类病毒检测技术培训于德才教学提纲.ppt

,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,类病毒检测技术培训于德才,类病毒结构示意图,The structure of viroid,马铃薯纺锤块茎类病毒热变性过程,Heat-denatured process diagram of PSTVd,三、类病毒结构,马铃薯纺锤块茎类病毒的五个功能区,The five functional domains of PSTVd,马铃薯类病毒中国分离株核苷酸序列,25 11 1 359,1,CCTGCACGTC GACGATT,G,GA,GCTC,ACAGGA ACCACGAG,TT,TAGTTCCGAG,Sac,P(25-11),51,GAACCAACTG CGGTTCCAAG GGCTAAACAC CCTCGCCCCG AAGCAAAGAA,101,AGATAGAGAA AAAGCGGTTC TCGGGAGCTT CAGTTGTTTC CACCGGGTAG,151,TAGCCGAAGC GACAGCGCAA AGGGGGCGAG GGGTGGTCCT GCGGGCGCGA,201 GGAAGGACAC CCGAAGAAAG GAAGGGTGAA AACCCTGTTT CGGCGGGAAT,251,TACTCCTGTC GGCCGCTGGG CACTCCCCAC CGTCCTTATT GCCAGTTCGC,301,TCCAGGTTTC CCCGGGGATC CCTGAAGCGC TCCTCCGAGC CGCCTTCTTT,35 26,351,TTTCTTTTCT GCTCAGGAGG TC,AGGTGTGA,ACG,GGTACC,C G,ATCTCTAGA,P(26-35),Kpn,401,GGATCCCCGG GTACCGAGCT CGAATTCGTA ATCATGGTCA TAGCTGTTTC,451 CTGTGTGAAA TTGTTATCCG CTCACAATTC CACACAACAT ACGAGCCGGA,501,AGCATAAAGT GTAAAGCCTG GGGTGCCTAA TGAGTGAGCT AACTCACATT,551,AATTGCGTTG CGCTCACTGC CCGCTTTCCA GTCGGGAAAC CTGTCGTGCC,601,AGCTGCATTA ATGAATCGGC CAACGCGCCG GGAGAGGCGG GTTGCGTATT,651 T,序列测定结果,PSTVd,致病区的位置和结构。图解表示,PSTVd-Intermediate,的完整结构，包括左未端区（,T,L,1-46/315-359），,致病区（47-73/286-314），中央保守区（74-120/240-285），可变区（121-148/212-239）和右未端区（,T,R,149-211）(Keese and symons,1985),premelting(PM),区1-3（,Steger,等，1984）和二级发夹结构1-111。下面为三个自然发生的,PSTVd,分离株系。水平箭头指出,PM1,和,VM,区域（,Schnlzer,等，1985），“星号”表示中间株系和强、弱系序列差异。此图引自,Owens et al(1996)。,T,L,Path,1(M),CGGAACUAAA CUCGUGGUUC CUGUGGUUCA CACCUGACCU,CCUGAG,CA,GA AAAGAAAAAA,2(M)CGGAACUAAA CUCGUGGUUC CUGUGGUUCA CACCUGACCU CCUGAGCA GA AAAGAAAAAA,3(M)CGGAACUAAA CUCGUGGUUC CUGUGGUUCA CACCUGACCU CCUGAGCA GA AAAGAAAAAA,4(I)CGGAACUAAA CUCGUGGUUC CUGUGGUUCA CACCUGACCU CCUGAGCA GA AAAGAAAAAA,5(S)CGGAACUAAA CUCGUGGUUC CUGUGGUUCA CACCUGACCU CCUGAGCA,A,GA AAAGAAAAAA,6(S)CGGAACUAAA CUCGUGGUUC CUGUGGUUCA CACCUGACCU CCUGAGCA GA AAAGAAAAAA,CCR,1(M),GAA,GGCGGCU,CGGAGGAGCG CUUCAGGGAU CCCCCGGGAA ACCUGGAGCG AACUGGCAAU,2(M)GAAGGCGGCU CGGAGGAGCG CUUCAGGGAU CCCCCGGGAA ACCUGGAGCG AACUGGCAAU,3(M)GAAGGCGGCU CGGAGGAGCG CUUCAGGGAU CCCCCGGGAA ACCUGGAGCG AACUGGCAAU,4(I)GAAGGCGGCU CGGAGGAGCG CUUCAGGGAU CCCCCGGGAA ACCUGGAGCG AACUGGCAA,AA,5(S)GAAGGCGGCU CGGAGGAGCG CUUCAGGGAU CCCCCGGGAA ACCUGGAGCG AACUGGCAAU,6(S)GAAGGCGGCU