Molecular Cloning,Expression And Functional Analysis Of BmFAD3-like And BmD6DES Genes From Silkworm,Bombyx Mori

Silkworm(Bombyx mori),is an important resource-based economic insect,also is an important mode of insects and experimental materials,silkworm pupa and silkworm pupa oil are important by-product of silkworm industry.The fatty acid composition of silkworm pupa oil and its synthetic mechanism research can provide theoretical basis for the comprehensive utilization of silkworm pupae.Study on genomic structure and function of silkworm can maintain silk industry sustained and stable development.Based on the database resources of the newest genome of silkworm,BmFAD3-like and BmD6 DES fatty acid desaturase genes were cloned and sequenced,then them were analysed by using the bioinformatics method.Expression profile,prokaryotic expression,Saccharomyces cerevisiae expression researches on the BmFAD3-like and BmD6 DES genes of B.mori had been carried out,Two genes mRNA relative transcription levels changing when silkworm pupa suffered low temperature induction,fungal infection and dsRNA-mediated RNAi treaments were studied.The obtained results were as follows: 1.Cloning and sequencing analysis the BmFAD3-like and BmD6 DES gene of silkwormUsing the conserved histidine structural domain sequence of fatty acid desaturase protein,a sequence homology search was conducted on genomic sequences of silkworms.Specific primers were designed,synthesized and cloned to 1 083 bp and 1 335 bp cDNA fragments from silkworm fresh pupae,which were named as BmFAD3-like and BmD6 DES respectively.Then two full length cDNA fatty acid desaturase genes were obtained by using RACE technology,The BmFAD3-like gene contained full length of 1 727 bp and 1 083 bp open reading frame(ORF),one 360 amino acids peptides encoded by it.The predicted molecular weight(MW)and isoelectric point(pI)were 41.5 KDa and 7.1,The BmD6 DES gene contained full length of 2 298 bp and 1 335 bp open reading frame(ORF),one 444 amino acids peptides encoded by it.The predicted molecular weight(MW)and isoelectric point(pI)were 51.7 KDa and 8.05.Two peptides encoded by BmFAD3-like and BmD6 DES genes have no signal peptide sequence.Based on conservative sequence of silkworm fatty acid desaturase,other insects fatty acid desaturase protein and other model organism fatty acid desaturase proteins,we had done the multiple sequence alignment and constructed phylogenetic tree of BmFAD3-like and BmD6 DES.The reported fatty acid desaturase of insects could not well gather into the same branch.Compared with the Δ9,Δ11and Δ12 three kinds of fatty acid desaturase,the BmFAD3-like and BmD6 DES genes had small similarity to each others.2.Expression patterns of the BmFAD3-like and BmD6 DES genesThe expression patterns of the Bm FAD3-like and BmD6 DES genes were determined by Semi-RT-PCR of different developments in silkworm: The BmFAD3-like and BmD6 DES genes were mainly concentrated in full developments silkworm(except eggs stage),But there are differences in the expression levels.The BmFAD3-like gene was determined significant expression from newly hatched larvae to 3rd sleep period,since 4th instar larvae lasted significantly higher expression,it was almost undetectable expression in the egg stage.The expression pattern of BmD6 DES was determined in different development was very similar with BmFAD3-like,The difference is that BmD6 DES gene was high expressed in the pupae stage,especially in the pupae metamorphosis,the expression levels was significantly higher than other periods,but it’s expression levels decreased significantly in the late moth,By analysis of the expression patterns of BmFAD3-like and BmD6 DES genes were detected in the 3 day of 5th instar larvae,particularly high levels in the ovaries,fat body,haemolymph,epidermis and low expression in midgut,silk gland,seminal vesicle.BmD6 DES gene was determined in the 3 day of 5th instar larvae tissues,particularly high expression levels in the fat body,epidermis,seminal vesicle and ovarian,in the midgut,silk gland,low expression levels in blood and lymph.In summary,silkworm fatty acid desaturase gene mainly expressed in the later stage of metamorphosis of adults-pupa-moth,it was suggested that they perhaps be related to the storage liposomes,reproductive development,parity and mating and pheromone synthesis of silkworm3.The prokaryotic expression of the BmFAD3-like and BmD6 DES genes of silkwormThe BmFAD3-like and BmD6 DES genes were expressed in E.coli expression system in vitro,target proteins were purificated by using Ni+ affinity chromatography column.To achieve efficient expression of the fusion protein we induced the protein by 1mmol/L(final concentration)IPTG 4 hours;Electrophoresis analysis showed that the BmFAD3-like and BmD6 DES gene encoding proteins were expressed in Escherichia coli t as inclusion body;The western blotting results suggested recombinant protein be reacted with prepared antibody well,and they could be found out after hybridization were two bright and single bands,which indicated that the prepared antibodies was specificity and could be used for subsequent experiments,also confirmed that the recombinant plasmids in E.coli BL21(DE3)in the successful expression,two molecular weight of about 44.3 KDa and 54.7 KDa protein was observed in the SDS-PAGE electrophoregram,which were similar the predicted molecular weight size..4 Saccharomyces cerevisiae expression of the BmFAD3-like and Bm D6 DES genes in silkwormAfter amplification,we digested BmFAD3-like and BmD6 DES genes fragment containing specific restricition sites,and subcloned into the expression vector pYES2.0,and then the recombinant vector was transformed into Saccharomyces cerevisiae.The recombinant strains were fermented by utilizing SC(Ura-)under low temperature(20 ℃),adding 2% raffinose induced purpose gene expression and linoleic acid as an exogenous substrates.Fatty acid composition of fermented product of recombinant engineering yeast were analysised by Gas chromatographic,the yeast with empty plasmid pYES2.0 was fermented as control.Compared with the control we found that the BmFAD3-like and BmD6 DES genes can be well expressed in yeast,We could judge pYBmFAD3 produced a new kind of fatty acid in the fermented product by the chromatographic peak figure,identified as α-linolenic acid(C18:3 Δ9,12,15),Its content was 2.8% of total fatty acids,similarly pYBmD6 DES produced a new kind of fatty acid in the fermented product by the chromatographic peak figure,identified as α-linolenic acid(C18:3 Δ6,9,12),Its content was 2.1% of total fatty acids.5.Function analysis of the BmFAD3-like and BmD6 DES genes in silkwormTo further explore the function of BmFAD3-like and BmD6 DES genes,we performed qRT-PCR to detected the two genes mRNA expression levels after silkworm pupa suffered low temperature induction,fungal infection and siRNA-mediated RNAi treatments.Compared with the control,the mRNA expression of BmFAD3-like and BmD6 DES genes in silkworms after 15% by induced at 0 ℃ after 36 h,expression levels of mRNA did not change significantly.It was hypothesized that low temperature could induce mRNA up-regulation,but could not significant increase protein(enzyme)activity.The mRNA expression levels of BmFAD3-like and BmD6 DES genes were up-regulated by about 54% at 25 ℃ after 6 h,the expression of mRNA raised more than 33% after 12 h.Due to the increase of infection degree and fungi metabolic consumption,the genes expression levels were only 20% less than the control.After siRNA mediated RNAi interference,the mRNA expression levesl of BmFAD3-like and BmD6 DES genes decreased about 5% after 12 h,the expression levels of mRNA decreased with the increase of time,then reached a maximum value-60% after 36 h.The mRNA expression of BmFAD3-like and BmD6 DES genes in silkworm pupa could effectively be down-regulated by siRNA-mediated RNAi,but this effect could not be detected long time.