry1C protein at the heading stage and at the tillering stage,the expression range of cry2A protein 6.52ug/g⁷.39ug/g.The lethal rates of stem borers of the second stage to the stem borer at the second instar larvae were all increased from 75%to 85%at the heading stage and at the tillering stage.The Bt protein expression The results showed that the expression of cry1C protein varied from 0.16ug/g to4.95ug/g and the expression of cry2A protein varied from 0.31ug/g to 7.39ug/g at heading stage and heading stage,The lethal rates of stem borers and stem borers of the second-stage rice stem borers ranged from 75%to 95%at heading stage.The lowest lethal threshold of cry1C protein content in leaves was less than or equal to 2.2ug/g,cry2A protein content of the minimum lethal threshold?6.52ug/g;CrylA protein content in the stem to the lowest Threshold?0.16ug/g,the minimum lethal threshold protein content cry2A?0.31ug/g.4?The flanking sequences were amplified by reverse PCR,and the amplified products were enriched by nested PCR.The products were sent to Genomics for first-generation sequencing and compared with Nipponbare genome.As a result,it was found that flanking sequences of cry1C The size of 1124bp was located on chromosome 11 of rice.The size of cry2A flanking fragment was 1628bp,which located on chromosome 12 of rice.5?The genome-wide background analysis of 800 SSR primers in the experimental and control groups was 95.60%?88.43%?93.52%?94.44%,respectively,in Changhui 121T,Changhui 891T,Changhui T025T and R205T,four restorers have basically restored the main characteristics of the reincarnated parents.6?According to the insertion sites of cry1C and cry2A,we designed the cross-intronic primers of flanking genes.The real-time PCR was used to detect the expression of 9 genes within 100Kb upstream and downstream of the insertion site of exogenous gene.The results showed that in experimental group and control group,There was no significant difference in the expression levels of 9 flanking genes between the upstream and downstream of cry1C and cry2A insertion sites.The results showed that there was no significant difference between photosynthetic characteristics and yield phenotypes in the experimental and control groups.The results further indicated that the cry1C and The insertion of cry2A did not change the expression level of flanking genes.The transgenic rice did not produce any unexpected effect on the traits.