美洲大蠊断肢再生相关Hedgehog信号通路基因及其dsRNA的应用的制作方法

本发明属于基因领域，涉及美洲大蠊断肢再生相关基因及其dsrna的应用。

背景技术：

蟑螂原产于非洲，好潮湿、温暖、黑暗的环境；喜欢躲藏在隐蔽的缝隙中；活动时间集中在夜晚，是世界性卫生害虫。美洲大蠊是蜚蠊科中体积最大的昆虫，它食性广泛，喜食糖和淀粉，污染食物、传播病菌和寄生虫，是世界性卫生害虫。在南方地区为室内优势品种，主要生存于下水道、暖气沟、厕所、浴室及酿造厂、酱品厂等阴暗潮湿的环境，容易传播疾病。

美洲大蠊具有避光性与聚集性，在饲养美洲大蠊时，在饲养缸中放置一些折叠纸板、纸筒以便遮光并供其栖息。美洲大蠊低龄若虫常常群聚在一起，这可能与分泌的信息素有关系，栖息时往往栖息在生活空间的最下层。他们一年中7、8和9月份的食量最大，也是他们繁殖最为旺盛的季节。他们也有一个特别的行为习性，即是互相残杀。即使在食物、饮水供应充足的条件下，仍频有互噬的现象出现，包括食卵、噬皮蜕、噬幼龄若虫和蜕皮若虫等情况。食卵现象比较多见，饲养缸中常见被食卵荚的残余部分，尾部夹持卵荚的雌虫被其他成虫追咬的情形也时有发生。但未发现若虫之间或成虫之间互食现象，所以它们仍能群聚在一起。

为了防止美洲大蠊，经常使用的化学农药和有机杀虫剂在害虫防治实践中表现出高效的杀虫效果，为害虫防治带来了革命性改变。但随着其长期大量使用，农药残留、水源和环境污染、生物链中断、生态失衡等一系列负面问题日益凸显。卫生环境的重视及蟑螂抗药性问题的出现，迫使我们要开发一种环境友好、高效、无毒的蟑螂防治方法，其中生物防治技术将为蟑螂防治提供一个新的防治方向。

技术实现要素：

本发明目的在于提供美洲大蠊断肢再生相关基因及其dsrna的应用。

本发明所采取的技术方案是：

定量检测hh、ptc、smo或/和cirna的试剂在制备促进美洲大蠊断肢再生的试剂中的应用。

定量检测hh、ptc、smo或/和cirna的试剂在制备防治美洲大蠊的试剂盒中的应用。

进一步的，所述定量检测hh、ptc、smo或/和cirna的试剂选自定量检测hh、ptc、smo或/和ci的rna转录水平的试剂。

进一步的，所述定量检测hh、ptc、smo或/和ci的rna转录水平的试剂选自定量检测hh、ptc、smo或/和ci的引物。

进一步的，所述定量检测hh、ptc、smo或/和ci的引物为：

hh：

hhqpcrf:5’-gagtccagagcgcacatcca-3’(seqidno.21)；

hhqpcrr:5’-tcctccgagaagcagccacc-3’(seqidno.22)；

ptc.：

ptcqpcrf:5’-atgcgtttctgagtgcg-3’(seqidno.23)；

ptcqpcrr:5’-gtaatttctgattggtggg-3’(seqidno.24)；

smo：

smoqpcrf:5’-gtaagatccgcaaggt-3’(seqidno.25)；

smoqpcrr:5’-cgtctgatggtggtaat-3’(seqidno.26)；

ci.：

ciqpcrf:5’-aatggagggctcgaagat-3’(seqidno.27)；

ciqpcrr:5’-tactgtcacggcgtaggtt-3’(seqidno.28)。

抑制hh、ptc、smo或/和ci表达的试剂在制备阻断美洲大蠊断肢再生的试剂中的应用。

进一步的，所述抑制hh、ptc、smo或/和ci表达的试剂为抑制hh、ptc、smo或/和cirna转录水平的试剂。

进一步的，所述抑制hh、ptc、smo或/和cirna转录水平的试剂选自沉默hh、ptc、smo或/和ci表达的sirna、沉默hh、ptc、smo或/和ci表达的shrna和沉默hh、ptc、smo或/和ci表达的dsrna的至少一种。

进一步的，所述沉默hh、ptc、smo或/和ci表达的dsrna选自以下序列：

靶向沉默hh基因的dsrna序列：

aacacccuuccagcaagcggucucaccgaggggcgcgugacccgcaaagacccucgcuucagggaucuggugcccaacuacaacgcagacaucguguucaaggacgaggaaggcacgggcgccgaccgccucaugacgcagaggugcaaggagaagcugaacacgcuggccaucucggugaugaaccaguggcccggcgugaagcugcgggugacggagggcugggacgaggaggggcaccacgccguggacucgcugcacuacgagggccgagccgucgacgucaccaccuccgacagggaucgagccaaguacggcaugcuggcgcgccucgccguggaggcgggcuucgacuggg(seqidno.17)；

靶向沉默ptc基因的dsrna序列：

uucgacgagcacuauauagaccaaauauuccuaaacauuaucccuugugccaucauuacuccauuagacuguuuuugggaagcuucgaagcuucugggaccagauuauccuguucaaauaccaggcgugggcuccaaggugcgcuggacgaaccuuaacccacaacaacuaauccaacaaaugaagacugucgaauugguauucccuuucgagacucucgaagacuacaugaaaagggcggggauaagcacgggcuaccaagagaagccgugucucgaccccuccgaccccgaaugcccgaccaccgcccccaacaaguuauccggacagauaccugacaucggggcagagcugacaggaggcugcuacggauaugcagccaaauauaugcacuggccugaggaccugguggugggcggagccacacgaaauaagacuggacacauacaaagggcgcgugcauugcagacuguggugcaacuaaugggagagagagagauguaugaauucuggucuaaaacguacaaaguccaucacuuggauuggacacaagaaaaagcugcuuuaauucucgagacauggcagaggc(seqidno.18)；

靶向沉默smo基因的dsrna序列：

acuuguaauuuucuauaucaauugcuguuuuuuggugucauguauuggaugguuggcacaguuuauuccuggugcacgugaaggcauuauaugccggaaagaugguacuuuaaggaugucagaaccuaauacuggagagaauuugucaugugugauaauauuuguauugguguauuacuucaucauggcugccuuugugugguuuguaauucucacguacacauggcacaucaguuuucaagcucuagguaaaauucaagagcgaauugauaagaaaagugcuuauuuucacaugguggcuuggucauugccucuaguucuuacuauuacaacuauggcacuaggggaaauagauggcgacaguguugcugguaucuguuuugugggauauguuaaucaugcugcacgagcaggauucuugcucguccccauuacaacaguacuuuuuguuggaggauacuuucucacuaggggucuuauaacacuggugagacuaaaaauuaacagucaagaaauuaucucagaaagagcaagugccaagauaagagaaaccauugugcgcaugggucucuuuucacuguuuacauuugucuuuguguu(seqidno.19)；

靶向沉默ci基因的dsrna序列：

gaucucgucaagcacaucaacaaugaccacauucaugccaacaagaagucguuugugugccgcugggaagacuguucaagggacgagaagcccuucaaagcgcaguauaugcugguggugcacaugcgacgacacacaggggagaagccccacaaauguacauuugaagggugcuacaaagccuauucgaggcuggagaaccugaagacgcaccucagaucucauacaggagaaaaacccuacaugugugaguauccuggaugcagcaaagccuucagcaaugcguccgaccgugccaaacaccagaaccgcacgcacuccaacgagaaaccguacguguguaaggccccgggcugcaccaagagguacacagaccccaguucgcugcggaagcacgugaagacagugcacggugccgaguucuaugccaacaagaagcacaagggagcuggcgacggcgccgaggauggcucuggaggggcgggaggccuacuggacagcagcccgcacagcgaggagaugaugagcggcaagacggcc(seqidno.20)。

一种促进美洲大蠊断肢再生的试剂盒，其特征在于，该试剂盒含有定量检测hh、ptc、smo或/和cirna的试剂。

进一步的，所述定量检测hh、ptc、smo或/和cirna的试剂为定量检测hh、ptc、smo或/和ci的rna转录水平的试剂。

进一步的，所述的定量检测hh、ptc、smo或/和cirna转录水平的试剂选自定量检测hh、ptc、smo或/和ci的引物。

进一步的，所述的定量检测hh、ptc、smo或/和ci的引物为：

hh：

hhqpcrf:5’-gagtccagagcgcacatcca-3’(seqidno.21)；

hhqpcrr:5’-tcctccgagaagcagccacc-3’(seqidno.22)；

ptc.：

ptcqpcrf:5’-atgcgtttctgagtgcg-3’(seqidno.23)；

ptcqpcrr:5’-gtaatttctgattggtggg-3’(seqidno.24)；

smo：

smoqpcrf:5’-gtaagatccgcaaggt-3’(seqidno.25)；

smoqpcrr:5’-cgtctgatggtggtaat-3’(seqidno.26)；

ci.：

ciqpcrf:5’-aatggagggctcgaagat-3’(seqidno.27)；

ciqpcrr:5’-tactgtcacggcgtaggtt-3’(seqidno.28)。

一种阻断美洲大蠊断肢再生的试剂盒，该试剂盒含有抑制hh、ptc、smo或/和ci表达的试剂。

进一步的，所述抑制hh、ptc、smo或/和ci表达的试剂为抑制hh、ptc、smo或/和ci的rna转录水平的试剂。

进一步的，所述抑制hh、ptc、smo或/和ci的rna转录水平的试剂选自沉默hh、ptc、smo或/和ci表达的sirna、沉默hh、ptc、smo或/和ci表达的shrna和沉默hh、ptc、smo或/和ci表达的dsrna的至少一种。

进一步的，所述的沉默hh、ptc、smo或/和ci的表达的dsrna选自以下序列：

靶向沉默hh基因的dsrna序列：

aacacccuuccagcaagcggucucaccgaggggcgcgugacccgcaaagacccucgcuucagggaucuggugcccaacuacaacgcagacaucguguucaaggacgaggaaggcacgggcgccgaccgccucaugacgcagaggugcaaggagaagcugaacacgcuggccaucucggugaugaaccaguggcccggcgugaagcugcgggugacggagggcugggacgaggaggggcaccacgccguggacucgcugcacuacgagggccgagccgucgacgucaccaccuccgacagggaucgagccaaguacggcaugcuggcgcgccucgccguggaggcgggcuucgacuggg(seqidno.17)；

