[Objective] In order to understand the mechanism of Pi-ta resistance and provide a basis for the development of more durable resistant varieties,the relationship between the genetic diversity in 3'-UTR and the resistance function of the Pi-ta gene were studied.[Method] A total of 137 Yunnan rice landraces were collected.DNA from seedling at the three-to four-leaf stage was extracted and the DNA coding sequences ofPi-ta gene 3'-UTR were amplified with designed primers in Yunnan rice landraces which possess high genetic diversity.The sequence from functionally key nucleoside 6 640 to the stop codon 6 675 was also amplified.Through bidirectional sequencing,DNA sequences in 3'-UTR region of all genetic varieties in Yunnan rice landraces were obtained and submitted to the GenBank.The genetic diversity of Pi-ta 3'-UTR region in Yunnan rice landraces was analyzed based on detecting polymorphic sites.The distribution of different haplotypes was analyzed in combination with the key resistance locus 6640 G in coding region.The network diagram of the relationship between different haplotypes was constructed based on maximum parsimony.[Result] The Pi-ta gene 3'-UTR of Yunnan rice landraces shows a high degree of genetic diversity.A total of 12 SNPs were found in the 3'-UTR with a length of 1.1 kb.The 137 rice landraces could be divided into 7 haplotypes by these SNPs.No recombination signal was found among haplotypes.The length of Pi-ta 3'-UTR coding region is 1 120 bp,5 times more than the average length of plant gene 3'-UTR (200 bp).The content of G+C is relatively low,40.43％,and there is no insertion or deletion polymorphism in the 3'-UTR coding region.There are many non-conservative potential polyA sites in the 3'-UTR sequence of Pi-ta,in addition,there is a very high frequency TTTT sequence in the 3'-UTR region of Pi-ta,which suggests that Pi-ta has a complex regulatory mechanism when terminate transcription.Analysis of the different transcripts of Pi-ta also showed that the 3'-UTR has complex and variable transcripts as contrasted to the coding sequence,indicating that the selective splicing of 3'-UTR may be related to resistance determination.The analysis of the haplotype distribution indicated that the 3'-UTR high polymorphism appeared only in the susceptible rice landraces,and all the resistant individuals share one haplotype.Interestingly,the only 3'-UTR resistant haplotype corresponds to the only resistant haplotype in the Pi-ta coding region,that is,the haplotype containing 6640 G is also the 3'-UTR resistant haplotype.This indicates that 3'-UTR is closely linked to the coding region,and is consistent and continuous in terms of function and selection pressure.The Pi-ta resistant haplotype range has been extended from the coding region to the 3'-UTR region,and when introducing Pi-ta into broad-spectrum-resistant varieties,it is necessary to ensure that the 3'-UTR region must have no other SNPs,all should be 3'-UTR resistant-haplotype-specific SNPs.[Conclusion] The 3'-UTR is closely linked to the encoding region in Pi-ta gene.In resistant rice landraces,the 3'-UTR suffered purifying selection,and maintained a single haplotype.Results of the study demonstrate that the 3'-UTR is indispensable for the resistance function of Pi-ta gene.%[目的]分析Pi-ta的3'-UTR区遗传变异与该基因抗性功能之间的关系,了解Pi-ta的抗性决定机制,为培养更持久的抗性品种提供依据.[方法]以遗传多样性极高的云南水稻地方品种为研究对象,收集了1 37个云南地方水稻品种.育苗后提取三叶一心期的水稻幼苗总DNA,设计引物扩增了Pi-ta的3'-UTR区的DNA序列,并扩增了关键功能位点6 640到终止密码子第6 675处这一段的DNA序列.通过双向序列测定获得了1 37条3-UTR区的DNA序列并提交至GenBank,通过变异位点检测分析云南水稻地方品种Pi-ta的3'-UTR区的遗传多样性程度,并基于最大简约法构建单倍型网络图,分析不同单倍型之间的谱系关系.同时,联合编码区关键抗病位点6 640的碱基状态对3'-UTR单倍型的分布进行分析,讨论3'-UTR区与Pi-ta抗性功能之间的关系.[结果]云南水稻地方品种Pi-ta的3'-UTR区呈现出高度的遗传多样性,长度为1.1kb的3'-UTR区共有12个SNP位点,由这些SNP可将137个品种划分成7个单倍型.不同单倍型之间没有重组的信号.Pi-ta的3'-UTR对应的DNA编码区长为1 120bp,是植物基因3'-UTR平均长度(200 bp)的5倍多,G+C含量相对较低,为40.43％,不存在插入或缺失导致的长度多态性.Pi-ta的3'-UTR序列中存在多个非保守的潜在polyA位点,此外,Pi-ta的3'-UTR区还存在非常高频率的TTTT序列,提示Pi-ta在转录终止时可能具有复杂的调控机制;而对Pi-ta的不同转录本的分析也表明3'-UTR对应于DNA编码区序列时呈现复杂多变的剪切方式,3'-UTR这种选择性拼接可能与抗性决定作用有关.对遗传多态的进一步分析表明,3'-UTR的SNP高度多态性都出现在感病品种中,所有抗性品种只共享一种单倍型.有趣的是,唯一的3'-UTR抗性单倍型与Pi-ta编码区唯一的抗性单倍型相对应,也即是6 640G所在单倍型也是3'-UTR唯一抗性单倍型.这表明3'-UTR与其编码区是紧密关联的,在功能上和所受到的选择压力方面是连续和一致的.Pi-ta的抗性单倍型区域已从编码区扩展到了3'-UTR区,在研制广谱抗性品种引入Pi-ta时需要同时保证其3'-UTR区不能有额外的SNP,必须是抗性单倍型特有的SNPs.[结论]Pi-ta的3'-UTR与其编码区紧密连锁,抗性品种的3'-UTR受到纯净化选择,维持单一单倍型,3'-UTR对于Pi-ta的抗性功能具有不可或缺的作用.
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机译：水稻抗稻瘟病基因Pi-ta和Pi-b多重PCR体系的构建与应用Establishment and Application of a Multiplex PCR System for the Detection of Blast Resistance Genes Pi-ta and Pi-b in Rice
