Genetic Analysis And Artificial Evolution Of Rapidly Evolving Rice Blast Resistance Genes

Rice(Oryza sativa)is one of the most important staple crops in the world and is not only a long history of cultivation,but also a broad geographical distribution.However,a wide variety of pathogens in nature continue to invade the rice,thus threatening the safe production of rice.Among them,rice blast with diverse physiological races and a very fast mutation rate is one of the most damaging disease faced by rice.Rice blast has a wide range of distribution,multiple onset period and grave consequences,which is the main cause of rice production loss in China.For now,the most economical and effective way to control rice blast is rice blast resistance breeding by use of resistance gene(R gene)in rice.However,the traditional resistance breeding methods and R gene cloning technology are time-consuming and laborious,which cannot meet current needs of resistance breeding and resistance gene cloning.As the resistance of resistant varieties is decreasing,the cloning and identification of rice blast resistence genes is becoming more and more difficult,and the available disease resistance resources for agricultural industry is becoming scarce.This problem has become the bottleneck of rice blast resistance breeding.Based on molecular evolution theory and the genetics of plant resistance genes,our laboratory had suggested that those NBS-LRR genes with rapid-evolving rate is the key to rice blast resistance.Based on this inference,a series of NBS-LRR loci with rapid evolutionary characteristics had been identified,among which several representative loci were surveyed then.The results confirmed the success of high-throughput cloning technology of rice blast resistance gene.This technique can effectively produce many R genes with rice blast resistance in a short time,which alleviates lack of rice blast resistance genes.In this study,four non-representative rapidly evolving loci and two non-rapidly evolving loci were selected for further survey.Then cloning and resistance identification of the target genes were carried out.And as a result,eight of the nine(-89%)cloned genes from the four rapidly evolving loci showed resistance to rice blast and all of the four cloned genes from two non-rapidly evolving loci didn't show any blast resistance.These results further demonstrated the effectiveness and feasibility of the high-throughput cloning technique.In addition,the genetic analysis of cloned genes indicated that high nucleotide polymorphisms and high non-synonymous mutation rates in coding regions contribute to blast resistance of the rapidly evolving loci.Insertions/deletions(InDels)in coding regions,changes in mRNA cleavage patterns,amino acid variation of important motifs in NBS region and changes in LRR core number are also the significant genetic characteristics of rapidly evolving NBS-LRR loci.These genetic features indicate that we can use artificial evolution technique to produce a large number of blast resistance genes with rich genetic variation.Based on this inspiration,we developed an artificial evolution technology system of rice blast resistance genes,which was based mainly on emerging molecular biology techniques such as synthetic biology techniques and gene artificial evolution techniques.This system has the strategy of "segmented cloning","in vitro recombination" and"stepwise assembly".In this study,three rapidly evolving loci(Pi37,Pi9 and AC134922)and two non-rapidly evolving loci(Os05g40150 and Os12g17410)were selected as targets for artificial evolution.Respectively,through sequence screening,vector construction,fragmented cloning,artificial mutagenesis,in vitro recombination and sequence assembly process,recombinants of each target gene were successfully obtained.At present,885 positive clones have been preliminarily obtained.Partial region of recombinant LRRs in 368 clones were sequenced.The sequence analysis showed that there were genetic differences between any two genes,and most of the genetic variation was derived from the existing variation in the donor sequences.So,artificial recombination experiment achieved the expected results.In the identification of rice blast resistance of 107 vectors,44 of them had certain resistance to rice blast,and some genes showed strong resistance to rice blast.Thus,the artificial evolution of rice blast resistance genes in this research is successful and feasible.This technology can not only greatly expand the rice blast resistance resources,but also provides a shortcut for the continuous acquisition of rice blast resistance genes.Artificial evolution of NBS-LRR genes has a good application prospects and provides theoretical guidance and technical support for the establishment and efficient use of rice resistance gene pool.