Identification Of Proteins Interacting With Silkworm Transcription Factor BR-C Using Yeast Two-Hybrid System

As is well known, two hormones,20-hydroxyecdysone (20E) and juvenile hormone (JH) coordinately orchestrate the growth and metamorphic development in insects. High JH titer in the early stage of each larval instar maintain the larval state while an increase of20E titer in the later stage of each larval instar initiates the larval molting for going to next instar. From the end of last larval instar to the end of pupal stage, high titer of20E triggers the molting for pupation and the formation of adult when JH is disappeared. So, the signaling pathways of20E and JH as well as their interaction have been becoming an interesting issue of the studies about insect metamorphosis.In insects, transcription factors Broad-Complex (BR-C), containing two conserved domains of C2H2zinc-finger domain and protein-protein interaction domain BTB, contribute to the cross-talk between20E and JH. Previous studies about insect BR-C concentrated on its zinc-finger domain and mainly focused on its response to20E and JH as well as roles on the transcriptional regulation of downstream targets. But, there is no report about the BTB domain and interacting proteins of BR-C proteins in insects. Here, we used silkworm as a model and identified the candidates of proteins interacting with silkworm BR-C protein by using the method of yeast Two-Hybrid system screening. The relative results will help us to well understand the roles of BR-C transcript factor in signaling pathways of20E and JH in insects.(1) Construction of silk gland cDNA library for yeast two-hybrid screeningIn silkworm, several recognized motifs for BR-C were predicted from the upstream genomic region of transcription initiation site of gene encoding silk proteins and regulators related to silk proteins biosynthesis, suggesting that BR-C may involve in regulating the biosynthesis of silk proteins in silk gland. So, we selected silk gland to construct the cDNA library for screening of proteins interacting with silkworm BR-C protein, using MatchmakerTM Gold Yeast Two-Hybrid System from Clontech. RT-PCR experiment showed that silkworm BR-C gene was expressed in the silk gland of the fifth instar larvae and prepupa. So, we extracted the total RNA from silkworm individuals on day1, day3, day5, and day of fifth instar, just wandering, and day1and day2of wandering. After a quality check, a mixture of equal amount of mRNAs from each developmental stage was used to construct a cDNA library. The titter of this silk gland cDNA library is estimated as1.28×l07cfu/ml, the average size of insert fragments is753bp, and the recombination rate was64.8%. These indicated that this silk gland cDNA library can be used to the screening of BR-C interacting proteins.(2) Construction of bait plasmid containing8TB domain of silkworm BR-CBTB domain of silkworm BR-C was used as bait for Yeast Two-Hybrid System Screening. Based on the protocol of MatchmakerTM Gold Yeast Two-Hybrid System, a cDNA fragment against BTB domain of silkworm BR-C was cloned into the pGBKT7vector and produced pGBKT7-BTB bait recombination plasmid. In addition, a test for autoactivation and toxicity showed that pGBKT7-BTB bait recombination plasmid has no autoactivation and toxicity for Y2HGold yeast on the each of three nutritional deficient medium plates including SD/-Trp, SD/-Trp/X-a-Gal, and SD/-Ttp/X-a-Gal/AbA. This showed that pGBKT7-BTB bait recombination plasmid can be used next screening of silk gland cDNA library.(3) Yeast Two-Hybrid System Screening the BR-C Interacting ProteinspGBKT7-BTB bait recombination plasmid was further used to screen silk gland cDNA library in order to get the proteins interacting with silkworm BR-C. Two controls were considered, namely Y2HGold [pGBKT7-53]×Y187[pGADT7-T] as positive control and Y2HGold [pGBKT7-Lam×Y187[pGADT7-T] as negative control. After two grounds screening on the plate of different nutritional deficient medium, namely SD/-Trp/-Leu/X-α-Gal/AbA and SD/Ade/-His/-Leu/-Trp/X-a-Gal/AbA, a total of54positive clones were indentified. The results of cloning and sequencing of these clones showed they encode for8genes.17positive clones encode for RACK1(Receptor of activated C kinase1). In addition, Serl、Fib-H、eiF3g and RpS16were also included in this set, which might be regarded them as candidates of proteins interacting with silkworm BR-C protein.(4) Co-localization analysis of RACK1of BR-C in silkworm cellRACK1can mediate the signaling of protein kinase C and induce a phosphorylation modification of targets through a protein-protein interaction between RACK1and its interacting targets. So the protein-protein interaction between RACK1and BR-C in silkworm should be investigated. The complete opening frame of silkworm RACK1and BR-C were cloned and then used to produce two recombination plasmids of pie2venus-RACK1and pie2venus-BR-C for their co-localization analysis. A co-localization analysis in BmN4cell showed silkworm BR-C protein located in nucleus; RACK1mainly located in cytoplasm, but also have a small amount of protein expression in nucleus, suggesting these two molecules can weakly co-localize in nucleus. It is required to confirm their interaction by using more methods Actually, previous studies showed RACK1is involved in the regulation of phosphorylation of its interacting proteins. So we speculated that an interaction can be taken place between RACK1and BR-C in silkworm, so this interaction may result in BR-C phosphorylation. But this is necessary for further confirmation.