中国野桑蚕DH-PBAN基因的分子克隆及序列分析
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摘要
滞育激素(DH)是诱导卵滞育的激素，最近的研究表明，DH活性可能广泛存在于昆虫中，但在已研究的昆虫中，其只在家蚕中作为滞育的调控子(Denlinger，2002)。说明DH在进化上比较特殊，因此研究DH，不仅有助于探明滞育机理，而且对DH的分子进化研究也有重要作用；对DH分子进化的研究可以反过来可以推动滞育机理的研究，两者相辅相成。滞育机理的研究为利用益虫如改造家蚕的滞育类型以及生物防治害虫奠定理论基础。因此DH的研究具有十分重要的意义。
家蚕分子生物学研究结果表明性信息素合成激活肽(PBAN)与DH为同一基因编码，该基因在翻译水平剪切成DH、PBAN及其它三种神经肽，这种加工方式在昆虫中是不多见的。PBAN是控制昆虫性信息合成的神经肽，在昆虫防治上具有广阔的应用前景，因此研究PBAN不仅具有理论上的意义，还具有潜在的应用价值。
由于家蚕起源于中国野桑蚕，故研究中国野桑蚕DH-PBAN基因，对探明作为重要经济昆虫和模式生物的家蚕DH-PBAN基因的功能、基因的进化以及家蚕起源与进化等均有重要意义。
为此：本研究重点在克隆获得中国野桑蚕DH-PBAN基因，并利用网上数据库资源进行序列分析及系统进化分析，为研究DH、PBAN及DH-PBAN基因功能奠定基础，同时研究野蚕与家蚕的进化。
本实验根据家蚕DH-PBAN基因设计引物，分段扩增中国野桑蚕DH-PBAN基因，主要结果如下：
1、首先建立了用特异引物从野桑蚕基因组中扩增目的基因或基因片段(尤其是单拷贝基因)比较稳定的PCR体系。该体系对于用特异引物从野桑蚕基因组中扩增其它基因或基因片段具有较好的借鉴作用。体系中影响扩增效果的关键因素是基因组DNA的纯度、浓度及引物浓度。
2、设计特异引物分段从中国野桑蚕基因组中扩增中国野桑蚕DH-PBAN基因，克隆、测序、拼接后首次获得了中国野桑蚕DH-PBAN基因全序列，序列全长5520bp，通过同源比对、结构分析，表明，该序列由6个外显子和5个内含子组成，编码DH、PBAN、α-SGNP、β-SGNP及γ-SGNP。中国野桑蚕DH-PBAN基因与家蚕DH-PBAN基因同源性为94％，DH-PBAN基因变化以序列的插入/缺失为主。与中国野桑蚕DH-PBAN相比，家蚕DH-PBAN第3内含子中插入了298bp长的片段，而第5内含子中却缺失了64bp长的片段。这种大片段的变化在DH-PBAN基因进化研究上是否具有意义，还有待进一步研究。中国野桑蚕DH-PBAN基因DNA序列比对结果说明DH-PBAN基因外显子在维持DH-PBAN的功能方面具有重要作用。
3、本试验利用DNAstar软件包的Protean(Nishikawa & Ooi，1987)对中国野桑蚕DH-PBAN氨基酸序列、生化性质等进行了分析，结果表明该多肽全长192，分子量为22271.46m. w.,1A(280)=1.29mg/mL，等电点为9.12，PH7时带电量为4.23。利用在线蛋白质结构分析软件Tmpred(Prediction of Transmembrane Regions and Orientation)进行蛋白质结构分析，表明前20多个氨基酸为引导肽。
4、对DH、PBAN及其基因cDNA、DNA序列进行同源比对，结果表明DH功能区域为C端六肽，PBAN功能区域为C端五肽。
i 根据DH＿PBAN及其基因cDNA序列构建的分子进化树反映中国野桑蚕、家蚕和日本野桑蚕三
者二间的关系主要有两类：一是中国野桑蚕和家蚕先聚在一起，然后再与日本野桑蚕聚在一起＿二
g？③④4＠M6＊③8＠X＄R－＠，M6RS＆8③R－＠，＠a＆8＊WB＃。g＠SS＆
聚在一起的情况。经综合分析，推测中国野桑蚕与日本野桑蚕共同起源于同一个祖先——吉野蚕＿
家蚕再从中国野桑蚕中分化出来，其进化路线示意图如下图所示。
＿日本野桑蚕
— —q野蚕一一一二一＼了一一中国野桑蚕
”家蚕
中国野桑蚕、家蚕与日本野桑在进化路线示意图
6、根据DH、PBAN及其基因cDNA、DNA序列所做的进化树结果，分析相关昆虫基同进化及
物种聚类情况，表明绝大多数与传统的形态聚类结果比较一致，但也有差异。推论可能是有些昆虫
所处环境比较特殊。
7、将以卵滞育的野桑蚕、家蚕与以蛹滞育的烟实夜蛾、棉铃虫的DH－PBAN进行比较，结果表
明，两种滞育类型昆虫DH－PBAN序列的差异主要集中在前24个氨基酸，即主要为引导肚部分。推
论DH－PBAN引导肽在决定昆虫滞育类型或决定DH的作用形式上可能具有重要的作DJ。
Diapause hormone (DH) is the hormone that can induce diapause of egg. DH activity may be widely distributed, only in Bombyx mori (silkworm), among the insects studied, has this peptide been captured as a regulator of diapause (Denlinger,2002). Therefore the studies on DH not only will help ascertain the mechanism of diapause, but also have great significance for the molecular evolutionary studies of DH. The studies on the mechanism of diapause will play basic role in utilizing beneficial insects such as reforming the diapause type of silkworm, and in biological control.