CGGAGGAGCG CUUCAGGGAU CCCCCGGGAA ACCUGGAGCG AACUGGCAA,AA,Var,T,R,1(M),AAGGACGGUG GGGAGUGCCC A GCGGCCG,AC AGGAGUAAUU CCCGCCGAAA CAGGGUUUUC,2(M)AAGGACGGUG GGGAGUGCCC A GCGGCCGAC AGGAGUAAUU CCCGCCGAAA CAGGGUUUUC,3(M)AAGGACGGUG GGGAGUGCCC A GCGGCCGAC AGGAGUAAUU CCCGCCGAAA CAGGGUUUUC,4(I)AAGGACGGUG GGGAGUGCCC A GCGGCCGAC AGGAGUAAUU CCCGCCGAAA CAGGGUUUUC,5(S)A GGACGGUG GGGAGUGCCC A,U,GCGGCCGAC AGGAGUAAUU CCCGCCGAAA CAGGGUUUUC,6(S)AAGGACGGUG GGGAGUGCCC A GCGGCCGAC AGGAGUAAUU CCCGCCGAAA CAGGGUUUUC,Var,1(M)ACCCUUCCUU UCUUCGGGUG UCCUUCCUCG,CGCCCGCAGG ACCACCCCUC GCCCCCUUU,G,2(M)ACCCUUCCUU UCUUCGGGUG UCCUUCCUCG CGCCCGCAGG ACCACCCCUC GCCCCCUUUG,3(M)ACCCUUCCUU UCUUCGGGUG UCCUUCCUCG CGCCCGCAGG ACCACCCCUC GCCCCCUUUG,4(I)ACCCUUCCUU UCUUCGGGUG UCCUUCCUCG CGCCCGCAGG ACCACCCCUC GCCCCCUUUG,5(S)ACCCUUCCUU UCUUCGGGUG UCCUUCCUCG CGCCCGCAGG ACCACCCCUC G,-,CCCCUUUG,6(S)ACCCUUCCUU UCUUCGGGUG UCCUUCCUCG CGCCCGCAGG ACCACCCCUC GCCCCCUUUG,CCR,Path,1(M),CGGUGUCGCU UCGGCUACUA CCCGGUGGAA ACAACUGAAG CUCCC,GAGAA CCGCUUUUUC,2(M)CGGUGUCGCU UC,A,GCUACUA CCCGGUGGAA ACAACUGAAG CUCCCGAGAA CCGCUUUUUC,3(M)CGGUGUCGCU UCGG,A,UAUUA CCCGGUGGAA ACAACUGAAG CUCCCGAGAA CCGCUUUUUC,4(I)CGGUGUCGCU UCGGCUACUA CCCGGUGGAA ACAACUGAAG CUCCCGAGAA CCGCUUUUUC,5(S)CGGUGUCGCU UCGGCUACUA CCCGGUGGAA ACAACUGAAG CUCCCGAGAA CCGCUUUUUC,6(S)CGGUGUCGCU UCG,A,CUACUA CCCGGUGGAA ACAACUGAAG CUCCCGAGAA CCGCUUUUUC,T,L,1(M),UCUAUCUUUC UUUG,CUUCGG,GGCGAGGGUG UUUAGCCCUU GGAACCGCAG UUGGUUCCU,2(M)UCUAUCUUUC UUUGCUUCGG GGCGAGGGUG UUUAGCCCUU GGAACCGCAG UUGGUUCCU,3(M)UCUAUCUUUC UUUGCUUCGG GGCGAGGGUG UUUAGCCCUU GGAACCGCAG UUGGUUCCU,4(I)UCUAUCUUAC -UUGCUUCGG GGCGAGGGUG UUUAGCCCUU GGAACCGCAG UUGGUUCCU,5(S)UCUAUCUUU,A,UUUGCUUCGG,C,GGCGAGGGUG UUUAGCCCUU GGAACCGCAG UUGGUUCCU,6(S)UCUAUCUU,-A,UGUGCUUC,A,G GGCGAGGGUG UUUAGCCCUU GGAACCGCAG UUGGUUCCU,PSTVd,中国分离株与已报道序列在一级结构上的差异,PSTVd,强、弱系及中间株系的核苷酸序列差异比较,Comparison with mild strain from China in nucleotide sequence of the isolates of the,mild,intermediate and severe strain of potato spindle tuber viroid(PSTVd),PSTVd,分离株系,PSTVd isolates,类病毒核酸长度,Viroid-nucleotide,Chain length,同中国弱株系相比核酸差异,Differences from“the mild strain”of China in sequence,变化位置,Position of the changed nuoleotide,Mild:,PSTVd-WA(,未例出),PSTVd-F(,未例出),PSTVd-India,PSTVd-WB,359,nt,358 