靶向沉默ptc基因的dsrna序列：

uucgacgagcacuauauagaccaaauauuccuaaacauuaucccuugugccaucauuacuccauuagacuguuuuugggaagcuucgaagcuucugggaccagauuauccuguucaaauaccaggcgugggcuccaaggugcgcuggacgaaccuuaacccacaacaacuaauccaacaaaugaagacugucgaauugguauucccuuucgagacucucgaagacuacaugaaaagggcggggauaagcacgggcuaccaagagaagccgugucucgaccccuccgaccccgaaugcccgaccaccgcccccaacaaguuauccggacagauaccugacaucggggcagagcugacaggaggcugcuacggauaugcagccaaauauaugcacuggccugaggaccugguggugggcggagccacacgaaauaagacuggacacauacaaagggcgcgugcauugcagacuguggugcaacuaaugggagagagagagauguaugaauucuggucuaaaacguacaaaguccaucacuuggauuggacacaagaaaaagcugcuuuaauucucgagacauggcagaggc(seqidno.18)；

靶向沉默smo基因的dsrna序列：

acuuguaauuuucuauaucaauugcuguuuuuuggugucauguauuggaugguuggcacaguuuauuccuggugcacgugaaggcauuauaugccggaaagaugguacuuuaaggaugucagaaccuaauacuggagagaauuugucaugugugauaauauuuguauugguguauuacuucaucauggcugccuuugugugguuuguaauucucacguacacauggcacaucaguuuucaagcucuagguaaaauucaagagcgaauugauaagaaaagugcuuauuuucacaugguggcuuggucauugccucuaguucuuacuauuacaacuauggcacuaggggaaauagauggcgacaguguugcugguaucuguuuugugggauauguuaaucaugcugcacgagcaggauucuugcucguccccauuacaacaguacuuuuuguuggaggauacuuucucacuaggggucuuauaacacuggugagacuaaaaauuaacagucaagaaauuaucucagaaagagcaagugccaagauaagagaaaccauugugcgcaugggucucuuuucacuguuuacauuugucuuuguguu(seqidno.19)；

靶向沉默ci基因的dsrna序列：

gaucucgucaagcacaucaacaaugaccacauucaugccaacaagaagucguuugugugccgcugggaagacuguucaagggacgagaagcccuucaaagcgcaguauaugcugguggugcacaugcgacgacacacaggggagaagccccacaaauguacauuugaagggugcuacaaagccuauucgaggcuggagaaccugaagacgcaccucagaucucauacaggagaaaaacccuacaugugugaguauccuggaugcagcaaagccuucagcaaugcguccgaccgugccaaacaccagaaccgcacgcacuccaacgagaaaccguacguguguaaggccccgggcugcaccaagagguacacagaccccaguucgcugcggaagcacgugaagacagugcacggugccgaguucuaugccaacaagaagcacaagggagcuggcgacggcgccgaggauggcucuggaggggcgggaggccuacuggacagcagcccgcacagcgaggagaugaugagcggcaagacggcc(seqidno.20)。

抑制hh、ptc、smo或/和ci表达的试剂在制备防治美洲大蠊的药物中的应用。

一种检测美洲大蠊断肢再生相关的基因，所述的基因为hh、ptc、smo或/和ci；所述的hh核苷酸序列如seqidno.1所示，ptc核苷酸序列如seqidno.2所示，smo核苷酸序列如seqidno.3所示，ci核苷酸序列如seqidno.4所示。

本发明的有益效果是：

本发明针对美洲大蠊具有顽强的断肢再生能力但又很难防治的现状，发现hh、ptc、smo和ci基因的表达水平和蟑螂断肢再生密切相关，揭示了hh、ptc、smo和ci基因具有影响美洲大蠊断肢再生的能力。还证明沉默hh、ptc、smo和ci基因的表达，能抑制美洲大蠊断肢再生的能力，即可以高效除去美洲大蠊害虫，又避免了环境污染，安全可靠。说明本发明hh、ptc、smo和ci基因可以作为美洲大蠊害虫防治的防治方向。

附图说明

图1a为断肢部位示意图；

图1b为hh、ptc、smo和ci基因dsrna处理对美洲大蠊断肢再生的影响；

图2为dsrna注射后，hh、ptc、smo和ci基因表达量变化。

具体实施方式

定量检测hh、ptc、smo或/和cirna的试剂在制备促进美洲大蠊断肢再生的试剂中的应用。

优选的，所述定量检测hh、ptc、smo或/和cirna的试剂选自定量检测hh、ptc、smo或/和ci的rna转录水平的试剂。

优选的，所述定量检测hh、ptc、smo或/和ci的rna转录水平的试剂选自定量检测hh、ptc、smo或/和ci的引物。

优选的，所述定量检测hh、ptc、smo或/和ci的引物为：

hh：

hhqpcrf:5’-gagtccagagcgcacatcca-3’(seqidno.21)；

hhqpcrr:5’-tcctccgagaagcagccacc-3’(seqidno.22)；

ptc.：

ptcqpcrf:5’-atgcgtttctgagtgcg-3’(seqidno.23)；

ptcqpcrr:5’-gtaatttctgattggtggg-3’(seqidno.24)；

smo：

smoqpcrf:5’-gtaagatccgcaaggt-3’(seqidno.25)；

smoqpcrr:5’-cgtctgatggtggtaat-3’(seqidno.26)；

ci.：

ciqpcrf:5’-aatggagggctcgaagat-3’(seqidno.27)；

ciqpcrr:5’-tactgtcacggcgtaggtt-3’(seqidno.28)。

抑制hh、ptc、smo或/和ci表达的试剂在制备阻断美洲大蠊断肢再生的试剂中的应用。

优选的，所述抑制hh、ptc、smo或/和ci表达的试剂为抑制hh、ptc、smo或/和cirna转录水平的试剂。

优选的，所述抑制hh、ptc、smo或/和cirna转录水平的试剂选自沉默hh、ptc、smo或/和ci表达的sirna、沉默hh、ptc、smo或/和ci表达的shrna和沉默hh、ptc、smo或/和ci表达的dsrna的至少一种。

优选的，所述沉默hh、ptc、smo或/和ci表达的dsrna选自以下序列：

靶向沉默hh基因的dsrna序列：

aacacccuuccagcaagcggucucaccgaggggcgcgugacccgcaaagacccucgcuucagggaucuggugcccaacuacaacgcagacaucguguucaaggacgaggaaggcacgggcgccgaccgccucaugacgcagaggugcaaggagaagcugaacacgcuggccaucucggugaugaaccaguggcccggcgugaagcugcgggugacggagggcugggacgaggaggggcaccacgccguggacucgcugcacuacgagggccgagccgucgacgucaccaccuccgacagggaucgagccaaguacggcaugcuggcgcgccucgccguggaggcgggcuucgacuggg(seqidno.17)；

靶向沉默ptc基因的dsrna序列：

uucgacgagcacuauauagaccaaauauuccuaaacauuaucccuugugccaucauuacuccauuagacuguuuuugggaagcuucgaagcuucugggaccagauuauccuguucaaauaccaggcgugggcuccaaggugcgcuggacgaaccuuaacccacaacaacuaauccaacaaaugaagacugucgaauugguauucccuuucgagacucucgaagacuacaugaaaagggcggggauaagcacgggcuaccaagagaagccgugucucgaccccuccgaccccgaaugcccgaccaccgcccccaacaaguuauccggacagauaccugacaucggggcagagcugacaggaggcugcuacggauaugcagccaaauauaugcacuggccugaggaccugguggugggcggagccacacgaaauaagacuggacacauacaaagggcgcgugcauugcagacuguggugcaacuaaugggagagagagagauguaugaauucuggucuaaaacguacaaaguccaucacuuggauuggacacaagaaaaagcugcuuuaauucucgagacauggcagaggc(seqidno.18)；

靶向沉默smo基因的dsrna序列：

acuuguaauuuucuauaucaauugcuguuuuuuggugucauguauuggaugguuggcacaguuuauuccuggugcacgugaaggcauuauaugccggaaagaugguacuuuaaggaugucagaaccuaauacuggagagaauuugucaugugugauaauauuuguauugguguauuacuucaucauggcugccuuugugugguuuguaauucucacguacacauggcacaucaguuuucaagcucuagguaaaauucaagagcgaauugauaagaaaagugcuuauuuucacaugguggcuuggucauugccucuaguucuuacuauuacaacuauggcacuaggggaaauagauggcgacaguguugcugguaucuguuuugugggauauguuaaucaugcugcacgagcaggauucuugcucguccccauuacaacaguacuuuuuguuggaggauacuuucucacuaggggucuuauaacacuggugagacuaaaaauuaacagucaagaaauuaucucagaaagagcaagugccaagauaagagaaaccauugugcgcaugggucucuuuucacuguuuacauuugucuuuguguu(seqidno.19)；

靶向沉默ci基因的dsrna序列：

gaucucgucaagcacaucaacaaugaccacauucaugccaacaagaagucguuugugugccgcugggaagacuguucaagggacgagaagcccuucaaagcgcaguauaugcugguggugcacaugcgacgacacacaggggagaagccccacaaauguacauuugaagggugcuacaaagccuauucgaggcuggagaaccugaagacgcaccucagaucucauacaggagaaaaacccuacaugugugaguauccuggaugcagcaaagccuucagcaaugcguccgaccgugccaaacaccagaaccgcacgcacuccaacgagaaaccguacguguguaaggccccgggcugcaccaagagguacacagaccccaguucgcugcggaagcacgugaagacagugcacggugccgaguucuaugccaacaagaagcacaagggagcuggcgacggcgccgaggauggcucuggaggggcgggaggccuacuggacagcagcccgcacagcgaggagaugaugagcggcaagacggcc(seqidno.20)。

一种促进美洲大蠊断肢再生的试剂盒，其特征在于，该试剂盒含有定量检测hh、ptc、smo或/和cirna的试剂。

优选的，所述定量检测hh、ptc、smo或/和cirna的试剂为定量检测hh、ptc、smo或/和ci的rna转录水平的试剂。

优选的，所述的定量检测hh、ptc、smo或/和cirna转录水平的试剂选自定量检测hh、ptc、smo或/和ci的引物。

优选的，所述的定量检测hh、ptc、smo或/和ci的引物为：

hh：

hhqpcrf:5’-gagtccagagcgcacatcca-3’(seqidno.21)；

hhqpcrr:5’-tcctccgagaagcagccacc-3’(seqidno.22)；

ptc.：

ptcqpcrf:5’-atgcgtttctgagtgcg-3’(seqidno.23)；

ptcqpcrr:5’-gtaatttctgattggtggg-3’(seqidno.24)；

smo：

smoqpcrf:5’-gtaagatccgcaaggt-3’(seqidno.25)；

smoqpcrr:5’-cgtctgatggtggtaat-3’(seqidno.26)；

ci.：

ciqpcrf:5’-aatggagggctcgaagat-3’(seqidno.27)；

ciqpcrr:5’-tactgtcacggcgtaggtt-3’(seqidno.28)。

一种阻断美洲大蠊断肢再生的试剂盒，该试剂盒含有抑制hh、ptc、smo或/和ci表达的试剂。

优选的，所述抑制hh、ptc、smo或/和ci表达的试剂为抑制hh、ptc、smo或/和ci的rna转录水平的试剂。

优选的，所述抑制hh、ptc、smo或/和ci的rna转录水平的试剂选自沉默hh、ptc、smo或/和ci表达的sirna、沉默hh、ptc、smo或/和ci表达的shrna和沉默hh、ptc、smo或/和ci表达的dsrna的至少一种。

