内蒙古中西部三种马铃薯病毒及病程相关蛋白的初步研究
摘要
马铃薯是世界性的高产作物,我国种植面积和产量均居世界第一位。马铃薯病毒病是马铃薯生产中的重要制约因素。马铃薯X病毒(Potato virus X,PVX)、马铃薯Y病毒(Potato virus Y,PVY)和马铃薯卷叶病毒(Potato leafroll virus,PLRV)是严重危害我国马铃薯生产的三种主要病毒。
根据PVX、PVY、PLRV外壳蛋白基因序列分别设计合成了三对特异性引物,对采自和林格尔县、呼和浩特市农场、武川县、四子王旗、商都县、多伦县等六个地区120份大田自然种植的马铃薯样品以RT-PCR方法进行三种病毒的检测并统计三种病毒的侵染方式。结果显示六个地区均检测到PVY和PLRV,检出率分别为45%和35%,PVX只在武川县和多伦县检测到,检出率为7.5%。六地区中以四子王旗样品带毒率最高,达到80%,其次为武川县和多伦县,均为65%。商都县、呼市农场和和林格尔县带毒率分别为60%、45%和30%。69个带毒样品当中PVY单独侵染样品占30.43%,PLRV单独侵染样品占21.74%。PVX与PVY复合侵染样品占8.70%,PVY与PLRV复合侵染样品占34.78%,而三种病毒复合侵染的样品仅占4.35%。未检测到PVX单独侵染和PVX与PLRV复合侵染的样品。
挑选扩增效果较好的PVX、PVY和PLRV片段进行测序,序列对比及聚类分析结果表明:PVY CP基因的遗传丰富度总体上高于PLRV,这两种病毒的CP基因类型倾向于按照地理环境因素进行分组。具有相似地理环境和气候特征地区的病毒CP基因序列相似度较高,不同地理环境和气候特征地区的病毒CP基因序列相似度较低。PVX、PVY在系统发育上未显示出特定规律性分组, PLRV在分组上则较为保守。
以三种马铃薯病毒检测均为阴性的马铃薯栽培苗为试材,使用汁液摩擦法接种PVY,在接种后的0h、6h、12h、24h、36h、48h、72h采取叶片检测了两种病程相关蛋白基因的表达情况。结果显示,PR6胰蛋白酶抑制剂基因在接毒后表达量一直处于常量状态,没有受到影响。茄属病程相关蛋白NtPrp27-like基因表达量从12h开始增加,36h达到最高,随后又降低至常量水平。
Potato is a staple food crop in the world and is widely grown in China. One of the important factors affecting potato quantity and quality is the diseases caused by several potato viruses. Potato virus X(PVX), potato virus Y(PVY), and potato leaf roll virus(PLRV) are the three viruses that result in decreased yield of potato in China.
In this study, three pairs of primers were designed and synthesized based on the nucleotide sequence of the coat protein(CP) gene of PVX, PVY and PLRV respectively. 120 samples were taken from Helingeer, Hohhot, Wuchuan, Siziwangqi, Shangdu and Duolun.RT-PCR was done to detect three kinds of potato virus infection and infection methods. The results shows that PVY and PLRV are detected in all six regions, the detection rates were 45% and 35%, PVX is detected only in Wuchuan and Duolun, the detection rate is 7.5%. Six regions with the highest rate of Siziwangqi infected samples, 80%, followed by Wuchuan and Duolun, both 65%. Shangdu, Hohhot, Helingeer has lower infected samoles and the infection rate was 60%, 45% and 30%. Among the 69 infected samples, PVY infection alone accounted for 30.43%, PLRV alone accounted for 21.74%. PVX and PVY infected samples combined accounted for 8.70%, PVY and PLRV infected samples combined accounted for 34.78%, while infection of three composite samples of the virus only 4.35%. PVX infected alone and infected comolesx with PVX and PLRV samples are not detected.
Determinae the sequence of PVY and PLRV, the results shows that PVY CP gene has higher genetic richness than PLRV. With similar geographic and climatic characteristics of the region the virus CP gene sequence has higher similarity, virus CP gene has similarity lower in different geographic regions.
