| With the increasing progress of society,rice cultivation method has changed radically,which realized the transformation from labor-dependent intensive type to the process of no-tillage seedling-throwing,direct sowing and mechanized production,and with it,herbicides has been widely used to solve the more serious weed hazard problem.The hybrid rice is recommended by the Food and Agriculture Organization of the United Nations as the preferred technology for ensuring world food security.However,the complicated seed production process and high labor cost are the obstacles to the sustainable development of hybrid rice and its introduction to the world.Therefore,breeding herbicide-resistant rice varieties can reduce the effects of herbicide application on rice growth and solve the high cost and purity problems of hybrid rice seeds,which has important practical significance.In this study,imidazolinone herbicides and glyphosate,two non-selective herbicides with nuclear single-gene controlled target-site resistance（TSR）,were selected as resistant screening herbicides.Through a combination of natural variation analysis,chemical mutagenesis treatment,and directed evolution based on protein structure,a imidazolinone herbicide resistance mutation,which was first reported in rice,as well as a novel mutation with enhanced glyphosate tolerance have been created.（1）Creation of imidazolinone herbicide-resistant rice germplasm by means of chemical mutagenesisThree types of resistance mutants,Ala179Val,Ser627Asn and Gly628Glu,were obtained by EMS mutagenesis treatment and pressure screening in M2 generation with high imazethapyr.Among them,Ser627Asn and Gly628Glu are the main resistance sites used in herbicide-resistant rice of foreign countries,and Ala179Val is the first resistance mutation reported in rice.The seeds of Ala179Val,Ser627Asn and Gly628Glu resistant mutant lines were coated with different concentrations of Doushile?.Ala179Val and Ser627Asn can germinal and grow normally under the herbicide concentration of 0.5%to 5%,and maintain the same growth trend as the control without herbicide coating.However,Gly628Glu could not germinate and grow normally at 3%and 5%concentrations,and the seedling growth of Gly628Glu at 0.5%and 1%concentrations was significantly worse than that of the control.The wild-type Huahang 31 could not germinate normally under any of these 4 concentrations of herbicide coating.In addition,for these three resistance genes,no matter in heterozygous or homozygous condition,the results of herbicide coating experiment with different concentration were consistent,this indicates that the combination of herbicide resistant germplasm（Ala179Val and Ser627Asn）with herbicide coating can realize the impurity removal and purity preservation of hybrid rice seed production.（2）Creation of rice germplasm not only resistant to imidazolinone herbicides but also sensitive to bendasone by CRISPR/Cas9 technology.Based on the imidazolinone herbicide resistance mutation Ala179Val,a new rice germplasm not only resistant to imidazolinone herbicides but also sensitive to bendasone was obtained by knocking out Os CYP81A6 gene using CRISPR/Cas9 technology.Under the condition of 0.5%Basagran?coating and 0.5%Basagran?+0.5%Doushile?simultaneously coating,the Oscyp81a6 knockout line could not germinate and grow normally,while the uncoated control line and resistant mutant line Huahang 31（Ala179Val）could grow normally and uniformly.Therefore,in the process of light and simplified seed production of hybrid rice,the hybrid F1seeds,which resistant to both imidazolinone herbicides and bendasone,can be created by crossing this novel germplasm（resistant to imidazolinone herbicides but also sensitive to bendasone）with any female parent（with natural bendazone resistance gene）.Since either parent is resistant to only one herbicide,the self-cross seed will die at the germination stage by using two herbicides coating,and all the normal growing rice were double resistant hybrids.So,it can be used in the impurity removal and purity preservation,light and simplified seed production of two-line hybrid rice in the future.（3）Creation of rice glyphosate-resistant rice germplasm based on EPSPS protein structure analysis and directed evolutionIn the case that neither natural mutation nor EMS mutagenesis could produce glyphosate-resistant rice,by creating defective E.coliΔaro A+ΔC-Plyase,a prokaryotic expression system for glyphosate resistance identification of Os EPSPS mutant was established in this study.Based on the structural analysis and rational design of EPSPS protein,a large number of Os EPSPS protein mutants were identified by using prokaryotic glyphosate resistance identification system.Finally,two key amino acids of the substrate entry channel were screened out.Through the identification of saturated mutations,it was confirmed that the two double mutations of N398S+D422F and N398C+D422F showed glyphosate tolerance,and this result was further confirmed by transgenic Arabidopsis thaliana and rice.These two sites are new resistance sites that have not been reported yet,and they are also the first resistance mutations obtained based on rational structural analysis of EPSPS protein,which provide a new idea for the development of glyphosate-resistant plants in the future.（4）The natural allelic variation,protein conservationism of Os PSPS and Os ALS were analyzed based on the resequencing data of 3000 rice linesOs EPSPS and Os ALS genes are highly conserved in natural resources of rice,and all the reported resistance mutations are located in the conserved regions of these two proteins.Unlike the resistance sites of EPSPS,which are located at the active center of the protein,the resistance sites of ALS are all located at the entry channel of the substrate.However,all the non-synonymous mutations in natural resources occurred in the non-conserved region of the protein,and after herbicide treatment,no resistance mutation was found in the 3K rice germplasm. |