优选的，所述的沉默hh、ptc、smo或/和ci的表达的dsrna选自以下序列：

靶向沉默hh基因的dsrna序列：

aacacccuuccagcaagcggucucaccgaggggcgcgugacccgcaaagacccucgcuucagggaucuggugcccaacuacaacgcagacaucguguucaaggacgaggaaggcacgggcgccgaccgccucaugacgcagaggugcaaggagaagcugaacacgcuggccaucucggugaugaaccaguggcccggcgugaagcugcgggugacggagggcugggacgaggaggggcaccacgccguggacucgcugcacuacgagggccgagccgucgacgucaccaccuccgacagggaucgagccaaguacggcaugcuggcgcgccucgccguggaggcgggcuucgacuggg(seqidno.17)；

靶向沉默ptc基因的dsrna序列：

uucgacgagcacuauauagaccaaauauuccuaaacauuaucccuugugccaucauuacuccauuagacuguuuuugggaagcuucgaagcuucugggaccagauuauccuguucaaauaccaggcgugggcuccaaggugcgcuggacgaaccuuaacccacaacaacuaauccaacaaaugaagacugucgaauugguauucccuuucgagacucucgaagacuacaugaaaagggcggggauaagcacgggcuaccaagagaagccgugucucgaccccuccgaccccgaaugcccgaccaccgcccccaacaaguuauccggacagauaccugacaucggggcagagcugacaggaggcugcuacggauaugcagccaaauauaugcacuggccugaggaccugguggugggcggagccacacgaaauaagacuggacacauacaaagggcgcgugcauugcagacuguggugcaacuaaugggagagagagagauguaugaauucuggucuaaaacguacaaaguccaucacuuggauuggacacaagaaaaagcugcuuuaauucucgagacauggcagaggc(seqidno.18)；

靶向沉默smo基因的dsrna序列：

acuuguaauuuucuauaucaauugcuguuuuuuggugucauguauuggaugguuggcacaguuuauuccuggugcacgugaaggcauuauaugccggaaagaugguacuuuaaggaugucagaaccuaauacuggagagaauuugucaugugugauaauauuuguauugguguauuacuucaucauggcugccuuugugugguuuguaauucucacguacacauggcacaucaguuuucaagcucuagguaaaauucaagagcgaauugauaagaaaagugcuuauuuucacaugguggcuuggucauugccucuaguucuuacuauuacaacuauggcacuaggggaaauagauggcgacaguguugcugguaucuguuuugugggauauguuaaucaugcugcacgagcaggauucuugcucguccccauuacaacaguacuuuuuguuggaggauacuuucucacuaggggucuuauaacacuggugagacuaaaaauuaacagucaagaaauuaucucagaaagagcaagugccaagauaagagaaaccauugugcgcaugggucucuuuucacuguuuacauuugucuuuguguu(seqidno.19)；

靶向沉默ci基因的dsrna序列：

gaucucgucaagcacaucaacaaugaccacauucaugccaacaagaagucguuugugugccgcugggaagacuguucaagggacgagaagcccuucaaagcgcaguauaugcugguggugcacaugcgacgacacacaggggagaagccccacaaauguacauuugaagggugcuacaaagccuauucgaggcuggagaaccugaagacgcaccucagaucucauacaggagaaaaacccuacaugugugaguauccuggaugcagcaaagccuucagcaaugcguccgaccgugccaaacaccagaaccgcacgcacuccaacgagaaaccguacguguguaaggccccgggcugcaccaagagguacacagaccccaguucgcugcggaagcacgugaagacagugcacggugccgaguucuaugccaacaagaagcacaagggagcuggcgacggcgccgaggauggcucuggaggggcgggaggccuacuggacagcagcccgcacagcgaggagaugaugagcggcaagacggcc(seqidno.20)。

抑制hh、ptc、smo或/和ci表达的试剂在制备防治美洲大蠊的药物中的应用。

一种检测美洲大蠊断肢再生相关的基因，所述的基因为hh、ptc、smo或/和ci；所述的hh核苷酸序列如seqidno.1所示，ptc核苷酸序列如seqidno.2所示，smo核苷酸序列如seqidno.3所示，ci核苷酸序列如seqidno.4所示。

下面结合具体实施例对本发明作进一步的说明。

实施例1断肢再生相关hedgehog信号通路基因的数据库搜索

基于美洲大蠊基因组数据库，采用生物信息学方法对美洲大蠊hedgehog信号通路中的hh、ptc、smo和ci基因进行搜索，经过序列分析及比对后，获得美洲大蠊hedgehog信号通路中的hh、ptc、smo和ci基因序列，分别为seqidno.1、seqidno.2、seqidno.3和seqidno.4。

hh基因的核苷酸序列如seqidno.1所示：

atgagggcgggggcgctgatgtggggggcgctgctgctgctggcggcgtcggcgtcgggctgcgggcccggacgagggggagggcgccgtcccgtgccccgcaagttgacccccctcgtgttcaaacagcacgtgcccaacgtaagcgaaaacacccttccagcaagcggtctcaccgaggggcgcgtgacccgcaaagaccctcgcttcagggatctggtgcccaactacaacgcagacatcgtgttcaaggacgaggaaggcacgggcgccgaccgcctcatgacgcagaggtgcaaggagaagctgaacacgctggccatctcggtgatgaaccagtggcccggcgtgaagctgcgggtgacggagggctgggacgaggaggggcaccacgccgtggactcgctgcactacgagggccgagccgtcgacgtcaccacctccgacagggatcgagccaagtacggcatgctggcgcgcctcgccgtggaggcgggcttcgactgggtgtactacgagtccagagcgcacatccactgctccgtcaagtcggagtcgtcgcaagcggccaagtcgggtggctgcttctcggaggagacgaccgtcgagacgtcgagcggcgcgcgtgtgcgcctggccgacctgcgagtcggagatcgcgtcctggcgctggacctggcgtcgggacgcctcacgtacagcgaagttgatgcagggagtgttcgcgccactcacgcgagagggcaccatcgtcgcggacggcgtcgtggcgtcgtgctacgcggtggtggacagtcagtggctcgcacacctcacctacgcgccttttag(seqidno.1)；

部分ptc基因的核苷酸序列如seqidno.2所示：

attacttggagtctaaaagacatgtgttactcgcccagtattcctgacttcgacgagcactatatagaccaaatattcctaaacattatcccttgtgccatcattactccattagactgtttttgggaagcttcgaagcttctgggaccagattatcctgttcaaataccaggcgtgggctccaaggtgcgctggacgaaccttaacccacaacaactaatccaacaaatgaagactgtcgaattggtattccctttcgagactctcgaagactacatgaaaagggcggggataagcacgggctaccaagagaagccgtgtctcgacccctccgaccccgaatgcccgaccaccgcccccaacaagttatccggacagatacctgacatcggggcagagctgacaggaggctgctacggatatgcagccaaatatatgcactggcctgaggacctggtggtgggcggagccacacgaaataagactggacacatacaaagggcgcgtgcattgcagactgtggtgcaactaatgggagagagagagatgtatgaattctggtctaaaacgtacaaagtccatcacttggattggacacaagaaaaagctgctttaattctcgagacatggcagaggcgattttcagaggaggttcgacggtccctaacaactgataatagcacggcatcttacaacatgtatgcattctcgactgtaacattaaacgatatactaagtcaattttcagagctaagcgtagtcaaagttgttgtcggatatatttttatgttggcatatgcaggagtctctctcctcaggtggcacgatccagttcgttcacaagctggtgtggggcttgctggagtactcttggtgtccatcacagtcgcagcaggtcttgggttctgtgctctgctgggcattgccttcaatgcttccaccacacaaattgtgccattcctcgcattgggccttggtgtcgatgatatgttcttgttgacacatacatatgcagagcaggaacaaggccagggaggttctcacaatcagtaccaacctgacgaacagaccggtgttgtgctcaagaggactggactcagtatcctcctcacctcactcagcaacatatgcgctttctttgctgctgccatcatccccatcccagcactgcgggtcttctcgttacaggctgcagtgctggtaatgttcaacctgggctgcacgctgctcgtcttcccagcagtggtcagtctcgatttaagaagaagacggtcaggccgtgtggacattctatgctgctgcctgcctgggggtcccacaggctctgaatggccttgctgtactctgcctaacgtcccaacaggccatctcgttcacgtgcctagaagacggagagggcaggttgtgccagaacctccaaggatgctccaaacaatcacgagagctctgccccccgacagacagcagactgttactgtgctggcacagccggcagctcctacacagccgcagacacccgaatgctgggtagggtccacaagagaactgttaacgctagatcctgagagggggcagggagctgttggaggttgtggcggccatcacatcatgcacgaatgttggagttggtcactcacaaggctagcagctacgcattatgcacctcttatcaccaaatcaccagtgaaggttgtgtcgatggtggtgctggtggtgatcctggcaggaagcgtttggggcattaccaaagtcacggacggcctggacttaactgacatagtccctcagaacacagacgagcatgcgtttctgagtgcgcagggtcggttcttcggcttctacaacatgtatgccgtgacgcagggtgactttgagtaccccaccaatcagaaattactgtatgaataccatgaggcattcatgcgcgtccctaacatcatcaagaacgatgatggtggtcttccagagttctggctcagtcttttccgggactggcttataggactccagaaggctttcgaccgagactggtctcgggggagtattacacaggaacggtggttctctaacgcatcagatgaggggatccttgcctacaagctgctggttcagactgggcatgtcgacaaccccatcgacaagtcacttattactcaggttaggctggttgatgcagaaggaatcatcaatcctaaagctttctacaactacctcagtgcctgggcatctaatgatgctttggcatacggagcttcgcaggcaaacctgcgaccagagccaaaatcctggtaccacgctacaaaagacttcgagctcaagatacccaagtccgcacctctggtgtacacccagctgcctttctatttacacggcctgggtgacacacaacacatcactaatcttattgctcaggtgcgggcgttgtgtcagaagttcgaagagcgaggactccccaactttccctcagggattcccttcctcttctgggagcaatacctcgggctacggcagtcgctgggtctggctttgctctgtgcattagctgcagtcttcctggtggtggctgtattgttgctcaatctgtgggctgcgaccctagtggtgctggccctggcagccatggtgcttcagctgcttggagtcatgggggccctcggaatcaagctcagtgcaatacctgctgtcctccttattgttgctgtgggcatcggcgtccacttcacagtacacatctgcctggggtttgtgacaagcataggaggaagagatcgacggacacgtctcgcattggagcagatgtttgcccctgtcactcacggagccctcacgacactgcttggtatctttatgctggccttttcagagtttgacttcattgtgaggtatttcttctacgtgctgcttgctctagtggtcgttggtctccttactggtctagtcttctttccagttttgctgtcgctaataggaccagctgccgaggttgtaccacatgagttccctgatcgcatttcgacacctacacctcctccatctcctgtgaggacgaggattggttcatgtcgggtagggaaggctccaccacagcccaggagaggtcacgggggtatgcagtgcaaccgcttacatgcagagccatccctcacgaccatcacagaggaagcgaactcatggcactcaactcatgagattgttgtacagcctgagctggtggtcgagactaccacttatggaacgcctcagactgcaagtcaaactacgggaggcagtggtgaaggttctggagcaggtcagacctcgacatctccccctcctcctccagggtctcagtcacacgtcaccacaaaggttacagccacaactctcgtcaaggttgaggtccacgcaccactgccaggagctgttgaacgtgatttgggaagcagcagtagcagcaataagagtcgctcaagtcgtcggagagactcaagctcgggatccgaccacgattcttcttcagataacaactcaggcaaacaaggtggtaccaccgcttcctag(seqidno.2)；

