大豆抗孢囊线虫3号生理小种的分子检测
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摘要
大豆孢囊线虫(SCN)(Heterodero glycines Ichinohe)是一种土传的定居性内寄生线虫，易引起大豆黄萎病。是农业生产上危害最大的病害之一。大豆孢囊线虫病生理小种多达十几种，在我国，大豆孢囊线虫病病原主要为3、4号生理小种。本研究用8个经常规鉴定为抗孢囊线虫3号生理小种的大豆品种(6个抗病，2个感病)为材料，据据甜菜抗孢囊线虫病基因的保守序列和已公布的大豆抗孢囊线虫病基因的相关序列设计了4对引物，组成16个组合，进行PCR特异扩增，从中筛选出一对特异引物，同时优化了PCR扩增反应体系中的一些影响因素，通过PCR扩增最终获得了一条600bp的特异片段，经PCR-Southorn blotting验证了该特异片段的同源性程度和真实性；最后对所扩增的特异片段进行克隆测序，并利用Blast软件在GonBank上进行序列相似性比较。利用这对特异引物对其他32个未经常规鉴定的大豆品种(30个商业品种和2个野生品种)进行了初步检测。本研究所得的主要研究结果如下：
1、建立了适合于大豆的PCR特异扩增反应体系：25μl的反应体系中，含模板DNA 50ng，10mol／L Tris-HCl(pH8.3)，50mmol／L KCl，2.0mol／L MgCl~(2+)，150μmol／L dNTPs，0.4μmol／L引物浓度，Taq酶1.5U。反应程序为：94℃预变性5min，再以94℃变性25s，59℃复性30s，72℃延伸60s进行35个循环，后在72℃保温10min。
2、获得一对适合用于大豆抗孢囊线虫3号生理小种分子检测的特异引物P_1P_5。利用该特异引物对8个经抗孢囊线虫3号生理小种常规鉴定的大豆品种进行PCR检测，检测结果与常规鉴定的吻合度达100％，PCR-Southern blotting进一步证实了该方法的可行性。
3、利用该引物组合对未经常规鉴定的32个品种进行PCR分子检测，初步获得抗病品种5个，即东农43、东农40567、东农410、油乌豆、野生种Ⅱ，该结果有待田间常规抗性鉴定进一步论证。
4、对特异片段进行克隆测序。应用Blast软件，与GenBank、EMBL、DDBJ、
福建农林大学2。。4局硕士学位论丈
PDB中所有序列进行同源性比较.结果发现，与大豆的似受体激酶
天月‘4(recePtor-like kinase RHG4 gene)基因(GenBank登录号为:AF5o65 18)的同
源性达到98%，与水稻中的似受体激酶基因mRNA的富含亮氛酸重复(o.sativa
mRNA for leucine rieh repeat reeePtor-like kinase)(Ge妞Bank登录号为:Y07748)的
同源性也达到%%，并将该序列登录到GenBank中，登录号为:AY5801 61.
Soybean cyst Nematode (SCN) (Heterodera glycines Ichinohe) is a kind of soilborn sedentary endoparasite nematode and the pathogen of soybean chlorsis. It has over ten physiological races. It is economically the most destructive pathogen affecting soybean culture. SCN Race 3. 4 are the predominant races that are the most damaging pathogens in ChinaJn this study, eight conventional identified soybean varieties (including 6 nematode resistant varieties to to SCN race3, 2 susceptible varieties to SCN race3) and thirty-two soybean varieties (30 cultivars and 2 wild soybean varieties) without conventional identification were tested. According to the declared sequences of cyst nematode resistant gene in sugar beet and soybean, four forward primers and four reverse primers were designed, 16 pairs of primers were composed, and a pair of primers was selected among them by PCR special amplification. Amplification conditions for PCR in soybean were optimized, the results showed that a special fragment of about 600bp was
amplified, and PCR-Southern blotting was further made in order to affirm the similarity and veracity. Then, the special fragment were cloned, sequenced and compared in GenBank by Blast software. Finally, 30 commerical cultivars of soybean and two wild soybean varieties were primarily detected by PCR. The results were summarized as followed:
1. An optimal reaction system for PCR in soybean was established: the PCR volume was 25祃 and contained the optimal composition included SOng of template DNA, 10mMTris-HCl(pH8.3), 50mM KC1, 2.0mM MgCl2+, ISOuMdNTPs, 0.4礛 primers, 1.5U Taq polymerase. The PCR reaction program was devised for 1 cycle of pre-denaturation at 94 C for 5min, 35 cycles of 25s at 94C (denaturation), 30s at 59C (annealing) and 60s at 72C(extension).in the last cycle,the reaction was kept for an additional 10 min at 72 C then cooled to 4 C.
2. A pair of primers, P1P5, were obtained for molecular detection of cyst nematode
Race3 in soybean. The results of PCR detection in soybean generally verified these of conventional identification, identify proportion was 100% by the pair primers. The feasibility of PCR detection was further testified by PCR-Southem blotting.
3. Among 32 soybean varieties without conventional identification, five varieties appeared a special fragment of about 600bp: Dongnong 43,Dongnong40S67, Dongnong-410, Youwudou and wild soybean variety II. The results need be tested further by field identification.
