家蚕Z染色体基因的剂量分析
摘要
剂量补偿效应(dosage compensation effect)是指在XY型性别决定的生物中平衡X连锁基因在两种性别中的有效剂量的遗传效应。不少生物,如哺乳类动物、果蝇、线虫等的剂量补偿机制已经研究的比较清楚。而目前有关家蚕剂量补偿机制的研究报道较少。家蚕的剂量补偿机制是其性别调控机制的重要部分,其研究对完善家蚕性别调控网络具有重要意义。
本实验室先前已制作了家蚕全基因组芯片,最近又完成了家蚕基因组精细图的绘制。为了确定家蚕中Z染色体上的基因是否存在剂量补偿,本研究利用家蚕全基因组芯片,检测了Z染色体基因在雌雄中的mRNA表达水平,分析家蚕剂量补偿。获得的主要结果如下:
1.鉴定了位于家蚕Z染色体上的579个基因。其中490个基因具有功能注释,57个为未知功能或假设蛋白等基因,还有32个基因与GenBank蛋白质数据库中的蛋白质无任何相似性。分析显示具有功能注释的基因主要功能为结合活性、催化活性和运输活性等。
2.利用家蚕全基因组芯片检测了家蚕雌雄不同组织或器官中z染色体基因的表达情况。采用的组织或器官包括精巢、卵巢、以及分雌雄的体壁、马氏管和头。为了更好地重复所获结果,精巢、卵巢、雌雄头和雌雄前中部丝腺分别进行了3次生物学重复,雌雄体壁和雌雄马氏管各进行了2次和4次生物学重复;同时,进行Cy5和Cy3荧光交换实验消除系统误差。检测结果发现Z染色体中314个基因在性腺中表达,180个在体壁中表达,186个在头中,155个在马氏管中和132个在前中部丝腺表达。
3.在性腺中表达的314个基因中78%的基因M:F的比值大于1,在体壁中,这比例为90%,在头中为85%,在马氏管中为74%,在前中部丝腺中为65%。这结果表明家蚕Z染色体基因在雌雄中表达量的比值大部分大于1,即家蚕Z染色体基因在雄中表达量整体上高于雌中表达量。t检验显示家蚕Z染色体基因在雄中表达量显著高于雌中,即家蚕缺乏剂量补偿机制来消除由于Z染色体数目在雌雄中差异而导致的基因表达差异。
4.采用M:F的比值将Z染色体基因分成三类:雄偏向性基因(M:F值≥1.5)、雌偏向性基因(M:F值＜0.5)和无偏向基因(M:F值在0.8到1.2之间)。结果发现,在体细胞中有94个雄偏向性基因,57个无偏向基因和6个雌偏向性基因。雄偏向性基因大多与结合活性、催化活性、运输、转录调控、分子传导、抗氧化剂活性、酶的调控和动力活性有关,而无偏向性基因缺乏酶调控活性和动力活性。对于雌偏向基因,它可能在雌特异的发育过程中发挥作用。
5.检测了雌雄基因表达量M:F比值在Z染色体上的分布。结果显示,M:F值随机地分布在Z染色体上,雄偏向性基因、雌偏向基因和无偏向基因的分布没有区域性。
6.分析了Z染色体基因M:F比值与基因表达量的相关性,发现雄偏向性基因表达量较低,无偏向性基因的表达量较高。这个结果表明了Z染色体基因M:F比值在某种程度上与基因的表达量具有相关性。
本研究利用家蚕全基因组芯片检测了Z染色体基因在雌雄中的表达情况,结果显示家蚕缺乏剂量补偿机制,同时发现家蚕Z染色体基因M:F比值与Z染色体序列本身无相关性,而与基因表达量具有相关性。这些结果对于我们研究家蚕Z染色体,完善家蚕性别调控网络具有重要意义。
Dosage compensation effect is a genetic effect in sex determination mechanism of male-heterogametic species,which making the level of most sex-linked gene products equal in two sexes.In some species,such as mammals,Drosophila melanogaster, Caenorhabditis elegans,etc.the mechanism of dosage compensation has been studied throughly up to now.However,we learned little about the molecular basis of the mechanism of dosage compensation in Bombyx mori.
Studies on the mechanism of dosage compensation is an important part for us to reveal the mechanism of the sex determination in Bombyx mori.In this research work, we performed a dosage analysis of Z chromosome in Bombyx mori by using microassay. We summaried the results we got as following:
1.Five hundred seventy nine genes which locate on the Z chromosome have been identified.Using these identified genes as queries,a further BLAST search have been performed with the protein sequence data in GenBank.The results indicate that 547 of the identified genes have homologues in the database,while 32 genes have no homologues.There are 490 genes with gene function annotation.Most of these genes function as binding proteins,catalytic protein and transporters,etc.