It is verified that pheromone biosynthesis activating neuropeptide (PBA.N) is encoded together with DM by the same gene. PBAN is a kind of neuropeptide that controls the biosynthesis of sex pheromone. Studies on PBAN have the theoretic signification find the latent practical prospect.
B. mori has origined from Bombyxmandarina China (Chinese wild silkworm), so the studies on DH-PBAN gene of B. mandarina C. have great significance to ascertain function and evolution of DH-PBAN gene of B. mori that is regarded as the model organism and to study origin and evolution of B.mori and so on.
This study aims to obtain DH-PBAN gene of B. mandarina C. , then analyze sequence and phylogenetics with databases online to study the function of DH, PBAN and DH-PBAN genes and the evolution of wild silkworm and silkworm.
According to the DH-PBAN gene sequences of B.mori , the primers are designed to amplify the DH-PBAN gene of B. mandarina C. in this study. The results are as follows:
1. The better reaction volumn to amplify aimed gene or gene fragment (especially the single-copy gene ) from genomic DNA of B. mandarina is established. This result will help amplify other genes or gene fragments from genomic DNA of B. mandarina C. with specific primers. The purity and content of genomic DNA and the contents of primers are the main factor that affecting the result of amplification.
2. The specific primers are designed to amplify DH-PBAN of B. mandarina C. from its genomic DNA. The DH-PBAN sequence of 5520bp from B. mandarina C. is obtained. The alignments and structure analysis result shows that this gene .which is consist of 6 exons and 5 introns, encodes DH, PBAN, a - SGNP, P - SGNP and Y-SGNP . Identity of DH-PBAN genes between B.mori and B. mandarina C. is 94%. Compared with DH-PBAN of B. mandarina C., 289bp-long-insertion is found in the 3rd intron of DH-PBAN of B. mori and 64bp-long-deletion is found in the 5th intron of it. It should be further studied whether it is significance for the study of DH-PBAN gene' s evolution of insertion/deletion. Alignment of DH-PBAN gene
between B. mori and B. mandarina show that extrons play important role on keeping the function of DH-PBAN gene.
3. The sequence and biochemical character are analyzed with Protean (Nishikawa & Ooi, 1987). This polypeptide is consist of 192 aa, 22271.46 m. w., 1A(280)=1. 29mg/mL Its isoelectric point is 9.12, and the charge at PH7 is 4. 23. The structure of protein is analyzed .The result shows that the leading peptides lies at the beginning of more than 20 amino acids of N-end.
4. Sequences of DH, PBAN and cDNA, DNA of their genes are aligned, the results shows that the functional region of DH lies in the six-peptide of C-end; and the functional region of PBAN lies in five-peptide of C-end.
