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【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Gen Bank,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 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poly A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。
【Objective】 The objective of this study was to analyze the relationship between Pi-ta 3’-UTR region genetic variation and the resistance function of Pi-ta, understand the mechanism of Pi-ta resistance and provide basis for cultivating more durable resistant varieties. 【Method】 A total of 137 Yunnan local rice cultivars were collected from Yunnan rice landraces with extremely high genetic diversity. After seedling raising, the total DNA of rice seedlings was extracted from clover-leaf stage. The primers were designed to amplify the DNA sequence of Pi-ta 3’-UTR region and amplify the key function site 6 640 to the stop codon 6 675 A piece of DNA sequence. The DNA sequences of 137 3’-UTR regions were obtained by two-way sequencing and submitted to Gen Bank. The degree of genetic diversity in the 3’-UTR region of Yunnan rice landraces Pi-ta was analyzed by mutation loci. Based on the maximum Simple method to construct haplotype network diagram, analysis of different haplotype relationship between pedigree. At the same time, the distribution of 3’-UTR haplotypes was analyzed by the base state of 6 640, the key disease resistance site in the joint coding region, to discuss the relationship between 3’-UTR region and Pi-ta resistance function. 【Result】 The 3’-UTR region of Pi-ta from Yunnan rice showed a high degree of genetic diversity. There were 12 SNPs in the 3’-UTR region of 1.1 kb in length, and 137 varieties could be divided by these SNPs Into 7 haplotypes. There is no recombination signal between different haplotypes. The DNA coding region of Pi-ta corresponding to 3’-UTR is 1 120bp, which is more than 5 times of the average length of plant gene 3’-UTR (200 bp) and relatively low G + C content of 40.43% Length polymorphism due to insertion or deletion. A number of non-conserved potential poly A sites exist in the 3’-UTR sequence of Pi-ta. In addition, there is still a very high frequency of TTTT sequences in the 3’-UTR region of Pi-ta, suggesting that Pi-ta May have a complex regulatory mechanism; analysis of different transcripts of Pi-ta also indicates that the 3’-UTR corresponds to a complex and changeable cleavage pattern in the coding region of the DNA. This alternative splicing of 3’-UTR may be possible And resistance to determine the role. Further analysis of the genetic polymorphism showed that the 3’-UTR highly polymorphic SNP occurred in susceptible varieties, and all resistant varieties only shared one haplotype. Interestingly, the only 3’-UTR resistant haplotype corresponded to the only haplotype resistance in the Pi-ta coding region, ie, the 6 640G haplotype was also the only resistance to 3’-UTR type. This indicates that the 3’-UTR is closely related to its coding region and is contiguous and consistent in terms of function and selection pressure. The resistance haplotype region of Pi-ta has been extended from the coding region to the 3’-UTR region. When introducing broad-spectrum resistant species into Pi-ta, it is necessary to ensure that there is no additional SNP in the 3’-UTR region. Is a haplotype-specific SNPs. 【Conclusion】 The 3’-UTR of Pi-ta is closely linked with its coding region. The 3’-UTR of the resistant species is purely selected to maintain a single haplotype, and the 3’-UTR has no function on Pi-ta resistance Or lack of effect.