2.RNAs of different tissues and organs of Dazao including anterior/median silk gland(A/MSG),testis,ovary,integument,malpighian tubule,and head were isolated seperately from female and male larvae on day 3 of the fifth instar.RNA samples were purified and subsequently used in the next step.Gene expression patterns of the 12 above samples have surveyed by genome-wide microarray.For tissue or organ testis, ovary,head of the female,and anterior/median silk gland(A/MSG)of the female,three biological repeats have been carried out,while 2 and 4 biological repeates for integument of the female and malpighian tubule of the female,respectively.Each experiment was performed as a dye-swap.The results of the microarray indicate that among the genes locating on the Z chromosome,there are 314,180,186,155,132 expressed in the gonad,integument,head,malpighian tubule and anterior/median silk gland(A/MSG)respectively.
3.We detected 579 genes on Z chromosome with different expression profiles,Of which,314 genes were expressed in gonads,180 genes in integuments,186 genes in heads,155 genes in Malpighian tubules,and 132 genes in anterior/median silk glands. Male:female(M:F)ratios of expression of genes on Z chromosome were calculated.In gonad,78%of 314 active genes expressed with a M:F ratio value more than 1,and 90% of 180 genes in integument,85%of 186 in head,74%of 155 in malpighian tubule,and 65%of 132 in A/MSG.The mass proportion of genes was with ratio value more than 1. The results showed that the Z chromosome gene expression level in male and female in various tissues or organs have reached a significant difference.The silkworm is female heterohomozygous(male ZZ,femal ZW).Silkworm Z chromosome gene expression in males was significantly higher than that of females.That is,silkworm body does not eliminate differences of the expression between male and female as a result of different number of chromosomes.There is no dosage compensation in silkworm.
4.Functional analysis of Z-linked genes was sorted by M:F ratios.To compare sets of different expression genes in sexes,we classified Z-linked genes into three categories by M:F ratios:male-biased genes(M:F≥1.5),female-biased genes(M:F＜0.5)and no-biased genes(M:F 0.8-1.2).In somatic tissues,there were 94 male-biased genes,57 no-biased genes,and 6 female-biased genes.The male-biased genes were enriched for binding activity,catalytic activity,transport,transtription regulation,molecular transduction,antioxidant activity,enzyme regulation and motor,which probably reflect some silkworm sex-linked characters.
5.We detected the distribution of region of M:F on the Z chromosome,the results showed that M:F value randomly distribute on Z chromosomes.Male-biased, female-biased or unbiased genes are not concentrated in a particular region of Z chromosome.
6.Correlation analysis of M:F ratio and the expression of genes,we found male-biased genes showed low expression,but no-biased genes had higher one.On Z chromosome,the ratio of M:F is to a certainity related with the gene expression.
In this study,we tested the expression of Z chromosome genes.The results suggested there are no dosage compensation in silkworm,and that M:F value is no correlated to Z chromosome sequence itself,but to their expresion level.The results is of great significance for us to study silkworm Z chromosome and improve our knowledge of sex-determining network.
本实验室先前已制作了家蚕全基因组芯片,最近又完成了家蚕基因组精细图的绘制。为了确定家蚕中Z染色体上的基因是否存在剂量补偿,本研究利用家蚕全基因组芯片,检测了Z染色体基因在雌雄中的mRNA表达水平,分析家蚕剂量补偿。获得的主要结果如下:
1.鉴定了位于家蚕Z染色体上的579个基因。其中490个基因具有功能注释,57个为未知功能或假设蛋白等基因,还有32个基因与GenBank蛋白质数据库中的蛋白质无任何相似性。分析显示具有功能注释的基因主要功能为结合活性、催化活性和运输活性等。
2.利用家蚕全基因组芯片检测了家蚕雌雄不同组织或器官中z染色体基因的表达情况。采用的组织或器官包括精巢、卵巢、以及分雌雄的体壁、马氏管和头。为了更好地重复所获结果,精巢、卵巢、雌雄头和雌雄前中部丝腺分别进行了3次生物学重复,雌雄体壁和雌雄马氏管各进行了2次和4次生物学重复;同时,进行Cy5和Cy3荧光交换实验消除系统误差。检测结果发现Z染色体中314个基因在性腺中表达,180个在体壁中表达,186个在头中,155个在马氏管中和132个在前中部丝腺表达。
3.在性腺中表达的314个基因中78%的基因M:F的比值大于1,在体壁中,这比例为90%,在头中为85%,在马氏管中为74%,在前中部丝腺中为65%。这结果表明家蚕Z染色体基因在雌雄中表达量的比值大部分大于1,即家蚕Z染色体基因在雄中表达量整体上高于雌中表达量。t检验显示家蚕Z染色体基因在雄中表达量显著高于雌中,即家蚕缺乏剂量补偿机制来消除由于Z染色体数目在雌雄中差异而导致的基因表达差异。
4.采用M:F的比值将Z染色体基因分成三类:雄偏向性基因(M:F值≥1.5)、雌偏向性基因(M:F值＜0.5)和无偏向基因(M:F值在0.8到1.2之间)。结果发现,在体细胞中有94个雄偏向性基因,57个无偏向基因和6个雌偏向性基因。雄偏向性基因大多与结合活性、催化活性、运输、转录调控、分子传导、抗氧化剂活性、酶的调控和动力活性有关,而无偏向性基因缺乏酶调控活性和动力活性。对于雌偏向基因,它可能在雌特异的发育过程中发挥作用。
5.检测了雌雄基因表达量M:F比值在Z染色体上的分布。结果显示,M:F值随机地分布在Z染色体上,雄偏向性基因、雌偏向基因和无偏向基因的分布没有区域性。
6.分析了Z染色体基因M:F比值与基因表达量的相关性,发现雄偏向性基因表达量较低,无偏向性基因的表达量较高。这个结果表明了Z染色体基因M:F比值在某种程度上与基因的表达量具有相关性。
本研究利用家蚕全基因组芯片检测了Z染色体基因在雌雄中的表达情况,结果显示家蚕缺乏剂量补偿机制,同时发现家蚕Z染色体基因M:F比值与Z染色体序列本身无相关性,而与基因表达量具有相关性。这些结果对于我们研究家蚕Z染色体,完善家蚕性别调控网络具有重要意义。
Dosage compensation effect is a genetic effect in sex determination mechanism of male-heterogametic species,which making the level of most sex-linked gene products equal in two sexes.In some species,such as mammals,Drosophila melanogaster, Caenorhabditis elegans,etc.the mechanism of dosage compensation has been studied throughly up to now.However,we learned little about the molecular basis of the mechanism of dosage compensation in Bombyx mori.