smo基因的核苷酸序列如seqidno.3所示：

atgaagatcttacataaggaaattatctatttaaaaaaatttcggctggcaatgcaaggattttttgcatcgaagttcgtcgtcttcgtattaactgctattattgagcctcttcatgtcgtgggagaatttagaggaaatgtgactgttttagtaagatccgcaaggtatgaaggagatcatgcattccttcgggacaaattttcatcagctgattccatcatattaccaccatcagacgggaaaggaccgtactgcagacacccagctcgttgtgaacaacttaattatacaacctgcatgggagttaaacttccttatacatctactacacttgatttggtggaaaatttaggtacacaggaactcgtacaggaaaaattacaacagtggcaaggattacgtcacattccgaaatgttggactgtcgttcaaccatttctctgtgctttatacatgcccaaatgtgagaatgggacagtggatctgccatcacaggaaatgtgcaaaatgacagtgggcccatgtcgaatactggaatcttacaaaagttggccatcgttcttcagatgtgacgacacagttagatttccaccaatgtgtaagaatgatgcccgagagctgaagtacaatactactagtcagtgccagagtcctttggtgccaactgacaatactctagcattctatgatggaatagaaggatgtggcattcagtgccaaaatccattgcttacagaagacgaacatagacagatccatcagttaattgcttgggcaggaaccatttgccttctttttaatatcttcacagtggtaacctttttcattgattggcgttctgcaaacaagtatccagcacttgtaattttctatatcaattgctgttttttggtgtcatgtattggatggttggcacagtttattcctggtgcacgtgaaggcattatatgccggaaagatggtactttaaggatgtcagaacctaatactggagagaatttgtcatgtgtgataatatttgtattggtgtattacttcatcatggctgcctttgtgtggtttgtaattctcacgtacacatggcacatcagttttcaagctctaggtaaaattcaagagcgaattgataagaaaagtgcttattttcacatggtggcttggtcattgcctctagttcttactattacaactatggcactaggggaaatagatggcgacagtgttgctggtatctgttttgtgggatatgttaatcatgctgcacgagcaggattcttgctcgtccccattacaacagtactttttgttggaggatactttctcactaggggtcttataacactggtgagactaaaaattaacagtcaagaaattatctcagaaagagcaagtgccaagataagagaaaccattgtgcgcatgggtctcttttcactgtttacatttgtctttgtgttggtgacctttgtgtgtcatgtgtacgaatttcatcatcacagtgactggagagaaagtttcagaaaattcatagtttgccgaattatgagcagtggtaatgcagacacaaagagatgtaagatagagtctcgaccaagtctctcaatgatgcaactgcatatattggtgatgttcttggcaggtgtcataatgtcatcatgggtatggacgagctccacagttgacacctggagaagatgtctcagaaggacttttaatactcaagcagaagaacccgtcagattgaagaagcataaagttattgctcaagcatttgcaaaaaggaagacattcaacaatgcaggtcgcctctccattagttttcatagcacccatgaggatcctgtaggtttaaattttgatatgaacagtattgcatcacaagacttcagctcaacctgggcagcagcattaccgaagctgatgacacgacgtggtgctcttattggtgttgccactggctcaaactcaagccagcatcgaaactcgatagactccgaaatcagttacagtgtgagaagggtgtcagttgaatcacgtcgccactcattggactcccaagtgtctgtacagattgctgaagtaacagcaacacgaaaagctgcagccactcctatgcctgcagcaagtactgcaactgtgtctggtggaaggggcagaaaaactcgcagtcgaagacaaagaagagagtttggacgtattcgaagtagtcgtgtgggacctcttttgattaggagaggcagtacaacatcacaagaaagtcagttaggagcacaaattctgtcagcacttactctagcaaatacaactcttccatcatttgttcccaatctaaagaaacgaaggactggcaatgcaggtttggacagctctaaagtcattctgccattcgccctgccagatcagagtcttgatgtaagtgatgaagaacaaaacagtgatgagaagatttctgggacaataaaaaaatttgatatctttctttgtagagaagctaatgatgtggatttggaggatattaatgacagtgatggggagatcagtgatggaatgccagcagaaaacgatatgaaagttgtaatagcaagtaagtttaatgaagatgagaatgatgacagaaatgatgatattaataataaacggaaaaacagaagtaccgaaattaatgcaaacactgtaagtgcaaatgatggcatccatattgaggaagatcagaaaaattccattgttgatatgggttcccatcatagtataagtgcagaagttgatgattatcgaagtagaacttcagagggaaacaggtatagaaataactatggcgattgtaaaaacataaatatacctgatgaagtgaaattgtatcttacagaaggctctgatgatgaacctccacaaaaattgaatttcgttccttctgtagatattggaggtgtttcagatggttccacatcattttgtcctgagctttctcatctagttgtgcagtcctcccattcaggtctgtcacaaggaagccctcataactttgatgcaagtaaaacgcgtgttatatcagttacaccctacaaccagagcaacaatgaaggacgtgaagttggaactcagaccacattgtcaatagactgtatcgagatgcaagaactaaaggggaaaaatataatacatccttcaccacgagcatgtgcaagccaaagtactcgggtatctccaccttcatcccggccttcagttcctcgatcatattctcacggtcattttgaaaagaatcagacttcacttgttaataaaatagctgataatgtgttatcaagtccagcaggtagagcactgaactacagttacaatgtacagcgtttagtgcaggaactttcagaaggcagtcatgctggacctccagttaggccggagaggagaaagagtgctcagggcaagaacagtgtaagaagtaagagtgctgataacaagaacgttgaattgcagtggaatccggttgcattgtga(seqidno.3)；

ci基因的核苷酸序列如seqidno.4所示：

atgacagcgagacgagcggcgcggcccgcgagtcatcttgtcacactgtcgcctgtctccatggcaaccctcgccgagagtcttcaactatttctagaatttggcccacagtcacttccaactggtggagtgagcggggagggactttccacctacttaccagccccggttatttccggcgagggcaccaacaaacatccataccgaacaccactccaacctaggagccctcgcaacgtaaacatcttgatccagacgccaccaacagcagaagtgagaaaatccacgttagagcattgggactccacggcagcagttaccaacttgtacgtctcagcactgtcacagctattgttgcccagcagtggctcaggttcgaatctctcggagctccaattcctggcggcgtcccggcgcgccgcggcggcagctgcaggggcggtgttgcccccgggggcagctggggccgcgggggccgccggggctgcagggggtggcgtggcagtaccaccaccacccccacatcatcccccgggccccgacttccaccccgcgtacaggctcaacccttacatggagcacctgtactcatctctccagcagcattctagtcctgccgcctctattcacggtttgggtctcccaccggactacctaggctcgcgcgcactgacagagctccagcctcctccttccactatcggtagctcggaaagaaagaaagaagaaagggaagatgatatagaagaaagaaggaagaaaagaaagaaggaagtaagtaaagcaagttccagacttacgagtcctcggccgagcagtttgcgacagagtcggaagagggccctgtcgtcatcgccatattcggactcgttcgacatcaattcgatgatacggttctctccgaactccctcgtctccatcgtgaacggatctcggagctccagtgccagtggttcatacggccacctctcagcaggggctatcagcccggctttgggcatgcaccccagcatggcgccccacctccagcagcttcaagcccaccttctacggactgcggcgggtagtttgctgccccctctgccaccccacccgcaggcacagacccaggccccgggagggctgttttctcttcctcaccatcccttgcagccctccacaggacattccatttctaaaacagagctgtcaggggatgcttgcaagaagctggactcacctgtttctagtagtgtcgtggaggcggacactagtgctcctggtggaatcagttcgcaccaacggaaggcgagaataaagagggaacctgtcactgccaatgccacaacagcgtctgtaggtgctggtgctgtgtccggagcattaggctcagactgcagtagcagtaagtgttcaccaggtggtgctgattctggcgatcctcttaaagatgaacctggcgacttcattgaaaccaactgccattggcgggactgtggacttgagtttcctacacaggacgatctcgtcaagcacatcaacaatgaccacattcatgccaacaagaagtcgtttgtgtgccgctgggaagactgttcaagggacgagaagcccttcaaagcgcagtatatgctggtggtgcacatgcgacgacacacaggggagaagccccacaaatgtacatttgaagggtgctacaaagcctattcgaggctggagaacctgaagacgcacctcagatctcatacaggagaaaaaccctacatgtgtgagtatcctggatgcagcaaagccttcagcaatgcgtccgaccgtgccaaacaccagaaccgcacgcactccaacgagaaaccgtacgtgtgtaaggccccgggctgcaccaagaggtacacagaccccagttcgctgcggaagcacgtgaagacagtgcacggtgccgagttctatgccaacaagaagcacaagggagctggcgacggcgccgaggatggctctggaggggcgggaggcctactggacagcagcccgcacagcgaggagatgatgagcggcaagacggccagtctgtccagccctagtgtcaagtcagaggaggccagttcgcctgcccagcagggaagtcctttgagtgtgactcagcatggcaacaatggtttagatgagccaatcagcgactcaaatgtgagcactaccaaccatgcgttagcagcagtggaggcagcagaagaatgggtggacgaggctgaagaacttgagctgccagagcttcacgtgagggctgtagtgggtggaggggcggaccccattggaccgccagcggaccatactgcacatcgcatgaggcggctgcaagctaaactacctccactaccaaatattgcaggtggccgacgagggattcatcttggcgacctgcataagcatttcactgatctcaaaatggagggctcgaagatctcaggcactgtgacacagacacagctgatagagctgcagattccactgggccctggccagaagcagctgcagcaacaggccaacctacgccgtgacagtaactccaccgtcagcacgtactacggcagcatgcggtcaggagacctgggctccagccgcagaagtagccaggcctcgcaggcttcagcgctgtcttgtgcaaggccaggcagtggccaggtgggctcgtcgtatgacccgatctcagccggcagctcacggaggtccagtcagctgagcaacacgggcggcataagtaccatggggcacctcagcagccacctccagcgcctgcactccagggcgcaggacccactctactgcacatccaacctcgtcctgcagacgcaaaacatgtccctgcaacaacaacggcgtcagcagctgcaacagcagttgtcagggcctggcaactggctgccacaaccaggagccaacaacagctccagcaaccccgtggacaccagacgcatgtcggagccatgcagagggggctctgataggtcaacacccccaccccgtcctcacagcgtggtcctgccgcccatcgcacgagcccaggagcaccaccctaaccaggaggtggtactggatgaagtcgccgaaggggagatggtggagaagaacctcgtgatcccagacgagatgctgcacttcctcagtcaggtgagccacctcacaggacgacattcctatgttattgttgttgttttctaa(seqidno.4)。

