水稻地方品种Pi-ta的3'-UTR遗传多态分析

doi:10.3864/j.issn.0578-1752.2017.15.001

摘要/Abstract

摘要： 【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137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137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1.1 kb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1 120 bp，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。

关键词: 水稻, 稻瘟病, 抗性基因, Pi-ta, UTR, 遗传多态

Abstract: 【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 of Pi-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.

Key words: Oryza sativa, rice blast, resistance gene, Pi-ta, UTR, genetic polymorphism

刘立娜, 杨静, 徐刘燕, 李成云. 水稻地方品种Pi-ta的3'-UTR遗传多态分析[J]. 中国农业科学, 2017, 50(15): 2851-2860.

LIU LiNa, YANG Jing, XU LiuYan, LI ChengYun. Genetic Diversity Analysis of Pi-ta Gene 3′-UTR in Rice Landraces[J]. Scientia Agricultura Sinica, 2017, 50(15): 2851-2860.

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