水稻品种抗稻瘟病等位基因鉴定及抗瘟新基因的定位和克隆

发布时间：2019-07-08 21:20

【摘要】：水稻是全世界最重要的粮食作物之.。在水稻生产中,病害的发生是威胁生产的重要问题。由真菌灰色巨角间座壳菌(Magnaporthe grisea)引起的稻瘟病作为最主要的病害之一,广泛发生于全世界各水稻产区。在我国稻瘟病位于水稻上三大病害之首,严重危害时致使水稻产量损失严重,尤其在晨露时间较长的丘陵地区或山区甚至经常出现绝收的现象,多年来一直是我国水稻产量及粮食安全的严重威胁。生产实践证明,培育持久抗性品种是稻瘟病防控的首要措施。本文通过对水稻主栽品种及育种亲本的抗瘟基因型进行鉴定,并通过对水稻品种抗稻瘟病新基因Pi65(t)的精细定位、克隆和载体构建,以期了解不同抗病基因在品种中的分布,为抗病品种选育及品种布局提供理论依据。1.辽宁省主栽水稻品种抗稻瘟病的等位基因型鉴定研究选取了辽宁省24份水稻材料,根据9个抗稻瘟病基因的保守区设计引物,扩增各品种的编码区序列,对扩增子序列进行对比分析,鉴定各基因在24个品种中的分布情况,并对部分抗病等位基因进行了功能验证。结果表明：辽宁省24个主栽水稻品种均不携带Pi36、Pi37、Pi21、Pit及其抗病等位基因,而Pid2、Pid3、Pita、Piz-t和Pil/Piks/Pikm/Pikp在24个品种中以不同突变类型及不同频率出现,其中,在2个品种中检测到Pid2的抗病等位基因及Pid3的抗病等位基因；4个品种检测到Pita的抗病等位基因,Pita的等位基因中新发现的几处碱基突变并未影响抗病基因的功能；所有品种中均无与Pik及其复等位基因完全一致的序列,但利用携带AVR-Pik的稻瘟病菌接种鉴定结果表明,辽粳454和沈农265携带的Pik等位基因可能仍具有抗病基因功能；在品种中检测到19种Piz-t的等位基因,接种结果表明大部分突变位点均使Piz-t的等位基因丧失了抗病功能。2.港育129品种抗稻瘟病新基因Pi65(t)的精细定位利用辽宁省采集的稻瘟病群体,对辽宁省110份水稻材料进行抗谱分析,结果筛选出连续两年在田间表现高抗(港育129)、高感(辽星1号)的品种,经基因型鉴定分析,高抗品种中不含有任何已知的抗病基因。利用港育129和辽星1号构建DH群体,通过SLAF-seq技术将候选区域定位至30.42-30.98M之间,继续通过在BC1F2代群体中筛选性状分离的个体(重组体),将候选基因进一步定位至30.42-30.85M之间,该区间内有21个基因,通过RT-PCR分析其中有4个基因在港源8号和辽星1号之间表达存在差异。而且在港育129接种稻瘟病菌后,4个基因表达量均显升高,说明4个基因可能与水稻抗性相关。4个基因分别为Os11g0691700、Os11g0691800、Os11g0691900、 Os11g0692100,均属于NBS-LRR类。由于NBS-LRR类基因是抗稻瘟病基因中常见的类型,因此认为这些基因很可能是在港育129中定位到的新基因。3.Pita、Pi9新等位基因及候选基因Osllg0691700的克隆和载体构建根据基因型鉴定的结果,水稻品种辽农979携带的Pi9等位基因和港源8号所携带的Pita等位基因,均为新的抗病等位基因。分别从两个品种中克隆基因,并将两个基因分别连接至表达载体；利用SLAF-seq技术,在品种港育129中定位到1个和抗病有关的基因Pi65(t),克隆该基因并连接至表达载体。试验结果表明,Pi9、Pita和Pi65(t)被成功克隆并连接至表达载体。
[Abstract]:Rice is the most important food crop in the world. In the production of rice, the occurrence of diseases is an important problem in the production of rice. Magnaporthe grisea, which is caused by Magnaporthe grisea, is one of the main diseases, and is widely used in various rice-producing areas in the world. It is a serious threat to rice yield and food security for many years. The production practice shows that the cultivation of durable resistant varieties is the primary measure of the prevention and control of the rice blast. The anti-blast genotypes of the rice varieties and their parents were identified, and the fine positioning, cloning and vector construction of the new gene Pi65 (t) of the rice variety were used to understand the distribution of the different disease-resistant genes in the variety. And provides a theoretical foundation for the breeding and the variety layout of the disease-resistant variety. In this paper,24 rice materials in Liaoning province were selected according to the allele-type identification of the rice blast resistance of the main rice varieties in Liaoning province, and the coding region sequences of each variety were amplified according to the conserved region design primers of 9 blast-resistant rice blast genes, and the amplicon sequences were compared and analyzed. The distribution of each gene in 24 varieties was identified, and the functional verification of some of the disease-resistant alleles was carried out. The results showed that Pid2, Pid3, Pita, Piz-t and Pil/ Piks/ Pikp/ Pikp appeared in 24 varieties with different mutation types and different frequencies, among which, Pid2, Pid3, Pita, Piz-t and Pil/ Piks/ Pik/ Pikp appeared in 24 varieties with different mutation types and different frequencies. The resistance allele of Pid2 and the disease-resistant allele of Pid3 were detected in two varieties. Four varieties of Pita's disease-resistant allele were detected, and several base mutations in Pita's allele did not affect the function of the disease-resistant gene. No sequence of Pik and its complex allele was found in all the varieties, but the identification of the rice blast fungus carrying the AVR-Pik showed that the Pik allele carried by the Liaoning and Shennong 265 may still have the disease-resistant gene function, and the alleles of the 19 Piz-t were detected in the variety. The results showed that most of the mutant sites had a loss of resistance to the allele of Piz-t. The rice blast population collected in Liaoning province was used for the fine positioning of the new gene Pi65 (t), which was collected in Liaoning province, and the resistance spectrum of 110 rice materials in Liaoning province was analyzed. The results showed that there were high anti-blast resistance (129) and high (LiaoXing 1) variety in the field for two consecutive years. Through the genotype identification analysis, no known disease-resistant gene is contained in the high-resistant variety. The candidate genes were further located between 30.42 and 30.98M by the SLLAF-seq technique, and the candidate genes were further located between 30.42 and 30.85 M. RT-PCR was used to analyze the difference of the expression of 4 genes between the port number 8 and the Liaoxing 1. The four genes may be related to the resistance of rice. The four genes are Os11g0691700, Os11g0691800, Os11g0691900 and Os11g0692100, all of which belong to NBS-LRR. Since the NBS-LRR class gene is a common type in the rice blast resistance gene, it is considered that these genes are most likely to be the new gene located in the port culture 129.3. The cloning and vector of the Pita, Pi9 new allele and the candidate gene Osllg0691700 are based on the results of the genotype identification, The Pi9 allele and the Pita allele carried by the rice variety, Liaonon 979, were all new disease-resistant alleles. The gene is cloned from two varieties, and the two genes are respectively connected to the expression vector, and one and a disease-related gene Pi65 (t) are positioned in the variety harbor culture 129 by using the SLAF-seq technique, and the gene is cloned and connected to the expression vector. The test results showed that Pi9, Pita and Pi65 (t) were successfully cloned and ligated to the expression vector.
【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S435.111.41

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