根据hh基因、ptc基因、smo基因或/和ci基因的核苷酸序列，应用dna序列比对，设计了一组灵敏度高且特异性强的引物，序列如下：

hh.t7引物：

hhf:5’-taatacgactcactataggttcaaacagcacgtgcccaac-3’(seqidno.5)；

hhr:5’-taatacgactcactataggagtggatgtgcgctctggac-3’(seqidno.6)；

ptc.t7引物:

ptcf:5’-taatacgactcactatagggacatgtgttactcgcccag-3’(seqidno.7)；

ptcr:5’-taatacgactcactataggttagggaccgtcgaacctcc-3’(seqidno.8)；

smot7引物：

smof:5’-taatacgactcactataggtgattggcgttctgcaaaca-3’(seqidno.9)；

smor:5’-taatacgactcactataggaattcgtacacatgacacaca-3’(seqidno.10)；ci.t7引物:

cif:5’-taatacgactcactatagggactgtggacttgagtttcc-3’(seqidno.11)；

cir:5’-taatacgactcactataggctctgacttgacactagggc-3’(seqidno.12)。

以美洲大蠊hh、ptc、smo和ci的cdna序列作为模板，分别用引物将相对应的靶序列进行扩增，引物两头都含有t7启动子，将pcr扩增产物克隆到ptopo载体中，分别获得含有hh、ptc、smo和ci靶序列的质粒，即名为ptopo-hh、ptopo-ptc、ptopo-smo和ptopo-ci的质粒。

实施例2dsrna的合成

靶向沉默hh基因的dsrna的制备方法：以ptopo-hh为模板，用两端含有t7启动子的引物seqidno.5和seqidno.6进行pcr扩增，然后将pcr扩增产物通过t7ribomax^tmexpressrnaisystem转录试剂盒(promega)合成dsrna，序列如seqidno.17所示；

靶向沉默ptc基因的dsrna的制备方法：以ptopo-ptc为模板，用两端含有t7启动子的引物seqidno.7和seqidno.8进行pcr扩增，然后以pcr产物为模板使用t7ribomax^tmexpressrnaisystem转录试剂盒(promega)进行dsrna的合成，dsrna的核苷酸序列为seqidno.18；

靶向沉默smo基因的dsrna的制备方法：以ptopo-smo为模板，用两端含有t7启动子的引物seqidno.9和seqidno.10进行pcr扩增，然后以pcr产物为模板使用t7ribomax^tmexpressrnaisystem转录试剂盒(promega)进行dsrna的合成，dsrna的核苷酸序列为seqidno.19；

靶向沉默ci基因的dsrna的制备方法：以ptopo-ci为模板，用两端含有t7启动子的引物seqidno.11和seqidno.12进行pcr扩增，然后以pcr产物为模板使用t7ribomax^tmexpressrnaisystem转录试剂盒(promega)进行dsrna的合成，dsrna的核苷酸序列为seqidno.20。

dsrna靶向沉默的hh基因的核苷酸序列如下所示：

aacacccttccagcaagcggtctcaccgaggggcgcgtgacccgcaaagaccctcgcttcagggatctggtgcccaactacaacgcagacatcgtgttcaaggacgaggaaggcacgggcgccgaccgcctcatgacgcagaggtgcaaggagaagctgaacacgctggccatctcggtgatgaaccagtggcccggcgtgaagctgcgggtgacggagggctgggacgaggaggggcaccacgccgtggactcgctgcactacgagggccgagccgtcgacgtcaccacctccgacagggatcgagccaagtacggcatgctggcgcgcctcgccgtggaggcgggcttcgactggg(seqidno.13)；

dsrna靶向沉默的ptc基因的核苷酸序列如下所示：

ttcgacgagcactatatagaccaaatattcctaaacattatcccttgtgccatcattactccattagactgtttttgggaagcttcgaagcttctgggaccagattatcctgttcaaataccaggcgtgggctccaaggtgcgctggacgaaccttaacccacaacaactaatccaacaaatgaagactgtcgaattggtattccctttcgagactctcgaagactacatgaaaagggcggggataagcacgggctaccaagagaagccgtgtctcgacccctccgaccccgaatgcccgaccaccgcccccaacaagttatccggacagatacctgacatcggggcagagctgacaggaggctgctacggatatgcagccaaatatatgcactggcctgaggacctggtggtgggcggagccacacgaaataagactggacacatacaaagggcgcgtgcattgcagactgtggtgcaactaatgggagagagagagatgtatgaattctggtctaaaacgtacaaagtccatcacttggattggacacaagaaaaagctgctttaattctcgagacatggcagaggc(seqidno.14)；

dsrna靶向沉默的smo基因的核苷酸序列如下所示：

acttgtaattttctatatcaattgctgttttttggtgtcatgtattggatggttggcacagtttattcctggtgcacgtgaaggcattatatgccggaaagatggtactttaaggatgtcagaacctaatactggagagaatttgtcatgtgtgataatatttgtattggtgtattacttcatcatggctgcctttgtgtggtttgtaattctcacgtacacatggcacatcagttttcaagctctaggtaaaattcaagagcgaattgataagaaaagtgcttattttcacatggtggcttggtcattgcctctagttcttactattacaactatggcactaggggaaatagatggcgacagtgttgctggtatctgttttgtgggatatgttaatcatgctgcacgagcaggattcttgctcgtccccattacaacagtactttttgttggaggatactttctcactaggggtcttataacactggtgagactaaaaattaacagtcaagaaattatctcagaaagagcaagtgccaagataagagaaaccattgtgcgcatgggtctcttttcactgtttacatttgtctttgtgtt(seqidno.15)；

dsrna靶向沉默的ci基因的核苷酸序列如下所示：

gatctcgtcaagcacatcaacaatgaccacattcatgccaacaagaagtcgtttgtgtgccgctgggaagactgttcaagggacgagaagcccttcaaagcgcagtatatgctggtggtgcacatgcgacgacacacaggggagaagccccacaaatgtacatttgaagggtgctacaaagcctattcgaggctggagaacctgaagacgcacctcagatctcatacaggagaaaaaccctacatgtgtgagtatcctggatgcagcaaagccttcagcaatgcgtccgaccgtgccaaacaccagaaccgcacgcactccaacgagaaaccgtacgtgtgtaaggccccgggctgcaccaagaggtacacagaccccagttcgctgcggaagcacgtgaagacagtgcacggtgccgagttctatgccaacaagaagcacaagggagctggcgacggcgccgaggatggctctggaggggcgggaggcctactggacagcagcccgcacagcgaggagatgatgagcggcaagacggcc(seqidno.16)。

靶向沉默hh基因的dsrna序列如seqidno.17所示：

aacacccuuccagcaagcggucucaccgaggggcgcgugacccgcaaagacccucgcuucagggaucuggugcccaacuacaacgcagacaucguguucaaggacgaggaaggcacgggcgccgaccgccucaugacgcagaggugcaaggagaagcugaacacgcuggccaucucggugaugaaccaguggcccggcgugaagcugcgggugacggagggcugggacgaggaggggcaccacgccguggacucgcugcacuacgagggccgagccgucgacgucaccaccuccgacagggaucgagccaaguacggcaugcuggcgcgccucgccguggaggcgggcuucgacuggg(seqidno.17)；

靶向沉默ptc基因的dsrna序列如seqidno.18所示：

uucgacgagcacuauauagaccaaauauuccuaaacauuaucccuugugccaucauuacuccauuagacuguuuuugggaagcuucgaagcuucugggaccagauuauccuguucaaauaccaggcgugggcuccaaggugcgcuggacgaaccuuaacccacaacaacuaauccaacaaaugaagacugucgaauugguauucccuuucgagacucucgaagacuacaugaaaagggcggggauaagcacgggcuaccaagagaagccgugucucgaccccuccgaccccgaaugcccgaccaccgcccccaacaaguuauccggacagauaccugacaucggggcagagcugacaggaggcugcuacggauaugcagccaaauauaugcacuggccugaggaccugguggugggcggagccacacgaaauaagacuggacacauacaaagggcgcgugcauugcagacuguggugcaacuaaugggagagagagagauguaugaauucuggucuaaaacguacaaaguccaucacuuggauuggacacaagaaaaagcugcuuuaauucucgagacauggcagaggc(seqidno.18)；

靶向沉默smo基因的dsrna序列如seqidno.19所示：

acuuguaauuuucuauaucaauugcuguuuuuuggugucauguauuggaugguuggcacaguuuauuccuggugcacgugaaggcauuauaugccggaaagaugguacuuuaaggaugucagaaccuaauacuggagagaauuugucaugugugauaauauuuguauugguguauuacuucaucauggcugccuuugugugguuuguaauucucacguacacauggcacaucaguuuucaagcucuagguaaaauucaagagcgaauugauaagaaaagugcuuauuuucacaugguggcuuggucauugccucuaguucuuacuauuacaacuauggcacuaggggaaauagauggcgacaguguugcugguaucuguuuugugggauauguuaaucaugcugcacgagcaggauucuugcucguccccauuacaacaguacuuuuuguuggaggauacuuucucacuaggggucuuauaacacuggugagacuaaaaauuaacagucaagaaauuaucucagaaagagcaagugccaagauaagagaaaccauugugcgcaugggucucuuuucacuguuuacauuugucuuuguguu(seqidno.19)；

靶向沉默ci基因的dsrna序列如seqidno.20所示：

gaucucgucaagcacaucaacaaugaccacauucaugccaacaagaagucguuugugugccgcugggaagacuguucaagggacgagaagcccuucaaagcgcaguauaugcugguggugcacaugcgacgacacacaggggagaagccccacaaauguacauuugaagggugcuacaaagccuauucgaggcuggagaaccugaagacgcaccucagaucucauacaggagaaaaacccuacaugugugaguauccuggaugcagcaaagccuucagcaaugcguccgaccgugccaaacaccagaaccgcacgcacuccaacgagaaaccguacguguguaaggccccgggcugcaccaagagguacacagaccccaguucgcugcggaagcacgugaagacagugcacggugccgaguucuaugccaacaagaagcacaagggagcuggcgacggcgccgaggauggcucuggaggggcgggaggccuacuggacagcagcccgcacagcgaggagaugaugagcggcaagacggcc(seqidno.20)。