Studies on the mechanism of dosage compensation is an important part for us to reveal the mechanism of the sex determination in Bombyx mori.In this research work, we performed a dosage analysis of Z chromosome in Bombyx mori by using microassay. We summaried the results we got as following:
1.Five hundred seventy nine genes which locate on the Z chromosome have been identified.Using these identified genes as queries,a further BLAST search have been performed with the protein sequence data in GenBank.The results indicate that 547 of the identified genes have homologues in the database,while 32 genes have no homologues.There are 490 genes with gene function annotation.Most of these genes function as binding proteins,catalytic protein and transporters,etc.
2.RNAs of different tissues and organs of Dazao including anterior/median silk gland(A/MSG),testis,ovary,integument,malpighian tubule,and head were isolated seperately from female and male larvae on day 3 of the fifth instar.RNA samples were purified and subsequently used in the next step.Gene expression patterns of the 12 above samples have surveyed by genome-wide microarray.For tissue or organ testis, ovary,head of the female,and anterior/median silk gland(A/MSG)of the female,three biological repeats have been carried out,while 2 and 4 biological repeates for integument of the female and malpighian tubule of the female,respectively.Each experiment was performed as a dye-swap.The results of the microarray indicate that among the genes locating on the Z chromosome,there are 314,180,186,155,132 expressed in the gonad,integument,head,malpighian tubule and anterior/median silk gland(A/MSG)respectively.
3.We detected 579 genes on Z chromosome with different expression profiles,Of which,314 genes were expressed in gonads,180 genes in integuments,186 genes in heads,155 genes in Malpighian tubules,and 132 genes in anterior/median silk glands. Male:female(M:F)ratios of expression of genes on Z chromosome were calculated.In gonad,78%of 314 active genes expressed with a M:F ratio value more than 1,and 90% of 180 genes in integument,85%of 186 in head,74%of 155 in malpighian tubule,and 65%of 132 in A/MSG.The mass proportion of genes was with ratio value more than 1. The results showed that the Z chromosome gene expression level in male and female in various tissues or organs have reached a significant difference.The silkworm is female heterohomozygous(male ZZ,femal ZW).Silkworm Z chromosome gene expression in males was significantly higher than that of females.That is,silkworm body does not eliminate differences of the expression between male and female as a result of different number of chromosomes.There is no dosage compensation in silkworm.
4.Functional analysis of Z-linked genes was sorted by M:F ratios.To compare sets of different expression genes in sexes,we classified Z-linked genes into three categories by M:F ratios:male-biased genes(M:F≥1.5),female-biased genes(M:F＜0.5)and no-biased genes(M:F 0.8-1.2).In somatic tissues,there were 94 male-biased genes,57 no-biased genes,and 6 female-biased genes.The male-biased genes were enriched for binding activity,catalytic activity,transport,transtription regulation,molecular transduction,antioxidant activity,enzyme regulation and motor,which probably reflect some silkworm sex-linked characters.
5.We detected the distribution of region of M:F on the Z chromosome,the results showed that M:F value randomly distribute on Z chromosomes.Male-biased, female-biased or unbiased genes are not concentrated in a particular region of Z chromosome.
6.Correlation analysis of M:F ratio and the expression of genes,we found male-biased genes showed low expression,but no-biased genes had higher one.On Z chromosome,the ratio of M:F is to a certainity related with the gene expression.
In this study,we tested the expression of Z chromosome genes.The results suggested there are no dosage compensation in silkworm,and that M:F value is no correlated to Z chromosome sequence itself,but to their expresion level.The results is of great significance for us to study silkworm Z chromosome and improve our knowledge of sex-determining network.
引文
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