Potato seedlings without virus were inoculation with PVY. Detect two patho genesis-related protein detection gene expression after inoculation of 0h, 6h, 12h, 24h, 36h, 48h, 72h. The results showed that, PR6 gene expression has been in a constant state after inoculation. The NtPRp27-like protein gene expression began to increase from 12h and achieve to the maximum in 36h, then reduced to a constant level.
根据PVX、PVY、PLRV外壳蛋白基因序列分别设计合成了三对特异性引物,对采自和林格尔县、呼和浩特市农场、武川县、四子王旗、商都县、多伦县等六个地区120份大田自然种植的马铃薯样品以RT-PCR方法进行三种病毒的检测并统计三种病毒的侵染方式。结果显示六个地区均检测到PVY和PLRV,检出率分别为45%和35%,PVX只在武川县和多伦县检测到,检出率为7.5%。六地区中以四子王旗样品带毒率最高,达到80%,其次为武川县和多伦县,均为65%。商都县、呼市农场和和林格尔县带毒率分别为60%、45%和30%。69个带毒样品当中PVY单独侵染样品占30.43%,PLRV单独侵染样品占21.74%。PVX与PVY复合侵染样品占8.70%,PVY与PLRV复合侵染样品占34.78%,而三种病毒复合侵染的样品仅占4.35%。未检测到PVX单独侵染和PVX与PLRV复合侵染的样品。
挑选扩增效果较好的PVX、PVY和PLRV片段进行测序,序列对比及聚类分析结果表明:PVY CP基因的遗传丰富度总体上高于PLRV,这两种病毒的CP基因类型倾向于按照地理环境因素进行分组。具有相似地理环境和气候特征地区的病毒CP基因序列相似度较高,不同地理环境和气候特征地区的病毒CP基因序列相似度较低。PVX、PVY在系统发育上未显示出特定规律性分组, PLRV在分组上则较为保守。
以三种马铃薯病毒检测均为阴性的马铃薯栽培苗为试材,使用汁液摩擦法接种PVY,在接种后的0h、6h、12h、24h、36h、48h、72h采取叶片检测了两种病程相关蛋白基因的表达情况。结果显示,PR6胰蛋白酶抑制剂基因在接毒后表达量一直处于常量状态,没有受到影响。茄属病程相关蛋白NtPrp27-like基因表达量从12h开始增加,36h达到最高,随后又降低至常量水平。
Potato is a staple food crop in the world and is widely grown in China. One of the important factors affecting potato quantity and quality is the diseases caused by several potato viruses. Potato virus X(PVX), potato virus Y(PVY), and potato leaf roll virus(PLRV) are the three viruses that result in decreased yield of potato in China.
In this study, three pairs of primers were designed and synthesized based on the nucleotide sequence of the coat protein(CP) gene of PVX, PVY and PLRV respectively. 120 samples were taken from Helingeer, Hohhot, Wuchuan, Siziwangqi, Shangdu and Duolun.RT-PCR was done to detect three kinds of potato virus infection and infection methods. The results shows that PVY and PLRV are detected in all six regions, the detection rates were 45% and 35%, PVX is detected only in Wuchuan and Duolun, the detection rate is 7.5%. Six regions with the highest rate of Siziwangqi infected samples, 80%, followed by Wuchuan and Duolun, both 65%. Shangdu, Hohhot, Helingeer has lower infected samoles and the infection rate was 60%, 45% and 30%. Among the 69 infected samples, PVY infection alone accounted for 30.43%, PLRV alone accounted for 21.74%. PVX and PVY infected samples combined accounted for 8.70%, PVY and PLRV infected samples combined accounted for 34.78%, while infection of three composite samples of the virus only 4.35%. PVX infected alone and infected comolesx with PVX and PLRV samples are not detected.
Determinae the sequence of PVY and PLRV, the results shows that PVY CP gene has higher genetic richness than PLRV. With similar geographic and climatic characteristics of the region the virus CP gene sequence has higher similarity, virus CP gene has similarity lower in different geographic regions.
Potato seedlings without virus were inoculation with PVY. Detect two patho genesis-related protein detection gene expression after inoculation of 0h, 6h, 12h, 24h, 36h, 48h, 72h. The results showed that, PR6 gene expression has been in a constant state after inoculation. The NtPRp27-like protein gene expression began to increase from 12h and achieve to the maximum in 36h, then reduced to a constant level.
引文
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