nt,359 nt,359 nt,UA,UCUGA,CA,CU,GA,309(致病区),309(致病区),255(中央保守区),258(中央保守区),253(中央保守区),PSTVd KF-6 mild type,359,nt,None,None,Severe:,PSTVd-F-SL,PSTVd S17-I-8,359,nt,359 nt,Ainsertion,Adeletion,Uinsertion,Cdeletion,GA,CA,Udeletion,C insertion,UAA,U deletion,CA,UG,GA,48-49（致病区）,122(可变区),140-141(可变区),232(可变区),254(中央保守守区),310(致病区）,311（致病区）,320-321(左未端区),120(中央保守区),309（致病区）,310（致病区）,312（致病区）,319(左未端区),Intermediate:,PSTVd-I-India,359,nt,UAA,UA,Udeletion,120(中央保守区),309(致病区),311(致病区),注：,From Herold et al.(1992);From Owens et al.(1992);From Singh et al.(1993);From Gross et al.(1981);and From Gora-Sochacka(1997).,四、类病毒鉴定,1、指示植物接种鉴定,1）,鲁特格番茄,2）新莨菪,2、电泳,1）垂直双向丙烯酰胺电泳（2-,D-PAGE）,2）,往返丙烯酰胺电泳（,R-PAGE）,3、,反转录,PCR（RT-PCR）,4、,核酸斑点杂交（,NASH）,往返丙烯酰胺电泳（,R-PAGE）,检测流程图,核酸提取,第一项电泳,反向电泳,固定,银染色,显色,结果判读,PSTVd,上样量：8,g,1.6 g,0.32 g,64,ng,13 ng,2.6 ng,520 pg,104,pg,最低检出率：13,ng,双向电泳检测,PSTVd,PSTVd,检测结果：,样品含有,PSTVd，,箭头所指为类病毒核酸带,RT-PCR,检测类病毒流程,引物设计,扩增产物分析,（）,以,cDN,为模板,扩增,制备,cDN,第一链,（反转录）,样品核酸提取,检测结果：,PCR,反应退火温度分别设定为55 和57，在57 下特异性好，可用来检测,PSTVd,434,bp,328bp,1 2 3 4 5,资料检索、收集、样品采集,类病毒病原鉴定:三种方法：,（,R-PAGE、,2-D-PAGE、,RT-PCR）,含,PSTVd pGEM-7Z,质粒,利用,HaeIII,对重组质粒进行酶切验证,鉴定结果：含,PSTVd,设计引物、对含,PSTVd,的,pGEM-7Z,质粒进行,PCR,扩增,利用生物素,biotin-11-dUTP,代替,dUTP,制备探针,进行核酸斑点杂交反应,NBT/BCIP,化学颜色反应,Flu,荧光素发光反应,试剂盒组装,生 产 应 用,核酸斑点杂交（）检测技术流程,探针制备,终止反应,预杂交,封闭,孵育,信号产生,杂交,漂洗,点样,尼龙膜的处理,500,ng 50 ng 5 ng 500 pg,50 pg,5 pg,1 2 3 4,200,pg 50 pg,200,pg 50 pg,NASH,方法检测,PSTVd,专化性,测试（颜色反应）,结果显示：,阳性样品有斑点，阴性样品不产生斑点。,反应专化性强。,核酸斑点杂交（）检测反应原理图,核酸斑点杂交（,NASH）,技术的研究,探针的制备,1、,Hae,单酶切含,PSTVd,单体的,pGEM-7Z,质粒,1 2 3,328,bp,Hae/7Z,空,Hae/7Z-PSTVd,(bp),正向插入 反向插入,1,657 657 657,2 458 458 561*,3 434 434 458,328*,369*,434,289,320*,289,267 289 267,174 267 174,142 174 142,102 142 125*,80 102 102,80 80,结果显示：,pGEM-7Z,重组质粒含有,PSTVd,单体，且为正向插入,2、引物筛选,根据已报导的,PSTVd,基因序列（,Genebank,登陆号,AY372400）,设计,两对引物对7,Z,质粒上的,PSTVd,单体目的片段进行,PCR,扩增,引物(,Primer),:,片段大小为154,bp,P(c):5CGA AGC CGA TGA TGGA ATA ACGTGG AGG ACT CG TGGA ATA ACG 3,与124-116同源,10,mM dNTP 2 l,引物(60,pM)2 l,模板(,P,7,z-PSTVd,单体)2,l,DEPC H,2,O 80.5 l,10PCR buffer 10 l,Taq,酶(,Promega,公司)2.5,l,Total 100 l,94 5分钟,94 2分钟,40 1.5分钟 5个循环,72 2分钟,55 1.5分钟,72 2分钟,72 10分钟,PCR,反应体系:热循环程序:,5个循环,此课件下载可自行编辑修改，仅供参考！感谢您的支持，我们努力做得更好！谢谢,