稻瘟病菌无毒基因AVR-Pii物理图谱的构建
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摘要
由子囊菌Magnaporthe grisea引起的稻瘟病是世界性的水稻病害之一，危害严重。防治该病的有效措施是栽培抗病品种，但更重要的方面是要广泛分析我国现有水稻品种的抗瘟基因型、了解田间菌群中无毒基因的种类、数量和分布并及时监测其变化。这将有助于了解病原菌小种的变异机理；有利于建立稻瘟病菌小种快速鉴定的分子技术，在实践上用于指导抗病品种的合理布局和轮换，以更有效地控制病害的发生。
本研究在张国珍博士获得与无毒基因AVR-Pii连锁的两个RAPD标记(并转化为SCAR标记)：OPI07_(700)和OPM10_(1314)的基础上进行染色体步移，旨在克隆AVR-Pii无毒基因。首先，采用PCR的方法对两个SCAR标记进行分析发现在SCAR标记OPM10_(1314)处菌株P131与Y34存在差异；不过，在SCAR标记OPI07_(700)处菌株P131与Y34不存在差异。用标记OPM10_(1314)的SCAR引物在菌株P131与Y34中分别扩增出了1314bp与1664bp的两条不同长度的DNA片段，测序后发现后者是在1314bp中插入了一段350bp的重复序列。基因组Southern杂交及数据库BLAST表明：标记OPI07_(700)在P131与Y34基因组是单拷贝的，SCAR标记OPM10_(1314)的1314bp中有重复序列。
对两个SCAR标记和AVR-Pii基因在染色体上的定位分析表明它们位于稻瘟病菌基因组第Ⅱ号染色体的端粒附近位置。其中无毒基因AVR-Pii较两个标记更靠近端粒，同时它们都在supercontigⅢ上(supercontigⅢ上一共有154个contigs，从Contig2.513到Contig2.360)，其中Contig2.360是在端粒位置。SCAR标记OPM10_(1314)被定位在Contig2.387上，OPI07_(700)被定位在Contig2.381上。
通过两个标记相向进行染色体步移，构建了两个SCAR标记区域的物理图谱，由3个含有最小重叠的TAC克隆覆盖了两个标记区域，分析两个标记间的物理距离大约为90Kb，根据两个标记间的遗传距离为3.16cM，可以推算出在该区域一个遗传单位相当于28Kb的物理距离。比Farman等发现的一个遗传单位相当于50Kb的物理距离要小；比Zhu等发现的一个遗传单位相当于41Kb的物理距离也小，而与AVR1-C039区域一个cM对应33.5Kb的物理距离相近。推测可能是由于该区域在染色体端粒的位置，重组事件发生的多的缘故。同时也可以知道近的SCAR标记OPI07_(700)到目的基因的距离约是186Kb。在此基础上向目的基因进行染色体步移，用4个含有最小重叠的TAC克隆向目的基因前进了约210Kb，由遗传图谱与物理图谱的对应关系知道已经覆盖了无毒基因AVR-Pii区域。这些工作为无毒基因AVR-Pii的克隆奠定了基础。
Firstly,the two SCAR markers OPI07700 and OPM101314 was analyzed,the result show that the strains of rice blast fungus P131 and Y34 have discrepancy where the place of the SCAR marker OPM10,314. The PCR amplification products are 1314 bp in P131 andl 664 bp in Y34,but nothing in 70-15 respectivly with the primer of the SCAR marker OPM101314.Then,the 1664 bp DNA band was cloned and sequenced.The result show that the sequence of 1664 is inserting a 350 bp repeated sequence into the sequence of 1314. There is no discrepancy between the strains of rice blast fungus P131,Y34 and 70-15 where the place of the SCAR marker OPI07700. Genomic Southern Blot and BLAST using the two SCAR markers OPI07700 and OPM101314.show that the marker OP107700 is a single copy in P131 and Y34 genome,and the marker PM101314 have some repeated sequence in P131 and Y34 genome.The markers and AVR-Pii were localized near the telomere on the SupercontigIII of the Chromosome II.The avirulence gene AVR-Pii is nearer the telomere than the two markers.
Chromosome walking were initiated from the two SCAR makers.Through screening the TAC genomic library and taking advantage of the rice blast DataBase, the physical map of the region of the two markers was constructed.Based on the contig maps from the chromosome walking,a minimum TAC tile containing 3 TAC clones was created.Then the physical distance between the two markers is about 90Kb.So the ratio between the physical and genetic distance is about 28Kb/cM.At the same time the genetic distance between the nearer SCAR maker OPI07700 and the avirulence gene AVR-Pii is 6.53 cM,so the physical distance is about 186Kb.With this knowledge we carry out chromosome walking toward the avirulence gene AVR-Pii. Through a minimum TAC tile containing 4 TAC clones we walking about 210Kb toward the avirulence gene.So the physical map of the region of the avirulence gene AVR-Pii was constructed.
