家蚕bHLH转录因子Bmdimm与Bmchip的相互作用

doi:10.3864/j.issn.0578-1752.2016.10.018

Abstract

Abstract: 【Objective】 Bmdimm is a member of the bHLH transcription factors family, which is one of key transcription factors that regulate the expression of fibroin heavy chain gene in the late stage of the fifth instar of Bombyx mori. Bmdimm was predicted to have a potential ubiquitination site of lysine residue K43, and therefore it is presumed that Bmdimm may be modified by ubiquitin and thus degraded. Bmchip belongs to the U-box subfamily of the B. mori ubiquitin ligase E3 family, which is expressed in the posterior silk gland during the fifth instar of B. mori. Studying on the interaction of Bmdimm and Bmchip will promote us to better understand the ubiquitylation of Bmdimm in vivo and its biological significance on the transcriptional regulation of fibroin heavy chain gene. 【Method】 The expression profiles of Bmdimm and Bmchip in the posterior silk gland of B. mori were analyzed based on the B. mori genome microarray database. The CDS sequences of Bmdimm and Bmchip were obtained by PCR cloning and gene synthesis, respectively. Multiple expression vectors were constructed and then transformed into E. coli to express the target proteins to screen the best expression conditions. The fused Bmdimm and Bmchip were purified using Ni²⁺ affinity chromatography, and then digested with protease. The fused tag and target proteins were separated via Ni²⁺ affinity chromatography again. The oligo states of Bmdimm and Bmchip in solution were assessed by gel filtration chromatography. The secondary structures of Bmdimm and Bmchip were studied by circular dichroism spectroscopy. The interaction of Bmdimm and Bmchip in vitro were investigated using Far-western blotting and Pull-down. The affinity of Bmdimm and Bmchip were determined by microscale thermophoresis.【Result】 The B. mori genome microarray expression profiles indicate both Bmdimm and Bmchip were expressed in the posterior silk gland during the fifth instar of B. mori. Multiple expression vectors of Bmdimm and Bmchip were constructed, and it was found that ppSUMO-Bmdimm and pET-32M·3C-Bmchip, 0.3 mmol·L^-1 IPTG and 16℃ were the best expression conditions. Then the recombinant Bmdimm and Bmchip was expressed and purified. Gel filtration analysis suggested both Bmdimm and Bmchip existed mainly as a dimer in solution. Circular dichroism spectroscopy showed both Bmdimm and Bmchip had α-helix structures. Far-western blotting and Pull-down assay suggested Bmdimm could interact with Bmchip in vitro. The dissociation constant K_D of Bmdimm and Bmchip was calculated to be (750±28.6) μmol·L^-1 using microscale thermophoresis.【Conclusion】 The bHLH transcription factor Bmdimm and B. mori ubiquitin ligase Bmchip are expressed in the posterior silk gland during the fifth instar of B. mori. Bmdimm and Bmchip have transient interaction in vitro, implying Bmdimm may be modified by ubiquitin via the interaction with Bmchip and thus down-regulated the transcription of fibroin heavy chain gene.

Key words: Bombyx mori, Bmdimm, Bmchip, interaction, ubiquitylation

ZHAO Peng, WANG Ye-jing, WEI Shu-guang, LIU Li-na, LI Zhen-zhen, ZHAO Ping, HE Hua-wei. Interaction of bHLH Transcription Factor Bmdimm and Bmchip in Bombyx mori[J].Scientia Agricultura Sinica, 2016, 49(10): 2027-2038.

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Interaction of bHLH Transcription Factor Bmdimm and Bmchip in Bombyx mori

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