4. Cloned and sequenced special fragment, compared similarity with all the sequences in GenBank, EMBL, DDBJ, PDB by Blast. The result showed that it has 98% similarity with Glycine max receptor-like kinase RHG4 (Rhg4) gene (GenBank accession NO. AF506518) and 96% similarity with O.sativa mRNA for leucine rich repeat receptor-like kinase (GenBank accession NO. Y07748). At last, direct submission of sequence data to GenBank, GenBank accession number for the nucleotide sequence is AY580161.
1、建立了适合于大豆的PCR特异扩增反应体系：25μl的反应体系中，含模板DNA 50ng，10mol／L Tris-HCl(pH8.3)，50mmol／L KCl，2.0mol／L MgCl~(2+)，150μmol／L dNTPs，0.4μmol／L引物浓度，Taq酶1.5U。反应程序为：94℃预变性5min，再以94℃变性25s，59℃复性30s，72℃延伸60s进行35个循环，后在72℃保温10min。
2、获得一对适合用于大豆抗孢囊线虫3号生理小种分子检测的特异引物P_1P_5。利用该特异引物对8个经抗孢囊线虫3号生理小种常规鉴定的大豆品种进行PCR检测，检测结果与常规鉴定的吻合度达100％，PCR-Southern blotting进一步证实了该方法的可行性。
3、利用该引物组合对未经常规鉴定的32个品种进行PCR分子检测，初步获得抗病品种5个，即东农43、东农40567、东农410、油乌豆、野生种Ⅱ，该结果有待田间常规抗性鉴定进一步论证。
4、对特异片段进行克隆测序。应用Blast软件，与GenBank、EMBL、DDBJ、
福建农林大学2。。4局硕士学位论丈
PDB中所有序列进行同源性比较.结果发现，与大豆的似受体激酶
天月‘4(recePtor-like kinase RHG4 gene)基因(GenBank登录号为:AF5o65 18)的同
源性达到98%，与水稻中的似受体激酶基因mRNA的富含亮氛酸重复(o.sativa
mRNA for leucine rieh repeat reeePtor-like kinase)(Ge妞Bank登录号为:Y07748)的
同源性也达到%%，并将该序列登录到GenBank中，登录号为:AY5801 61.
Soybean cyst Nematode (SCN) (Heterodera glycines Ichinohe) is a kind of soilborn sedentary endoparasite nematode and the pathogen of soybean chlorsis. It has over ten physiological races. It is economically the most destructive pathogen affecting soybean culture. SCN Race 3. 4 are the predominant races that are the most damaging pathogens in ChinaJn this study, eight conventional identified soybean varieties (including 6 nematode resistant varieties to to SCN race3, 2 susceptible varieties to SCN race3) and thirty-two soybean varieties (30 cultivars and 2 wild soybean varieties) without conventional identification were tested. According to the declared sequences of cyst nematode resistant gene in sugar beet and soybean, four forward primers and four reverse primers were designed, 16 pairs of primers were composed, and a pair of primers was selected among them by PCR special amplification. Amplification conditions for PCR in soybean were optimized, the results showed that a special fragment of about 600bp was
amplified, and PCR-Southern blotting was further made in order to affirm the similarity and veracity. Then, the special fragment were cloned, sequenced and compared in GenBank by Blast software. Finally, 30 commerical cultivars of soybean and two wild soybean varieties were primarily detected by PCR. The results were summarized as followed:
1. An optimal reaction system for PCR in soybean was established: the PCR volume was 25祃 and contained the optimal composition included SOng of template DNA, 10mMTris-HCl(pH8.3), 50mM KC1, 2.0mM MgCl2+, ISOuMdNTPs, 0.4礛 primers, 1.5U Taq polymerase. The PCR reaction program was devised for 1 cycle of pre-denaturation at 94 C for 5min, 35 cycles of 25s at 94C (denaturation), 30s at 59C (annealing) and 60s at 72C(extension).in the last cycle,the reaction was kept for an additional 10 min at 72 C then cooled to 4 C.
2. A pair of primers, P1P5, were obtained for molecular detection of cyst nematode
Race3 in soybean. The results of PCR detection in soybean generally verified these of conventional identification, identify proportion was 100% by the pair primers. The feasibility of PCR detection was further testified by PCR-Southem blotting.
3. Among 32 soybean varieties without conventional identification, five varieties appeared a special fragment of about 600bp: Dongnong 43,Dongnong40S67, Dongnong-410, Youwudou and wild soybean variety II. The results need be tested further by field identification.
4. Cloned and sequenced special fragment, compared similarity with all the sequences in GenBank, EMBL, DDBJ, PDB by Blast. The result showed that it has 98% similarity with Glycine max receptor-like kinase RHG4 (Rhg4) gene (GenBank accession NO. AF506518) and 96% similarity with O.sativa mRNA for leucine rich repeat receptor-like kinase (GenBank accession NO. Y07748). At last, direct submission of sequence data to GenBank, GenBank accession number for the nucleotide sequence is AY580161.
引文
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