5. The phylogeny analysis are investigated using the sequences of DH, PBAN and cDNA, DNA of their genes. The clusters are based on all kinds of sequences, and they can be divided into two kinds: one is B. mandarina China is clustered with B. mori first, then clustered with B. mandarina Japan, and the other is B. mandarina China is clustered with B. mandarina Japan first, then clustered with B. mori . It can be referred that B. mandarina China and B. mandarina
Japan origin from the ancestor in common----old wild silkworm, and B. mori diverges from
B. mandarina China, the sketch map of evolutionary route is showed below.
6. The phylogenetic trees are reconstructed based on the sequences of
家蚕分子生物学研究结果表明性信息素合成激活肽(PBAN)与DH为同一基因编码，该基因在翻译水平剪切成DH、PBAN及其它三种神经肽，这种加工方式在昆虫中是不多见的。PBAN是控制昆虫性信息合成的神经肽，在昆虫防治上具有广阔的应用前景，因此研究PBAN不仅具有理论上的意义，还具有潜在的应用价值。
由于家蚕起源于中国野桑蚕，故研究中国野桑蚕DH-PBAN基因，对探明作为重要经济昆虫和模式生物的家蚕DH-PBAN基因的功能、基因的进化以及家蚕起源与进化等均有重要意义。
为此：本研究重点在克隆获得中国野桑蚕DH-PBAN基因，并利用网上数据库资源进行序列分析及系统进化分析，为研究DH、PBAN及DH-PBAN基因功能奠定基础，同时研究野蚕与家蚕的进化。
本实验根据家蚕DH-PBAN基因设计引物，分段扩增中国野桑蚕DH-PBAN基因，主要结果如下：
1、首先建立了用特异引物从野桑蚕基因组中扩增目的基因或基因片段(尤其是单拷贝基因)比较稳定的PCR体系。该体系对于用特异引物从野桑蚕基因组中扩增其它基因或基因片段具有较好的借鉴作用。体系中影响扩增效果的关键因素是基因组DNA的纯度、浓度及引物浓度。
2、设计特异引物分段从中国野桑蚕基因组中扩增中国野桑蚕DH-PBAN基因，克隆、测序、拼接后首次获得了中国野桑蚕DH-PBAN基因全序列，序列全长5520bp，通过同源比对、结构分析，表明，该序列由6个外显子和5个内含子组成，编码DH、PBAN、α-SGNP、β-SGNP及γ-SGNP。中国野桑蚕DH-PBAN基因与家蚕DH-PBAN基因同源性为94％，DH-PBAN基因变化以序列的插入/缺失为主。与中国野桑蚕DH-PBAN相比，家蚕DH-PBAN第3内含子中插入了298bp长的片段，而第5内含子中却缺失了64bp长的片段。这种大片段的变化在DH-PBAN基因进化研究上是否具有意义，还有待进一步研究。中国野桑蚕DH-PBAN基因DNA序列比对结果说明DH-PBAN基因外显子在维持DH-PBAN的功能方面具有重要作用。
3、本试验利用DNAstar软件包的Protean(Nishikawa & Ooi，1987)对中国野桑蚕DH-PBAN氨基酸序列、生化性质等进行了分析，结果表明该多肽全长192，分子量为22271.46m. w.,1A(280)=1.29mg/mL，等电点为9.12，PH7时带电量为4.23。利用在线蛋白质结构分析软件Tmpred(Prediction of Transmembrane Regions and Orientation)进行蛋白质结构分析，表明前20多个氨基酸为引导肽。
4、对DH、PBAN及其基因cDNA、DNA序列进行同源比对，结果表明DH功能区域为C端六肽，PBAN功能区域为C端五肽。
i 根据DH＿PBAN及其基因cDNA序列构建的分子进化树反映中国野桑蚕、家蚕和日本野桑蚕三
者二间的关系主要有两类：一是中国野桑蚕和家蚕先聚在一起，然后再与日本野桑蚕聚在一起＿二
g？③④4＠M6＊③8＠X＄R－＠，M6RS＆8③R－＠，＠a＆8＊WB＃。g＠SS＆
聚在一起的情况。经综合分析，推测中国野桑蚕与日本野桑蚕共同起源于同一个祖先——吉野蚕＿
家蚕再从中国野桑蚕中分化出来，其进化路线示意图如下图所示。
＿日本野桑蚕
— —q野蚕一一一二一＼了一一中国野桑蚕
”家蚕
中国野桑蚕、家蚕与日本野桑在进化路线示意图
6、根据DH、PBAN及其基因cDNA、DNA序列所做的进化树结果，分析相关昆虫基同进化及
物种聚类情况，表明绝大多数与传统的形态聚类结果比较一致，但也有差异。推论可能是有些昆虫
所处环境比较特殊。
7、将以卵滞育的野桑蚕、家蚕与以蛹滞育的烟实夜蛾、棉铃虫的DH－PBAN进行比较，结果表
明，两种滞育类型昆虫DH－PBAN序列的差异主要集中在前24个氨基酸，即主要为引导肚部分。推
论DH－PBAN引导肽在决定昆虫滞育类型或决定DH的作用形式上可能具有重要的作DJ。
Diapause hormone (DH) is the hormone that can induce diapause of egg. DH activity may be widely distributed, only in Bombyx mori (silkworm), among the insects studied, has this peptide been captured as a regulator of diapause (Denlinger,2002). Therefore the studies on DH not only will help ascertain the mechanism of diapause, but also have great significance for the molecular evolutionary studies of DH. The studies on the mechanism of diapause will play basic role in utilizing beneficial insects such as reforming the diapause type of silkworm, and in biological control.