实施例3dsrna活体注射影响美洲大蠊断肢再生

为了验证筛选出的hedgehog信号通路中的hh、ptc、smo和ci基因在美洲大蠊断肢再生过程中所发挥的功能，对刚断肢的美洲大蠊进行dsrna注射的方式来实现。采用不靶向美洲大蠊任何基因的ck作为dsrna注射处理的阴性对照。

步骤如下：首先挑选刚蜕皮的美洲大蠊幼虫(d0)，继续培养一天使其适应周围环境，然后对其进行第一次dsrna注射(d1)，24小时后对美洲大蠊的一侧后肢进行截肢手术处理(d2)，就是保留完整的基节和转节，将腿节、胫节和跗节一起剪掉(见图1a)，然后在接下来的d4和d7天进行另外两次的dsrna干扰，每次注射3ug。注射步骤如下：将美洲大蠊幼虫麻醉后用胶带固定，然后使用显微注射方法将dsrna溶液注射到美洲大蠊的腹部。注射后持续观察dsrna处理对幼虫断肢再生的影响直到下次蜕皮。

结果发现hh、ptc、smo和ci基因被沉默后都显著影响美洲大蠊的断肢再生能力，新生成的腿无法正常行使爬行功能，分别出现明显畸形或部分缺失，结果如图1b。证明hh、ptc、smo和ci基因和美洲大蠊的断肢再生能力存在关系。

实施例4hedgehog信号通路hh、ptc、smo和ci基因沉默检测

在注射第二次dsrna后的24h检测hh、ptc、smo和ci沉默效率，提取美洲大蠊腿节、胫节和跗节的总rna，每组设置3个生物学重复，并反转录成第一链cdna，然后以此为模板，进行real-timepcr检测目的基因(hh、ptc、smo和ci)和管家基因(β-actin)的相对表达量，使用2^-△△ct法计算目的基因(hh、ptc、smo和ci)的沉默效率。

以β-actin做内参。所用qrt-pcr引物为：

hh：

hhqpcrf:5’-gagtccagagcgcacatcca-3’(seqidno.21)；

hhqpcrr:5’-tcctccgagaagcagccacc-3’(seqidno.22)；

ptc.：

ptcqpcrf:5’-atgcgtttctgagtgcg-3’(seqidno.23)；

ptcqpcrr:5’-gtaatttctgattggtggg-3’(seqidno.24)；

smo：

smoqpcrf:5’-gtaagatccgcaaggt-3’(seqidno.25)；

smoqpcrr:5’-cgtctgatggtggtaat-3’(seqidno.26)；

ci.：

ciqpcrf:5’-aatggagggctcgaagat-3’(seqidno.27)；

ciqpcrr:5’-tactgtcacggcgtaggtt-3’(seqidno.28)。

结果如图2所示，结果发现dsrna注射后，可以明显降低hh、ptc、smo和ci基因的表达量，达到沉默hh、ptc、smo和ci基因的目的。

综上所述，蟑螂断肢再生的一个重要过程就是再生腿的塑型，而hedgehog信号通路包括多个重要的成形素基因，如hh、ptc、smo和ci等，该通路某个基因的缺失将使得蟑螂无法进行正常的附肢再生，影响其正常行走能力，利用其重要生物学功能来进行害虫防治，省时省力，安全高效。dsrna注射到美洲大蠊的体腔，结果表明合成的dsrna可以特异性地沉默美洲大蠊hh、ptc、smo和ci基因的mrna表达，并导致美洲大蠊出现断肢再生。

