水稻白叶枯病抗性基因Xa31(t)的精细定位及候选基因的克隆
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摘要
水稻白叶枯病(Bacterial Blight, BB)是一种严重的细菌性病害。自1884年在日本第一次被发现以来,水稻白叶枯病已在南纬20度至北纬58度的世界范围内被观察到。在我国,除新疆外,各稻区均有发生,华东、华中和华南发生普遍。引起水稻白叶枯病的黄单胞杆菌(Xanthomonas oryzae pv. Oryzae,Xoo)为革兰氏阴性菌,它通过气孔或伤口入侵宿主木质部(维管束)进行繁殖,沿叶脉产生灰褐色至白色病斑。由黄单胞杆菌引起的水稻白叶枯病在流行年份可导致水稻减产20~30%,严重时高达50%。
到目前为止,已被确定的水稻白叶枯病抗性基因至少有36个,23个显性基因,13个隐性基因,编号已排到Xa36(t)。王春台等在云南地方品种‘扎昌龙’(Za Chang Long,ZCL)中发现了一个新的抗性基因,并命名为Xa31(t),定位于水稻4号染色体的G235和C600两个分子标记之间,物理距离相差100 Kb。本研究对Xa31(t)进行进一步精细定位,筛选克隆目的区域的候选基因。获得了以下主要研究结果:
1.构建了一个含有294个单株的ZCL/IRBB1的F2代群体,根据目的基因在染色体上的目的区域,以NCBI公布的水稻品种日本晴(Oryza Sativa L. SSP. Japonica cv. Nipponbare)基因组序列为基础,选取相应的BAC或PAC克隆,在各克隆均匀选取序列片段,与水稻9311 (Oryza Sativa L.SSP. Indica)基因组序列进行BLAST比对,找到无同源或同源性较低的区域,用Primer5.0共设计PCR引物46对。最后筛选到5对多态性PCR分子标记,用于对作图群体的遗传分析。将Xa31(t)进一步定位到约20 Kb内。
2.在抗性基因Xa31(t)的目标区域,根据网上公布的日本晴的基因组序列,用生物信息学的方法预测候选基因,然后对候选基因进行BLAST分析,根据含有水稻白叶枯病抗性结构域和ORF,确定了5个候选基因。对这5个候选基因设计引物,进行长片段PCR扩增,得到了两个候选基因的全长序列,构建TA克隆载体进行测序,筛选正确的克隆;以pCMABIA1300构建的表达载体,转化水稻感病品种日本晴和台北309,已获得了部分阳性植株,其抗性正在鉴定之中。
Bacterial blight (BB) of rice is a kind of serious bacterial diseases in rice. Since it was first found at 1884 in Japan, BB has been observed in worldwide rice-planting area from the south latitude 20 degrees to north latitude 58 degrees. In China, BB occurs in every rice planting areas except Xinjiang Province. BB is caused by a kind of Gram-negative bacteria, Xanthomonas (Xanthomonas oryzae pv. Oryzae,Xoo) through the stoma or wound xylem (vascular bundle) of host rice plant to reproduce taupe and white disease spot along leaf vein turns. .Rice bacterial blight disease can cause the 20~30% loss of rice output in pop year, even up to 50% in serious occurring year.
Up to now, there are at least 36 BB resistance genes, 23 dominant genes and 13 recessive genes, in rice have been identified, Wang et al. discoered a new resistance gene named Xa31(t) from ZCL, a regional rice variety from Yunnan province in southwest China. It was mapped on rice chromosome 4, between G235 and C600 the two molecular markers, and the Physical distance is 100 Kb. The aim of this research is to further make a fine map of Xa31(t) and to clone its candidate genes. The main results are as follows:
1. A F2 population with 294 individual plants from a cross of ZCL×IRBB1 has been constructed. Fouty six pairs of PCR primers disigned by PrePrimer 5.0 based on sequence of Nipponbare and 9311 genomes within the mapped target gene (Xa31(t)) region on chromosome 4 are used for polymorphic analysis between the two parents of ZCL and IRBB1, and 5 pairs of polymophic PCR molecular markers has been selected. Using these 5 markers screen the 294 individual plants of the F2 population from a cross of ZCL×IRBB1. Genetic analysis are finally limiting Xa31(t) within a region of about 20 Kb.