It is verified that pheromone biosynthesis activating neuropeptide (PBA.N) is encoded together with DM by the same gene. PBAN is a kind of neuropeptide that controls the biosynthesis of sex pheromone. Studies on PBAN have the theoretic signification find the latent practical prospect.
B. mori has origined from Bombyxmandarina China (Chinese wild silkworm), so the studies on DH-PBAN gene of B. mandarina C. have great significance to ascertain function and evolution of DH-PBAN gene of B. mori that is regarded as the model organism and to study origin and evolution of B.mori and so on.
This study aims to obtain DH-PBAN gene of B. mandarina C. , then analyze sequence and phylogenetics with databases online to study the function of DH, PBAN and DH-PBAN genes and the evolution of wild silkworm and silkworm.
According to the DH-PBAN gene sequences of B.mori , the primers are designed to amplify the DH-PBAN gene of B. mandarina C. in this study. The results are as follows:
1. The better reaction volumn to amplify aimed gene or gene fragment (especially the single-copy gene ) from genomic DNA of B. mandarina is established. This result will help amplify other genes or gene fragments from genomic DNA of B. mandarina C. with specific primers. The purity and content of genomic DNA and the contents of primers are the main factor that affecting the result of amplification.
2. The specific primers are designed to amplify DH-PBAN of B. mandarina C. from its genomic DNA. The DH-PBAN sequence of 5520bp from B. mandarina C. is obtained. The alignments and structure analysis result shows that this gene .which is consist of 6 exons and 5 introns, encodes DH, PBAN, a - SGNP, P - SGNP and Y-SGNP . Identity of DH-PBAN genes between B.mori and B. mandarina C. is 94%. Compared with DH-PBAN of B. mandarina C., 289bp-long-insertion is found in the 3rd intron of DH-PBAN of B. mori and 64bp-long-deletion is found in the 5th intron of it. It should be further studied whether it is significance for the study of DH-PBAN gene' s evolution of insertion/deletion. Alignment of DH-PBAN gene
between B. mori and B. mandarina show that extrons play important role on keeping the function of DH-PBAN gene.
3. The sequence and biochemical character are analyzed with Protean (Nishikawa & Ooi, 1987). This polypeptide is consist of 192 aa, 22271.46 m. w., 1A(280)=1. 29mg/mL Its isoelectric point is 9.12, and the charge at PH7 is 4. 23. The structure of protein is analyzed .The result shows that the leading peptides lies at the beginning of more than 20 amino acids of N-end.
4. Sequences of DH, PBAN and cDNA, DNA of their genes are aligned, the results shows that the functional region of DH lies in the six-peptide of C-end; and the functional region of PBAN lies in five-peptide of C-end.
5. The phylogeny analysis are investigated using the sequences of DH, PBAN and cDNA, DNA of their genes. The clusters are based on all kinds of sequences, and they can be divided into two kinds: one is B. mandarina China is clustered with B. mori first, then clustered with B. mandarina Japan, and the other is B. mandarina China is clustered with B. mandarina Japan first, then clustered with B. mori . It can be referred that B. mandarina China and B. mandarina
Japan origin from the ancestor in common----old wild silkworm, and B. mori diverges from