以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含本发明的保护范围之内。

sequencelisting

<110>华南师范大学

<120>美洲大蠊断肢再生相关hedgehog信号通路基因及其dsrna的应用

<130>

<160>28

<170>patentinversion3.5

<210>1

<211>831

<212>dna

<213>人工序列

<400>1

atgagggcgggggcgctgatgtggggggcgctgctgctgctggcggcgtcggcgtcgggc60

tgcgggcccggacgagggggagggcgccgtcccgtgccccgcaagttgacccccctcgtg120

ttcaaacagcacgtgcccaacgtaagcgaaaacacccttccagcaagcggtctcaccgag180

gggcgcgtgacccgcaaagaccctcgcttcagggatctggtgcccaactacaacgcagac240

atcgtgttcaaggacgaggaaggcacgggcgccgaccgcctcatgacgcagaggtgcaag300

gagaagctgaacacgctggccatctcggtgatgaaccagtggcccggcgtgaagctgcgg360

gtgacggagggctgggacgaggaggggcaccacgccgtggactcgctgcactacgagggc420

cgagccgtcgacgtcaccacctccgacagggatcgagccaagtacggcatgctggcgcgc480

ctcgccgtggaggcgggcttcgactgggtgtactacgagtccagagcgcacatccactgc540

tccgtcaagtcggagtcgtcgcaagcggccaagtcgggtggctgcttctcggaggagacg600

accgtcgagacgtcgagcggcgcgcgtgtgcgcctggccgacctgcgagtcggagatcgc660

gtcctggcgctggacctggcgtcgggacgcctcacgtacagcgaagttgatgcagggagt720

gttcgcgccactcacgcgagagggcaccatcgtcgcggacggcgtcgtggcgtcgtgcta780

cgcggtggtggacagtcagtggctcgcacacctcacctacgcgccttttag831

<210>2

<211>3540

<212>dna

<213>人工序列

<400>2

attacttggagtctaaaagacatgtgttactcgcccagtattcctgacttcgacgagcac60

tatatagaccaaatattcctaaacattatcccttgtgccatcattactccattagactgt120

ttttgggaagcttcgaagcttctgggaccagattatcctgttcaaataccaggcgtgggc180

tccaaggtgcgctggacgaaccttaacccacaacaactaatccaacaaatgaagactgtc240

gaattggtattccctttcgagactctcgaagactacatgaaaagggcggggataagcacg300

ggctaccaagagaagccgtgtctcgacccctccgaccccgaatgcccgaccaccgccccc360

aacaagttatccggacagatacctgacatcggggcagagctgacaggaggctgctacgga420

tatgcagccaaatatatgcactggcctgaggacctggtggtgggcggagccacacgaaat480

aagactggacacatacaaagggcgcgtgcattgcagactgtggtgcaactaatgggagag540

agagagatgtatgaattctggtctaaaacgtacaaagtccatcacttggattggacacaa600

gaaaaagctgctttaattctcgagacatggcagaggcgattttcagaggaggttcgacgg660

tccctaacaactgataatagcacggcatcttacaacatgtatgcattctcgactgtaaca720

ttaaacgatatactaagtcaattttcagagctaagcgtagtcaaagttgttgtcggatat780

atttttatgttggcatatgcaggagtctctctcctcaggtggcacgatccagttcgttca840

caagctggtgtggggcttgctggagtactcttggtgtccatcacagtcgcagcaggtctt900

gggttctgtgctctgctgggcattgccttcaatgcttccaccacacaaattgtgccattc960

ctcgcattgggccttggtgtcgatgatatgttcttgttgacacatacatatgcagagcag1020

gaacaaggccagggaggttctcacaatcagtaccaacctgacgaacagaccggtgttgtg1080

ctcaagaggactggactcagtatcctcctcacctcactcagcaacatatgcgctttcttt1140

gctgctgccatcatccccatcccagcactgcgggtcttctcgttacaggctgcagtgctg1200

gtaatgttcaacctgggctgcacgctgctcgtcttcccagcagtggtcagtctcgattta1260

agaagaagacggtcaggccgtgtggacattctatgctgctgcctgcctgggggtcccaca1320

ggctctgaatggccttgctgtactctgcctaacgtcccaacaggccatctcgttcacgtg1380

cctagaagacggagagggcaggttgtgccagaacctccaaggatgctccaaacaatcacg1440

agagctctgccccccgacagacagcagactgttactgtgctggcacagccggcagctcct1500

acacagccgcagacacccgaatgctgggtagggtccacaagagaactgttaacgctagat1560

cctgagagggggcagggagctgttggaggttgtggcggccatcacatcatgcacgaatgt1620

tggagttggtcactcacaaggctagcagctacgcattatgcacctcttatcaccaaatca1680

ccagtgaaggttgtgtcgatggtggtgctggtggtgatcctggcaggaagcgtttggggc1740

attaccaaagtcacggacggcctggacttaactgacatagtccctcagaacacagacgag1800

catgcgtttctgagtgcgcagggtcggttcttcggcttctacaacatgtatgccgtgacg1860

cagggtgactttgagtaccccaccaatcagaaattactgtatgaataccatgaggcattc1920

atgcgcgtccctaacatcatcaagaacgatgatggtggtcttccagagttctggctcagt1980

cttttccgggactggcttataggactccagaaggctttcgaccgagactggtctcggggg2040

agtattacacaggaacggtggttctctaacgcatcagatgaggggatccttgcctacaag2100

ctgctggttcagactgggcatgtcgacaaccccatcgacaagtcacttattactcaggtt2160

aggctggttgatgcagaaggaatcatcaatcctaaagctttctacaactacctcagtgcc2220

tgggcatctaatgatgctttggcatacggagcttcgcaggcaaacctgcgaccagagcca2280

aaatcctggtaccacgctacaaaagacttcgagctcaagatacccaagtccgcacctctg2340

gtgtacacccagctgcctttctatttacacggcctgggtgacacacaacacatcactaat2400

cttattgctcaggtgcgggcgttgtgtcagaagttcgaagagcgaggactccccaacttt2460

ccctcagggattcccttcctcttctgggagcaatacctcgggctacggcagtcgctgggt2520

ctggctttgctctgtgcattagctgcagtcttcctggtggtggctgtattgttgctcaat2580

ctgtgggctgcgaccctagtggtgctggccctggcagccatggtgcttcagctgcttgga2640

gtcatgggggccctcggaatcaagctcagtgcaatacctgctgtcctccttattgttgct2700

gtgggcatcggcgtccacttcacagtacacatctgcctggggtttgtgacaagcatagga2760

ggaagagatcgacggacacgtctcgcattggagcagatgtttgcccctgtcactcacgga2820

gccctcacgacactgcttggtatctttatgctggccttttcagagtttgacttcattgtg2880

aggtatttcttctacgtgctgcttgctctagtggtcgttggtctccttactggtctagtc2940

ttctttccagttttgctgtcgctaataggaccagctgccgaggttgtaccacatgagttc3000

cctgatcgcatttcgacacctacacctcctccatctcctgtgaggacgaggattggttca3060

tgtcgggtagggaaggctccaccacagcccaggagaggtcacgggggtatgcagtgcaac3120

cgcttacatgcagagccatccctcacgaccatcacagaggaagcgaactcatggcactca3180

actcatgagattgttgtacagcctgagctggtggtcgagactaccacttatggaacgcct3240

cagactgcaagtcaaactacgggaggcagtggtgaaggttctggagcaggtcagacctcg3300

acatctccccctcctcctccagggtctcagtcacacgtcaccacaaaggttacagccaca3360

actctcgtcaaggttgaggtccacgcaccactgccaggagctgttgaacgtgatttggga3420

agcagcagtagcagcaataagagtcgctcaagtcgtcggagagactcaagctcgggatcc3480

gaccacgattcttcttcagataacaactcaggcaaacaaggtggtaccaccgcttcctag3540

<210>3

<211>3501

<212>dna

<213>人工序列

<400>3

atgaagatcttacataaggaaattatctatttaaaaaaatttcggctggcaatgcaagga60

ttttttgcatcgaagttcgtcgtcttcgtattaactgctattattgagcctcttcatgtc120

gtgggagaatttagaggaaatgtgactgttttagtaagatccgcaaggtatgaaggagat180

catgcattccttcgggacaaattttcatcagctgattccatcatattaccaccatcagac240

gggaaaggaccgtactgcagacacccagctcgttgtgaacaacttaattatacaacctgc300

atgggagttaaacttccttatacatctactacacttgatttggtggaaaatttaggtaca360

caggaactcgtacaggaaaaattacaacagtggcaaggattacgtcacattccgaaatgt420

tggactgtcgttcaaccatttctctgtgctttatacatgcccaaatgtgagaatgggaca480

gtggatctgccatcacaggaaatgtgcaaaatgacagtgggcccatgtcgaatactggaa540

tcttacaaaagttggccatcgttcttcagatgtgacgacacagttagatttccaccaatg600

tgtaagaatgatgcccgagagctgaagtacaatactactagtcagtgccagagtcctttg660

gtgccaactgacaatactctagcattctatgatggaatagaaggatgtggcattcagtgc720

caaaatccattgcttacagaagacgaacatagacagatccatcagttaattgcttgggca780

ggaaccatttgccttctttttaatatcttcacagtggtaacctttttcattgattggcgt840

tctgcaaacaagtatccagcacttgtaattttctatatcaattgctgttttttggtgtca900

tgtattggatggttggcacagtttattcctggtgcacgtgaaggcattatatgccggaaa960

gatggtactttaaggatgtcagaacctaatactggagagaatttgtcatgtgtgataata1020

tttgtattggtgtattacttcatcatggctgcctttgtgtggtttgtaattctcacgtac1080

acatggcacatcagttttcaagctctaggtaaaattcaagagcgaattgataagaaaagt1140

gcttattttcacatggtggcttggtcattgcctctagttcttactattacaactatggca1200

ctaggggaaatagatggcgacagtgttgctggtatctgttttgtgggatatgttaatcat1260

gctgcacgagcaggattcttgctcgtccccattacaacagtactttttgttggaggatac1320

tttctcactaggggtcttataacactggtgagactaaaaattaacagtcaagaaattatc1380

tcagaaagagcaagtgccaagataagagaaaccattgtgcgcatgggtctcttttcactg1440

tttacatttgtctttgtgttggtgacctttgtgtgtcatgtgtacgaatttcatcatcac1500

agtgactggagagaaagtttcagaaaattcatagtttgccgaattatgagcagtggtaat1560

gcagacacaaagagatgtaagatagagtctcgaccaagtctctcaatgatgcaactgcat1620

atattggtgatgttcttggcaggtgtcataatgtcatcatgggtatggacgagctccaca1680

gttgacacctggagaagatgtctcagaaggacttttaatactcaagcagaagaacccgtc1740

agattgaagaagcataaagttattgctcaagcatttgcaaaaaggaagacattcaacaat1800

gcaggtcgcctctccattagttttcatagcacccatgaggatcctgtaggtttaaatttt1860

gatatgaacagtattgcatcacaagacttcagctcaacctgggcagcagcattaccgaag1920

ctgatgacacgacgtggtgctcttattggtgttgccactggctcaaactcaagccagcat1980

cgaaactcgatagactccgaaatcagttacagtgtgagaagggtgtcagttgaatcacgt2040

cgccactcattggactcccaagtgtctgtacagattgctgaagtaacagcaacacgaaaa2100

gctgcagccactcctatgcctgcagcaagtactgcaactgtgtctggtggaaggggcaga2160

aaaactcgcagtcgaagacaaagaagagagtttggacgtattcgaagtagtcgtgtggga2220

cctcttttgattaggagaggcagtacaacatcacaagaaagtcagttaggagcacaaatt2280

ctgtcagcacttactctagcaaatacaactcttccatcatttgttcccaatctaaagaaa2340

cgaaggactggcaatgcaggtttggacagctctaaagtcattctgccattcgccctgcca2400

gatcagagtcttgatgtaagtgatgaagaacaaaacagtgatgagaagatttctgggaca2460

ataaaaaaatttgatatctttctttgtagagaagctaatgatgtggatttggaggatatt2520

aatgacagtgatggggagatcagtgatggaatgccagcagaaaacgatatgaaagttgta2580

atagcaagtaagtttaatgaagatgagaatgatgacagaaatgatgatattaataataaa2640

cggaaaaacagaagtaccgaaattaatgcaaacactgtaagtgcaaatgatggcatccat2700

attgaggaagatcagaaaaattccattgttgatatgggttcccatcatagtataagtgca2760

gaagttgatgattatcgaagtagaacttcagagggaaacaggtatagaaataactatggc2820

gattgtaaaaacataaatatacctgatgaagtgaaattgtatcttacagaaggctctgat2880

gatgaacctccacaaaaattgaatttcgttccttctgtagatattggaggtgtttcagat2940

ggttccacatcattttgtcctgagctttctcatctagttgtgcagtcctcccattcaggt3000

ctgtcacaaggaagccctcataactttgatgcaagtaaaacgcgtgttatatcagttaca3060

ccctacaaccagagcaacaatgaaggacgtgaagttggaactcagaccacattgtcaata3120

gactgtatcgagatgcaagaactaaaggggaaaaatataatacatccttcaccacgagca3180

tgtgcaagccaaagtactcgggtatctccaccttcatcccggccttcagttcctcgatca3240

tattctcacggtcattttgaaaagaatcagacttcacttgttaataaaatagctgataat3300

gtgttatcaagtccagcaggtagagcactgaactacagttacaatgtacagcgtttagtg3360

caggaactttcagaaggcagtcatgctggacctccagttaggccggagaggagaaagagt3420

gctcagggcaagaacagtgtaagaagtaagagtgctgataacaagaacgttgaattgcag3480

tggaatccggttgcattgtga3501

<210>4

<211>3192

<212>dna

<213>人工序列

<400>4

atgacagcgagacgagcggcgcggcccgcgagtcatcttgtcacactgtcgcctgtctcc60

atggcaaccctcgccgagagtcttcaactatttctagaatttggcccacagtcacttcca120

actggtggagtgagcggggagggactttccacctacttaccagccccggttatttccggc180

gagggcaccaacaaacatccataccgaacaccactccaacctaggagccctcgcaacgta240

aacatcttgatccagacgccaccaacagcagaagtgagaaaatccacgttagagcattgg300

