Cloning And Functional Analysis Of TRNA-isopentenyltransferase Gene(BmIPT)in Silkworm,Bombyx Mori

Bombyx mori is an important economic insect with many advantages,such as large progeny size,fast growth,clear genetic background and rich genetic resources.There were important theoretical and practical values for the study of protein synthesis efficiency in Bombyx mori.Transfer RNA played a role as an adapter in the biosynthesis of proteins,which canconnect the information of messager RNA to the machinery of protein synthesis.Many reports on tRNA have demonstrated that most organisms will use 1%-10%of their encoded genes on tRNA modifications to finely regulate tRNA activity.Modification of A37 to i~6A is a classical example of tRNA modifications.Studies have shown that the isoprenyl modification of adenine at position 37significantly affects the accuracy and efficiency of protein translation.Prenylation refers to the transfer of the dimethylallyl group on dimethylallyl pyrophosphate to the adenine nucleotide at position 37,which is catalyzed by tRNA isopentenyltransferase.This study focused on exploring the effect of prenylation of adenine at position 37 of tRNA in Bombyx mori.Here,for the first time,the candidate tRNA isopentenyltransferase genes have beenidentified in silkworm Bombyx mori.The functional tRNA isopentenyltransferase gene have been confirmed by complementing the anti-suppressor phenotype of a mutant that lacks MOD5,the intrinsic tRNA isopentenyltransferase gene.Significant defective phenotypes of silk synthesis and secretion were obtained when the functional tRNA isopentenyltransferase was down-regulated by RNA interference.PiggyBac transposon-mediated transgenic technology was employed to investigate the effect of overexpressing silkworm tRNA isopentenyltransferase gene.It was interesting that both the whole body weight and cocoon shell rate were decreased when overexpression of tRNA isopentenyltransferase was driven by a universal cytoplasmic actin4 promoter.The main findings are as follows:1.Cloning,bioinformatics analysis,expression analysis and evolutionary analysis of silkworm tRNA isopentenyltransferase(BmIPT)geneTo identify the candidate sequence of tRNA isopentenyltransferase in the B.mori genome,a homology search of the B.mori genomic sequence databasewas performed.One predicted ORF with 1482bp was shown to be most likely tRNA isopentenyltransferase gene.RT-PCR showed that there were three bands for the candidate silkworm tRNA isopentenyltransferase gene.The results by sequencing revealed that all the three bands came from the same genome locus and they were three alternative splicings for the candidate silkworm tRNA isopentenyltransferase.Then they were named as BmIPT1,BmIPT2 and BmIPT3.The open reading frame(ORF)of BmIPT1,BmIPT2 and BmIPT3 gene is 1482bp,981 bp and 1212bp,respectively while BmIPT1,BmIPT2 and BmIPT3 has 7,5 and 6 exons,respectively.Multiple sequence alignments showed that the BmIPT1 protein containsATP/GTP binding site,DMAPP binding site and zinc finger domain,which are typical structural domains for a conserved tRNA-isopentenyltransferase gene.The predicted protein sequence of BmIPT2 and BmIPT3 lack the typical zinc finger domain and ATP/GTP binding site,respectively.Microarray data were used to analyze the spatio-temporal expression pattern of silkworm tRNA isopentenyltransferase.The results showed that silkworm tRNA isopentenyltransferase expressed in all the tissue/organ samples investigated.The four tissue/organs with highest abundance of silkworm tRNA isopentenyltransferase were testis,ovary,hemocyte and posterior silk gland.The expression of silkworm tRNA isopentenyltransferase increases highly at the stage of the sixth day of fifth instar,60h after wanding and adult.In addition,the results showed that the expression of silkworm tRNA isopentenyltransferase increased significantly from the first day of fifth instar to the fourth day of fifth instar in the middle and posterior part of silk gland.It is interesting that tRNA isopentenyltransferase expressed significantly higher in Bombyx mori than Bombyx mandarina for all the three tissue/organ samples(midgut,silk gland and fat body)investigated.2.Functional complementarity verification of silkwormtRNA-isopentenyltransferase in a mutant yeast(MT-8)that lack mod5In order to determine which band(s)of silkworm tRNA isopentenyltransferase candidate could complement the loss-of-suppression phenotype of MT-8,the cDNA of BmIPT1,BmIPT2 and BmIPT3 was introduced into yeast expression vector pAG306GAL-ccdB-TAP by Gateway cloning,and then transformed into the S.cerevisiae mutant strain MT-8.The recombinant yeast strains were named as BmIPT1-MT8,BmIPT2-MT8,BmIPT3-MT8,respectively.As a consequence of the functional IPT gene,BmIPT1-MT8 keeps a pink-white colour when growing on rich media(YPD),whereas BmIPT2-MT8,BmIPT3-MT8 and MT-8 accumulates a red pigment.The results also showed that only BmIPT1-MT8 was able to restore lysine independence.To determine if the complementation of the phenotype was accompanied by restored synthesis of i~6A,total tRNA was extracted and enzymatically degraded.The results of HPLC separations showed that BmIPT1-MT8 appear a peak at the position of i~6A,while BmIPT2-MT8,BmIPT3-MT8 and MT-8 contained no peak at the position of i~6A.3.RNAi-based fuctional study of tRNA isopentenyltransferase in Silkworm,Bombyx moriDouble strand RNAs were synthesized by in vitro transcription using T7 RNA polymerase.Ds BmIPT and dsEGFP were separately injected into the second gastropod with a dose of 40ug per silkworm.The expression of BmIPT was significantly lower in the dsBmIPT treated group than that in the control group injected with dsEGFP.60%silkworm could not spin silk and the other 40%spinned a significant lower amount silk in the dsBmIPT treated group,while all the silkworms spinned a normal amount of silk in the dsEGFP treated group.Insects were dissected at different stage after wandering.The middle silk gland was full of materials and obviously larger in the dsBmIPT treated group,while all the parts of silk gland were hollow and atrophic in the dsBmIPT group at 48 hours after wandering.The midgut and silk glands of the larvae could not degenerate in the ds BmIPT treated group,whereas midgut and silk glands of the larvae degenerate and could not be detected in the dsEGFP treated group at 24 hour of the pupa stage.All the pupae could not transform into adults and died at the end of pupa stage in Ds BmIPT treated group.The results showed tRNA isopentenyltransferase played an important role for a normal growth and development in Bombyx mori.4.Study on overexpression of tRNA isopentenyltransferase in Silkworm,Bombyx moriTo explore the effect of overexpression of BmIPT1 gene on silkworms,three transgenic overexpression vectors were constructed.They are pBac 3×P3EGFP[hr3A4-BmIPT-Ser1PA],pBac 3×P3 EGFP[hr3 Ser 1-BmIPT-Ser1PA]and pBac3×P3 EGFP[hr3 FibH-BmIPT-SV40],in which BmIPT1 was driven by a universal cytoplasmic actin4 promoter,a middle silk gland-specific Sercin 1 promotor and a posterior silk gland-specific FibH promoter,respectively.Currently,three transgenic positive lines of pBac 3×P3 EGFP[hr3A4-BmIPT-Ser1PA]were obtained.They were named T1,T2 and T3,respectively.The transgene insertion sites for both T1 and T2located in the intergenic region of chromosome 22,while transgene insertion sites for T3 located in the intergenic region of chromosome 7.The expression of BmIPT was significantly enhanced in all three transgenic lines.However,the whole body weight and the cocoon mass were both decreased compared with the non-transgenic lines.