B. mandarina China, the sketch map of evolutionary route is showed below.
6. The phylogenetic trees are reconstructed based on the sequences of
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曾锦标．1978．家蚕与野蚕的杂交及染色体的初步观察．蚕桑通报．(4)：21～24
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郑重．1990．节肢动物的滞育研究．台湾海峡，9(2)：48～60
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Begun, DJ. 1994. Evolutionary inferences from DNA variation at the 6-phosphogluconate dehydrogenase locus in natural population of Drosophila; selection and geographic diffentiation. Genetics,136:155～171
Cho S,Mitchell A,Regier JC, et al. 1995.A highly conserved nuclear gene for low-level phylogenetics:Elongation Faetor-1 a recovers morphology-based tree from heliothine moths. Mol.Biol.Evol,12:650～656
Danforth, BN and Ji S.Q.1998. Elongation factor-1 a occurs as two copies in bees:implications for phylogenetic analysis of EF-1 a sequences in insects.Mol.Biol.Evol.(1):338～341
Danks HV. 1987. Insect Dormancy: An Ecological Perspective. Ottawa: Biol. Survey Can
Davis MT, Vakharia VN, Henry J, et al. 1992.Molecular cloning of the pheromone biosynthesis-activating neuropeptide in Helicoverpa zea. Proc. Natl. Acad. Sci. U.S.A,89(1):142～146
Denlinger DL.1985.Hormonal control of diapause. In Comprehensive Insect Physiology, Biochemistry and Pharmacology, ed. GA Kerkut, LI Gilbert, 8:353-412. Oxford: Pergamon Denlinger DL, Giebultowicz JM, Saunders DS. 2001. Insect Timing: Circadian Rhythmicity to Seasonality. Amsterdam: Elsevier
Denlinger DL, 2002. Regulation of diapause. Annual Review of Entomology. 47:93～123
Dickson RC. 1949.Factors governing the induction of diapause in the oriental fruit moth. Ann Ent Soc Am, 42:511～537
Friedrich M and Tautz D.1997.Evolution and Phylogeny of the Diptera:a molecular phylogenetic analysis using 28S rDNA sequences.Syst.Biol.,46(4):674～698
Fukuda S.1951.The production of diapause eggs by transplanting the suboesophageal ganglion in the silkworm. Proc.Jap.Acad.,27:672～676
Hasegawa K.1951.Studies on the voltinism in the silkworm, Bombyx with special reference to organs containing determination of voltinism. Proc. Jap.Acad.,27:667～671
Hasegawa K.1957.The diapause hormone of the silkworm, Bombyx mori.Nature,179:1300～1301
Henneguy LF.1904.Mophologie, reproduction, Embryogenie. Les Insects.Paris
Hwang JS, Lee JS, Goo TW, et al.1999.The comparative molecular study between Bombycidae and Saturniidae based on mtDNA RFLP and cytochrome oxidase Ⅰ gene sequences:implication for molecularevolution.Zeitschrift fur Naturforschung.Section C, Biosciences,54:7～8
Isobe M,Hasegawa K,Goto T.1973.Isolation of the diapause hormone from the silkworm, Bombyx mori. J.Inseet Physiol.19:1221～1239
Kawano T,Kataoka H,Nagasawa H et al.1992.cDNA cloning and sequence determination of the pheromone biosynthesis activating neuropeptide of the silkworm, Bombyx mori. Biochem Riophys Res Common,189:221～226
Lavenzeau L et al.19(?)6.The endocrine ccntrol of diapause in inseets. Ir.:P(?)rchet M et al(eds). Advances in invertebrate reproduction.(4):69～78
Lee KY, Denlinger DL. 1997. A role for ecdysteroids in the induction and maintenance of the phaxate first instar diapause of the gypsy moth, Lymantria dispar. J. Insect Physiol.,43: 289～96
Menn J and Borkovec A B.1989.Insect Neuropeptides:potential new insect control agents. J.Agric.Food Chem.,37:271～278
Meola RW, Adkisson PL.1977.Release of prothoracicotropic hormone and potentiation of developmental ability during diapause in the bollworm, Heliothis zea. J. Insect Physiol.,23:683～88
Michael SC, Soowon C and Felix AHS.2000.The current state of insect molecular systematics:a
thriving tower of Babel.Annu. Rev.Entomol.,45:1～54
Mitchell A, Cho S, Regier JC, et al. 1997. Phylogenetics utility of elongation factor-1 a in Noctuoidea(Insecta:Lepidoptera):The limits of synomymous substitution. Mol.Biol.Evol.,14(4):381～390
Nagasawa H,Kuniyoshi H,Arima R, et al. 1994. Structure and activity of Bombyx PBAN. Arch Insect Biochem Phys.,25:261～270
Pinyarat W, Shimada T,Xu WH, et al. 1995. Linkage analysis of the gene encoding the preet, sor protein of DH-PBAN in the silkworm, Bombyx mori. Genet. Res., (Cambridge),65:105～111
Raina AK, Jaffe H, Kempe T G, et al. 1989. Identification of a neuropeptide hormone that regulates sex pheromone production in female moths. Scieace,244:796～798
Raina AK, Klun JA. 1984. Brain factor control of the sex pheromone production in the female corn earworm moth. Science,225:531～533
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