gactccacggcagcagttaccaacttgtacgtctcagcactgtcacagctattgttgccc360

agcagtggctcaggttcgaatctctcggagctccaattcctggcggcgtcccggcgcgcc420

gcggcggcagctgcaggggcggtgttgcccccgggggcagctggggccgcgggggccgcc480

ggggctgcagggggtggcgtggcagtaccaccaccacccccacatcatcccccgggcccc540

gacttccaccccgcgtacaggctcaacccttacatggagcacctgtactcatctctccag600

cagcattctagtcctgccgcctctattcacggtttgggtctcccaccggactacctaggc660

tcgcgcgcactgacagagctccagcctcctccttccactatcggtagctcggaaagaaag720

aaagaagaaagggaagatgatatagaagaaagaaggaagaaaagaaagaaggaagtaagt780

aaagcaagttccagacttacgagtcctcggccgagcagtttgcgacagagtcggaagagg840

gccctgtcgtcatcgccatattcggactcgttcgacatcaattcgatgatacggttctct900

ccgaactccctcgtctccatcgtgaacggatctcggagctccagtgccagtggttcatac960

ggccacctctcagcaggggctatcagcccggctttgggcatgcaccccagcatggcgccc1020

cacctccagcagcttcaagcccaccttctacggactgcggcgggtagtttgctgccccct1080

ctgccaccccacccgcaggcacagacccaggccccgggagggctgttttctcttcctcac1140

catcccttgcagccctccacaggacattccatttctaaaacagagctgtcaggggatgct1200

tgcaagaagctggactcacctgtttctagtagtgtcgtggaggcggacactagtgctcct1260

ggtggaatcagttcgcaccaacggaaggcgagaataaagagggaacctgtcactgccaat1320

gccacaacagcgtctgtaggtgctggtgctgtgtccggagcattaggctcagactgcagt1380

agcagtaagtgttcaccaggtggtgctgattctggcgatcctcttaaagatgaacctggc1440

gacttcattgaaaccaactgccattggcgggactgtggacttgagtttcctacacaggac1500

gatctcgtcaagcacatcaacaatgaccacattcatgccaacaagaagtcgtttgtgtgc1560

cgctgggaagactgttcaagggacgagaagcccttcaaagcgcagtatatgctggtggtg1620

cacatgcgacgacacacaggggagaagccccacaaatgtacatttgaagggtgctacaaa1680

gcctattcgaggctggagaacctgaagacgcacctcagatctcatacaggagaaaaaccc1740

tacatgtgtgagtatcctggatgcagcaaagccttcagcaatgcgtccgaccgtgccaaa1800

caccagaaccgcacgcactccaacgagaaaccgtacgtgtgtaaggccccgggctgcacc1860

aagaggtacacagaccccagttcgctgcggaagcacgtgaagacagtgcacggtgccgag1920

ttctatgccaacaagaagcacaagggagctggcgacggcgccgaggatggctctggaggg1980

gcgggaggcctactggacagcagcccgcacagcgaggagatgatgagcggcaagacggcc2040

agtctgtccagccctagtgtcaagtcagaggaggccagttcgcctgcccagcagggaagt2100

cctttgagtgtgactcagcatggcaacaatggtttagatgagccaatcagcgactcaaat2160

gtgagcactaccaaccatgcgttagcagcagtggaggcagcagaagaatgggtggacgag2220

gctgaagaacttgagctgccagagcttcacgtgagggctgtagtgggtggaggggcggac2280

cccattggaccgccagcggaccatactgcacatcgcatgaggcggctgcaagctaaacta2340

cctccactaccaaatattgcaggtggccgacgagggattcatcttggcgacctgcataag2400

catttcactgatctcaaaatggagggctcgaagatctcaggcactgtgacacagacacag2460

ctgatagagctgcagattccactgggccctggccagaagcagctgcagcaacaggccaac2520

ctacgccgtgacagtaactccaccgtcagcacgtactacggcagcatgcggtcaggagac2580

ctgggctccagccgcagaagtagccaggcctcgcaggcttcagcgctgtcttgtgcaagg2640

ccaggcagtggccaggtgggctcgtcgtatgacccgatctcagccggcagctcacggagg2700

tccagtcagctgagcaacacgggcggcataagtaccatggggcacctcagcagccacctc2760

cagcgcctgcactccagggcgcaggacccactctactgcacatccaacctcgtcctgcag2820

acgcaaaacatgtccctgcaacaacaacggcgtcagcagctgcaacagcagttgtcaggg2880

cctggcaactggctgccacaaccaggagccaacaacagctccagcaaccccgtggacacc2940

agacgcatgtcggagccatgcagagggggctctgataggtcaacacccccaccccgtcct3000

cacagcgtggtcctgccgcccatcgcacgagcccaggagcaccaccctaaccaggaggtg3060

gtactggatgaagtcgccgaaggggagatggtggagaagaacctcgtgatcccagacgag3120

atgctgcacttcctcagtcaggtgagccacctcacaggacgacattcctatgttattgtt3180

gttgttttctaa3192

<210>5

<211>40

<212>dna

<213>人工引物

<400>5

taatacgactcactataggttcaaacagcacgtgcccaac40

<210>6

<211>39

<212>dna

<213>人工引物

<400>6

taatacgactcactataggagtggatgtgcgctctggac39

<210>7

<211>39

<212>dna

<213>人工引物

<400>7

taatacgactcactatagggacatgtgttactcgcccag39

<210>8

<211>39

<212>dna

<213>人工引物

<400>8

taatacgactcactataggttagggaccgtcgaacctcc39

<210>9

<211>39

<212>dna

<213>人工引物

<400>9

taatacgactcactataggtgattggcgttctgcaaaca39

<210>10

<211>40

<212>dna

<213>人工引物

<400>10

taatacgactcactataggaattcgtacacatgacacaca40

<210>11

<211>39

<212>dna

<213>人工引物

<400>11

taatacgactcactatagggactgtggacttgagtttcc39

<210>12

<211>39

<212>dna

<213>人工引物

<400>12

taatacgactcactataggctctgacttgacactagggc39

<210>13

<211>358

<212>dna

<213>人工序列

<400>13

aacacccttccagcaagcggtctcaccgaggggcgcgtgacccgcaaagaccctcgcttc60

agggatctggtgcccaactacaacgcagacatcgtgttcaaggacgaggaaggcacgggc120

gccgaccgcctcatgacgcagaggtgcaaggagaagctgaacacgctggccatctcggtg180

atgaaccagtggcccggcgtgaagctgcgggtgacggagggctgggacgaggaggggcac240

cacgccgtggactcgctgcactacgagggccgagccgtcgacgtcaccacctccgacagg300

gatcgagccaagtacggcatgctggcgcgcctcgccgtggaggcgggcttcgactggg358

<210>14

<211>589

<212>dna

<213>人工序列

<400>14

ttcgacgagcactatatagaccaaatattcctaaacattatcccttgtgccatcattact60

ccattagactgtttttgggaagcttcgaagcttctgggaccagattatcctgttcaaata120

ccaggcgtgggctccaaggtgcgctggacgaaccttaacccacaacaactaatccaacaa180

atgaagactgtcgaattggtattccctttcgagactctcgaagactacatgaaaagggcg240

gggataagcacgggctaccaagagaagccgtgtctcgacccctccgaccccgaatgcccg300

accaccgcccccaacaagttatccggacagatacctgacatcggggcagagctgacagga360

ggctgctacggatatgcagccaaatatatgcactggcctgaggacctggtggtgggcgga420

gccacacgaaataagactggacacatacaaagggcgcgtgcattgcagactgtggtgcaa480

ctaatgggagagagagagatgtatgaattctggtctaaaacgtacaaagtccatcacttg540

gattggacacaagaaaaagctgctttaattctcgagacatggcagaggc589

<210>15

<211>600

<212>dna

<213>人工序列

<400>15

acttgtaattttctatatcaattgctgttttttggtgtcatgtattggatggttggcaca60

gtttattcctggtgcacgtgaaggcattatatgccggaaagatggtactttaaggatgtc120

agaacctaatactggagagaatttgtcatgtgtgataatatttgtattggtgtattactt180

catcatggctgcctttgtgtggtttgtaattctcacgtacacatggcacatcagttttca240

agctctaggtaaaattcaagagcgaattgataagaaaagtgcttattttcacatggtggc300

ttggtcattgcctctagttcttactattacaactatggcactaggggaaatagatggcga360

cagtgttgctggtatctgttttgtgggatatgttaatcatgctgcacgagcaggattctt420

gctcgtccccattacaacagtactttttgttggaggatactttctcactaggggtcttat480

aacactggtgagactaaaaattaacagtcaagaaattatctcagaaagagcaagtgccaa540

gataagagaaaccattgtgcgcatgggtctcttttcactgtttacatttgtctttgtgtt600

<210>16

<211>540

<212>dna

<213>人工序列

<400>16

gatctcgtcaagcacatcaacaatgaccacattcatgccaacaagaagtcgtttgtgtgc60

cgctgggaagactgttcaagggacgagaagcccttcaaagcgcagtatatgctggtggtg120

cacatgcgacgacacacaggggagaagccccacaaatgtacatttgaagggtgctacaaa180

gcctattcgaggctggagaacctgaagacgcacctcagatctcatacaggagaaaaaccc240

tacatgtgtgagtatcctggatgcagcaaagccttcagcaatgcgtccgaccgtgccaaa300

caccagaaccgcacgcactccaacgagaaaccgtacgtgtgtaaggccccgggctgcacc360

aagaggtacacagaccccagttcgctgcggaagcacgtgaagacagtgcacggtgccgag420

ttctatgccaacaagaagcacaagggagctggcgacggcgccgaggatggctctggaggg480

gcgggaggcctactggacagcagcccgcacagcgaggagatgatgagcggcaagacggcc540

<210>17

<211>358

<212>dna

<213>人工序列

<400>17

aacacccuuccagcaagcggucucaccgaggggcgcgugacccgcaaagacccucgcuuc60

agggaucuggugcccaacuacaacgcagacaucguguucaaggacgaggaaggcacgggc120

gccgaccgccucaugacgcagaggugcaaggagaagcugaacacgcuggccaucucggug180

augaaccaguggcccggcgugaagcugcgggugacggagggcugggacgaggaggggcac240

cacgccguggacucgcugcacuacgagggccgagccgucgacgucaccaccuccgacagg300

gaucgagccaaguacggcaugcuggcgcgccucgccguggaggcgggcuucgacuggg358

<210>18

<211>589

<212>dna

<213>人工序列

<400>18

uucgacgagcacuauauagaccaaauauuccuaaacauuaucccuugugccaucauuacu60

ccauuagacuguuuuugggaagcuucgaagcuucugggaccagauuauccuguucaaaua120

ccaggcgugggcuccaaggugcgcuggacgaaccuuaacccacaacaacuaauccaacaa180

augaagacugucgaauugguauucccuuucgagacucucgaagacuacaugaaaagggcg240

gggauaagcacgggcuaccaagagaagccgugucucgaccccuccgaccccgaaugcccg300

accaccgcccccaacaaguuauccggacagauaccugacaucggggcagagcugacagga360

ggcugcuacggauaugcagccaaauauaugcacuggccugaggaccugguggugggcgga420

gccacacgaaauaagacuggacacauacaaagggcgcgugcauugcagacuguggugcaa480

cuaaugggagagagagagauguaugaauucuggucuaaaacguacaaaguccaucacuug540

gauuggacacaagaaaaagcugcuuuaauucucgagacauggcagaggc589

<210>19

<211>600

<212>dna

<213>人工序列

<400>19

acuuguaauuuucuauaucaauugcuguuuuuuggugucauguauuggaugguuggcaca60

guuuauuccuggugcacgugaaggcauuauaugccggaaagaugguacuuuaaggauguc120

agaaccuaauacuggagagaauuugucaugugugauaauauuuguauugguguauuacuu180

caucauggcugccuuugugugguuuguaauucucacguacacauggcacaucaguuuuca240

agcucuagguaaaauucaagagcgaauugauaagaaaagugcuuauuuucacaugguggc300

uuggucauugccucuaguucuuacuauuacaacuauggcacuaggggaaauagauggcga360

caguguugcugguaucuguuuugugggauauguuaaucaugcugcacgagcaggauucuu420

gcucguccccauuacaacaguacuuuuuguuggaggauacuuucucacuaggggucuuau480

aacacuggugagacuaaaaauuaacagucaagaaauuaucucagaaagagcaagugccaa540

gauaagagaaaccauugugcgcaugggucucuuuucacuguuuacauuugucuuuguguu600

<210>20

<211>540

<212>dna

<213>人工序列

<400>20

gaucucgucaagcacaucaacaaugaccacauucaugccaacaagaagucguuugugugc60

cgcugggaagacuguucaagggacgagaagcccuucaaagcgcaguauaugcugguggug120

cacaugcgacgacacacaggggagaagccccacaaauguacauuugaagggugcuacaaa180

gccuauucgaggcuggagaaccugaagacgcaccucagaucucauacaggagaaaaaccc240

uacaugugugaguauccuggaugcagcaaagccuucagcaaugcguccgaccgugccaaa300

caccagaaccgcacgcacuccaacgagaaaccguacguguguaaggccccgggcugcacc360

aagagguacacagaccccaguucgcugcggaagcacgugaagacagugcacggugccgag420

uucuaugccaacaagaagcacaagggagcuggcgacggcgccgaggauggcucuggaggg480

gcgggaggccuacuggacagcagcccgcacagcgaggagaugaugagcggcaagacggcc540

<210>21

<211>20

<212>dna

<213>人工引物

<400>21

gagtccagagcgcacatcca20

<210>22

<211>20

<212>dna

<213>人工引物

<400>22

tcctccgagaagcagccacc20

<210>23

<211>17

<212>dna

<213>人工引物

<400>23

atgcgtttctgagtgcg17

<210>24

<211>19

<212>dna

<213>人工引物

<400>24

gtaatttctgattggtggg19

<210>25

<211>16

<212>dna

<213>人工引物

<400>25

gtaagatccgcaaggt16

<210>26

<211>17

<212>dna

<213>人工引物

<400>26

cgtctgatggtggtaat17

<210>27

<211>18

<212>dna

<213>人工引物

<400>27

aatggagggctcgaagat18

<210>28

<211>19

<212>dna

<213>人工引物

<400>28

tactgtcacggcgtaggtt19