2. According to the sequence of about 20 Kb in the region of resistance gene Xa31(t) located in the Nipponbare genomics, 5 candidate genes were predicted by bioinformatics analysis. and full-length cDNAs of 2 candidate genes had been obtained from ZCL. The ORFs of two candidate genes were recombined with the expression vector pCMABIA1300, respectively and introduced into rice callus of Nipponbare and TB309 by the means of Agrobacterium-mediated transformation. A few of positive plants have been identified and the resistance analysis on these plant are being studied now.
到目前为止,已被确定的水稻白叶枯病抗性基因至少有36个,23个显性基因,13个隐性基因,编号已排到Xa36(t)。王春台等在云南地方品种‘扎昌龙’(Za Chang Long,ZCL)中发现了一个新的抗性基因,并命名为Xa31(t),定位于水稻4号染色体的G235和C600两个分子标记之间,物理距离相差100 Kb。本研究对Xa31(t)进行进一步精细定位,筛选克隆目的区域的候选基因。获得了以下主要研究结果:
1.构建了一个含有294个单株的ZCL/IRBB1的F2代群体,根据目的基因在染色体上的目的区域,以NCBI公布的水稻品种日本晴(Oryza Sativa L. SSP. Japonica cv. Nipponbare)基因组序列为基础,选取相应的BAC或PAC克隆,在各克隆均匀选取序列片段,与水稻9311 (Oryza Sativa L.SSP. Indica)基因组序列进行BLAST比对,找到无同源或同源性较低的区域,用Primer5.0共设计PCR引物46对。最后筛选到5对多态性PCR分子标记,用于对作图群体的遗传分析。将Xa31(t)进一步定位到约20 Kb内。
2.在抗性基因Xa31(t)的目标区域,根据网上公布的日本晴的基因组序列,用生物信息学的方法预测候选基因,然后对候选基因进行BLAST分析,根据含有水稻白叶枯病抗性结构域和ORF,确定了5个候选基因。对这5个候选基因设计引物,进行长片段PCR扩增,得到了两个候选基因的全长序列,构建TA克隆载体进行测序,筛选正确的克隆;以pCMABIA1300构建的表达载体,转化水稻感病品种日本晴和台北309,已获得了部分阳性植株,其抗性正在鉴定之中。
Bacterial blight (BB) of rice is a kind of serious bacterial diseases in rice. Since it was first found at 1884 in Japan, BB has been observed in worldwide rice-planting area from the south latitude 20 degrees to north latitude 58 degrees. In China, BB occurs in every rice planting areas except Xinjiang Province. BB is caused by a kind of Gram-negative bacteria, Xanthomonas (Xanthomonas oryzae pv. Oryzae,Xoo) through the stoma or wound xylem (vascular bundle) of host rice plant to reproduce taupe and white disease spot along leaf vein turns. .Rice bacterial blight disease can cause the 20~30% loss of rice output in pop year, even up to 50% in serious occurring year.
Up to now, there are at least 36 BB resistance genes, 23 dominant genes and 13 recessive genes, in rice have been identified, Wang et al. discoered a new resistance gene named Xa31(t) from ZCL, a regional rice variety from Yunnan province in southwest China. It was mapped on rice chromosome 4, between G235 and C600 the two molecular markers, and the Physical distance is 100 Kb. The aim of this research is to further make a fine map of Xa31(t) and to clone its candidate genes. The main results are as follows:
1. A F2 population with 294 individual plants from a cross of ZCL×IRBB1 has been constructed. Fouty six pairs of PCR primers disigned by PrePrimer 5.0 based on sequence of Nipponbare and 9311 genomes within the mapped target gene (Xa31(t)) region on chromosome 4 are used for polymorphic analysis between the two parents of ZCL and IRBB1, and 5 pairs of polymophic PCR molecular markers has been selected. Using these 5 markers screen the 294 individual plants of the F2 population from a cross of ZCL×IRBB1. Genetic analysis are finally limiting Xa31(t) within a region of about 20 Kb.
2. According to the sequence of about 20 Kb in the region of resistance gene Xa31(t) located in the Nipponbare genomics, 5 candidate genes were predicted by bioinformatics analysis. and full-length cDNAs of 2 candidate genes had been obtained from ZCL. The ORFs of two candidate genes were recombined with the expression vector pCMABIA1300, respectively and introduced into rice callus of Nipponbare and TB309 by the means of Agrobacterium-mediated transformation. A few of positive plants have been identified and the resistance analysis on these plant are being studied now.
引文
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