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1、样品采自大庆地区，样品为克新13号,二、类病毒种类,已知序列类病毒种类及其分类 The variants of viroid known sequence and their classification,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 GACG
2、ATTGGA GCTC ACAGGA ACCACGAGTT 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 TACTCCT
3、GTC GGCCGCTGGG CACTCCCCAC CGTCCTTATT GCCAGTTCGC 301 TCCAGGTTTC CCCGGGGATC CCTGAAGCGC TCCTCCGAGC CGCCTTCTTT 35 26 351 TTTCTTTTCT GCTCAGGAGG TCAGGTGTGA ACGGGTACCC GATCTCTAGA P(26-35) Kpn 401 GGATCCCCGG GTACCGAGCT CGAATTCGTA ATCATGGTCA TAGCTGTTTC 451 CTGTGTGAAA TTGTTATCCG CTCACAATTC CACACAACAT ACGAGCCG
4、GA 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（TL,1-46/315-359），致病区（47-73/286-314），中央保守区（74-120/240-285）
5、，可变区（121-148/212-239）和右未端区（TR, 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)。,TL Path 1(M) CGGAACUAAA CUCGUGGUUC CUGUGGUUCA CACCUGACCU CCUGAGCA GA AAAGAAAAAA 2(M) CGGAACUAAA
6、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 CCUGAGCAAGA AAAGAAAAAA 6(S) CGGAACUAAA CUCGUGGUUC C
7、UGUGGUUCA CACCUGACCU CCUGAGCA GA AAAGAAAAAA CCR 1(M) GAAGGCGGCU 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 C
8、CCCCGGGAA ACCUGGAGCG AACUGGCAAAA 5(S) GAAGGCGGCU CGGAGGAGCG CUUCAGGGAU CCCCCGGGAA ACCUGGAGCG AACUGGCAAU 6(S) GAAGGCGGCU CGGAGGAGCG CUUCAGGGAU CCCCCGGGAA ACCUGGAGCG AACUGGCAAAA Var TR 1(M) AAGGACGGUG GGGAGUGCCC A GCGGCCGAC AGGAGUAAUU CCCGCCGAAA CAGGGUUUUC 2(M) AAGGACGGUG GGGAGUGCCC A GCGGCCGAC AGGAGU
9、AAUU 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 AUGCGGCCGAC AGGAGUAAUU CCCGCCGAAA CAGGGUUUUC 6(S) AAGGACGGUG GGGAGUGCCC A GCGGCCGAC AGGAGUAAUU CCCGCCG
10、AAA CAGGGUUUUC Var 1(M) ACCCUUCCUU UCUUCGGGUG UCCUUCCUCG CGCCCGCAGG ACCACCCCUC GCCCCCUUUG 2(M) ACCCUUCCUU UCUUCGGGUG UCCUUCCUCG CGCCCGCAGG ACCACCCCUC GCCCCCUUUG 3(M) ACCCUUCCUU UCUUCGGGUG UCCUUCCUCG CGCCCGCAGG ACCACCCCUC GCCCCCUUUG 4(I) ACCCUUCCUU UCUUCGGGUG UCCUUCCUCG CGCCCGCAGG ACCACCCCUC GCCCCCUU
11、UG 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 CUCCCGAGAA CCGCUUUUUC 2(M) CGGUGUCGCU UCAGCUACUA CCCGGUGGAA ACAACUGAAG CUCCCGAGAA CCGCUUUUUC 3(M)
12、 CGGUGUCGCU UCGGAUAUUA CCCGGUGGAA ACAACUGAAG CUCCCGAGAA CCGCUUUUUC 4(I) CGGUGUCGCU UCGGCUACUA CCCGGUGGAA ACAACUGAAG CUCCCGAGAA CCGCUUUUUC 5(S) CGGUGUCGCU UCGGCUACUA CCCGGUGGAA ACAACUGAAG CUCCCGAGAA CCGCUUUUUC 6(S) CGGUGUCGCU UCGACUACUA CCCGGUGGAA ACAACUGAAG CUCCCGAGAA CCGCUUUUUC TL 1(M) UCUAUCUUUC U
13、UUGCUUCGG 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) UCUAUCUUUA UUUGCUUCGGC GGCGAGGGU
14、G UUUAGCCCUU GGAACCGCAG UUGGUUCCU 6(S) UCUAUCUU -A UGUGCUUCAG 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 tube
15、r viroid(PSTVd),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：13ng,双向电泳
