Functional analysis of BmClock, BmCry-α, and BmCry-β, circadian clock genes found in the genome of the silkworm, Bombyx mori

在家蚕EST数据库中发现了果蝇生物钟基因clock （BmClock）的同源基因。测定BmClock cDNA的核苷酸序列。cDNA全长7521 bp，编码585个氨基酸序列，包含3个保守结构域，即基本螺旋-环-螺旋、PAS-1和PAS-2。BmClock mRNA表达具有昼夜节律性，mRNA表达高峰出现在夜间。我们还在家蚕EST数据库中发现了哺乳动物隐色素基因的同源物（BmCry-α）。该cDNA全长4509 bp，编码了一个完整的730个氨基酸的蛋白。aa序列分别保守了与DNA、MTHF和FAD结合的结构域。在ESTs中还发现了另一种隐色素样序列，它是果蝇隐色素基因的同源物。cDNA全长1587 bp。它编码308个氨基酸残基。对大脑的RT-PCR分析表明，这两种色素mrna都以昼夜节律的方式表达，其峰值出现在夜间。另一种隐色素（BmCry-γ）作为EST被发现，另外还发现了DNA 6-4光解酶基因家族（包括隐色素）的成员。利用基因芯片技术研究了家蚕幼虫脂肪体的昼夜节律。我们发现11个基因以昼夜节律方式表达mrna。另一方面，我们还进行了多伏特菌株与双伏特菌株的杂交实验，发现除了性连锁基因外，常染色体基因也调节光周期。

An orthologue for clock (BmClock), a Drosophila circadian clock gene, was found in the Bombyx mori EST database. The nucleotide sequence of the BmClock cDNA was determined. The full length cDNA was 7521 bp in length and encoded a putative 585 amino acid sequence, which contained three conserved domains, a basic helix-loop-helix, PAS-1, and PAS-2. BmClock mRNA was expressed with circadian rythmicity, and the mRNA peak was observed in the evening.We also found an orthologue (BmCry-α) of mammalian cryptochrome genes in the B. mori EST database. The cDNA was 4509 bp in length and encoded a complete 730-amino acid protein. The aa sequence conserved the domains for binding to DNA, MTHF, and FAD, respectively. Another kind of cryptochrome-like sequence was also found in the ESTs, it was an orthologue of the Drosophila cryptochrome gene. The length of cDNA was 1587 bp. It encoded 308 amino acid residues. RT-PCR analyzes of the brains demonstrated that both two chryptochrome mRNAs are expressed in a circadian fashion, and their peak appeared in the night. Another kind of cryptochrome (BmCry-γ) has just been found i as an EST. Additionally a member of the DNA 6-4 photolyase gene family (including cryptochromes) was found.The cDNA microarray was utilized to find circadian rhythm in the larval fat body of B. mori. We found 11 genes that expressed mRNAs in the circadian fashion. On the other hand, we also did a crossing experiment between a polyvoltine strain and a bivoltine strain, and found that not only sex-linked gene but also autosomal gene(s) regulates the photoperiodism.