Cloning and expression analysis of <i>BmYki</i> gene in silkworm, <i>Bombyx mori</i>

<div><p>The transcriptional coactivator Yorkie(Yki), is a critical downstream effector of the Hippo(Hpo) signaling pathway that controls organ size through the regulation of cell proliferation and apoptosis. During the past ten years the biological function of Yki has been studied extensively in <i>Drosophila</i> and a few other insects, however, little is known about it in the silkworm, <i>Bombyx mori</i>, a major research model of lepidopteran insect. Here, we describe the isolation, characterization and expression of the <i>B</i>. <i>mori</i> Yki ortholog, BmYki. The coding sequence of the <i>BmYki</i> was 1314 bp in length, encoding a protein of 437 amino acids containing two conserved WW domains. <i>BmYki</i> transcripts were ubiquitous but not abundant in all detected tissues and developmental stages. Comparatively, it was expressed at pretty high level in silk glands and at the stage of fifth-instar day-3 larvae. Overexpression of <i>BmYki</i> in cultured <i>B</i>. <i>mori</i> embryonic cells significantly promoted transcription of genes associated with cell proliferation and apoptosis, indicating that <i>BmYki</i> functions in the regulation of organ growth-related biological processes. Interestingly, transcription of silk protein-coding genes and transcription factors regulating the synthesis of silk proteins was downregulated remarkably, suggesting that <i>BmYki</i> was involved in the regulation of silk protein synthesis. This study provides new insights into the role of <i>BmYki</i> in Hpo pathway regulation in silkworm.</p></div

Localization of BmYki-EGFP fusion proteins in BmN cells.

<p>The coding sequence of the BmYki with the stop codon deleted was fused to EGFP and transiently transfected into the BmN cells. Subcelluar localization of the expressed BmYki-EGFP fusion proteins was observed by fluorescence microscopy.</p

Expression profiles of <i>BmYki</i> in <i>B</i>. <i>mori</i>.

<p>(A)mRNA levels of <i>BmYki</i> in different tissues of <i>Dazao</i> strain. HD(head); EP(epidermis); FB (fat body); TR (trachea); HA(hemocyte); MG(midgut); MT(malpighian tubule); TE(testis); OV(ovary); ASG(anterior silk gland); MSG(middle silk gland); PSG(posterior silk gland); SG (total silk gland). (B)mRNA levels of <i>BmYki</i> at different developmental stages of <i>Dazao</i> strain. 1L-1, 2L-1, 3L-1, and 4L-1(day-1 of the first, second, third, and fourth larval instar); 5L-1, 5L-3, 5L-5, and 5L-7(day-1, 3, 5, and 7 of the fifth instar), P-1(day-1 of the pupal stage), MM-1 and FM-1(day-1 of the male and female moth). Relative mRNA levels of <i>BmYki</i> against <i>sw22934</i> are shown. Error bars represent mean ±SD of three samples.</p

mRNA levels of silk protein synthesis-related genes in BmE cells overexpressing <i>BmYki</i>.

<p>Expression of silk protein synthesis-related genes in BmE cells were quantified by qRT-PCR. Relative mRNA levels are indicated as the ratios of mRNA levels between the target gene and <i>sw22934</i>. Error bars represent mean ±SD of three samples.</p

mRNA levels of Yki target genes in BmE cells overexpressing <i>BmYki</i>.

<p>Expression of <i>B</i>. <i>mori</i> genes homologous to known targets of <i>Drosophila</i> Yki. Relative mRNA levels are indicated as the ratios of mRNA levels between the target gene and <i>sw22934</i>. Error bars represent mean ±SD of three samples.</p

Alignment amino acid sequences of BmYki of three <i>B</i>. <i>mori</i> strains.

<p>Identical amino acid residues among <i>Dazao</i>, <i>Nistari</i> and <i>LH</i> strains are shaded in black. The WW domains are underlined in red.</p

Primer sequences.

<p>Primer sequences.</p