At one time, according to the rice blast DataBase we choose three locations that be likely to include the avirulence gene AVR-Pii to initiate chromosome walking.then three congtig maps were constructed.the second and the third congtig map can overlap.But the relationship of the first congtig map and followed contig maps and 4 minimum TAC tile need further research.
During the chromosome walking no further marker was found.
本研究在张国珍博士获得与无毒基因AVR-Pii连锁的两个RAPD标记(并转化为SCAR标记)：OPI07_(700)和OPM10_(1314)的基础上进行染色体步移，旨在克隆AVR-Pii无毒基因。首先，采用PCR的方法对两个SCAR标记进行分析发现在SCAR标记OPM10_(1314)处菌株P131与Y34存在差异；不过，在SCAR标记OPI07_(700)处菌株P131与Y34不存在差异。用标记OPM10_(1314)的SCAR引物在菌株P131与Y34中分别扩增出了1314bp与1664bp的两条不同长度的DNA片段，测序后发现后者是在1314bp中插入了一段350bp的重复序列。基因组Southern杂交及数据库BLAST表明：标记OPI07_(700)在P131与Y34基因组是单拷贝的，SCAR标记OPM10_(1314)的1314bp中有重复序列。
对两个SCAR标记和AVR-Pii基因在染色体上的定位分析表明它们位于稻瘟病菌基因组第Ⅱ号染色体的端粒附近位置。其中无毒基因AVR-Pii较两个标记更靠近端粒，同时它们都在supercontigⅢ上(supercontigⅢ上一共有154个contigs，从Contig2.513到Contig2.360)，其中Contig2.360是在端粒位置。SCAR标记OPM10_(1314)被定位在Contig2.387上，OPI07_(700)被定位在Contig2.381上。
通过两个标记相向进行染色体步移，构建了两个SCAR标记区域的物理图谱，由3个含有最小重叠的TAC克隆覆盖了两个标记区域，分析两个标记间的物理距离大约为90Kb，根据两个标记间的遗传距离为3.16cM，可以推算出在该区域一个遗传单位相当于28Kb的物理距离。比Farman等发现的一个遗传单位相当于50Kb的物理距离要小；比Zhu等发现的一个遗传单位相当于41Kb的物理距离也小，而与AVR1-C039区域一个cM对应33.5Kb的物理距离相近。推测可能是由于该区域在染色体端粒的位置，重组事件发生的多的缘故。同时也可以知道近的SCAR标记OPI07_(700)到目的基因的距离约是186Kb。在此基础上向目的基因进行染色体步移，用4个含有最小重叠的TAC克隆向目的基因前进了约210Kb，由遗传图谱与物理图谱的对应关系知道已经覆盖了无毒基因AVR-Pii区域。这些工作为无毒基因AVR-Pii的克隆奠定了基础。
Firstly,the two SCAR markers OPI07700 and OPM101314 was analyzed,the result show that the strains of rice blast fungus P131 and Y34 have discrepancy where the place of the SCAR marker OPM10,314. The PCR amplification products are 1314 bp in P131 andl 664 bp in Y34,but nothing in 70-15 respectivly with the primer of the SCAR marker OPM101314.Then,the 1664 bp DNA band was cloned and sequenced.The result show that the sequence of 1664 is inserting a 350 bp repeated sequence into the sequence of 1314. There is no discrepancy between the strains of rice blast fungus P131,Y34 and 70-15 where the place of the SCAR marker OPI07700. Genomic Southern Blot and BLAST using the two SCAR markers OPI07700 and OPM101314.show that the marker OP107700 is a single copy in P131 and Y34 genome,and the marker PM101314 have some repeated sequence in P131 and Y34 genome.The markers and AVR-Pii were localized near the telomere on the SupercontigIII of the Chromosome II.The avirulence gene AVR-Pii is nearer the telomere than the two markers.
Chromosome walking were initiated from the two SCAR makers.Through screening the TAC genomic library and taking advantage of the rice blast DataBase, the physical map of the region of the two markers was constructed.Based on the contig maps from the chromosome walking,a minimum TAC tile containing 3 TAC clones was created.Then the physical distance between the two markers is about 90Kb.So the ratio between the physical and genetic distance is about 28Kb/cM.At the same time the genetic distance between the nearer SCAR maker OPI07700 and the avirulence gene AVR-Pii is 6.53 cM,so the physical distance is about 186Kb.With this knowledge we carry out chromosome walking toward the avirulence gene AVR-Pii. Through a minimum TAC tile containing 4 TAC clones we walking about 210Kb toward the avirulence gene.So the physical map of the region of the avirulence gene AVR-Pii was constructed.
At one time, according to the rice blast DataBase we choose three locations that be likely to include the avirulence gene AVR-Pii to initiate chromosome walking.then three congtig maps were constructed.the second and the third congtig map can overlap.But the relationship of the first congtig map and followed contig maps and 4 minimum TAC tile need further research.
During the chromosome walking no further marker was found.
引文
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2．张国珍．(2002) 博士论文：稻瘟病菌对三个水稻品种无毒性的遗传分析及分子标记
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