Gene Analysis Of Myofibrillar Calcium-binding Protein And Related Protein In Bombyx Mandarian

Wild silkworm(Bombyx mandarina M.) is a pest of sericulture,and the silkworm (Bombyx mori L.) is an economic insect.They have evolved from the ancient wild silkworm with different selection pressure.Now the silkworm and wild silkworm are different in many aspects,while the differences in moving behaviors is interesting.To study the flight mechanism of wild silkworm,this paper compares and analyzes the muscle contraction regulatory protein gene of Bombyx mandarina and Bombyx mori by bioinformatics methods.We have cloned cDNA of troponin C,the four isoforms Of troponin I and myosin light chain 2 from Bombyx mandarina,analysed the gene sequence and structure,discussed the tissue expression pattern of the gene.The results provide a reference for further study of the muscle system of wild silkworm,and provide relevant genetic information for exploring the differences in flight capacity between the wild silkworm and silkworm.The results are as follows:1 Gene cloning and sequence analysis of troponin C in Bombyx mandarina Troponin C(TnC) is the Ca²⁺ binding subunit of troponin complex.There were many studies about it in Drosophila melanogaster and Hetian.We cloned the troponin C gene of Bombyx mandarina,which is nearly 851 bp in full-length,including 100 bp of the 5`un-transtated region(5`-UTR),477 bp of the protein coding region,stop codon TAA and 271 bp of 3`un-translated region.The gene encodes 159 amino acids.The peptide chain carries more negative charge that can bind Ca<sup>2+</sup> and contains conservative amino acid sequence(DX[DN]X[DSG]X6E)of TnC protein.The result of sequence analysis showes that the homology of TnC amino acid sequence in Bombyx mandarina and TnC73F(NP<sub>5</sub>24122) in Drosophila is 82%,higher than that of other isoforms of Drosophila melanogaster TnC.And the wild silkworm TnC has the same amino acid sequence with Bombyx mori TnC. 2 Gene cloning and sequence analysis of Bombyx mandarina troponin IIn many insects,many troponin I(TnI) isoforms are encoded by a single gene.The main function of TnI is to inhibit the formation of actomyosin.Taking mRNA from ephebic Bombyx mandarina as templates,the cDNA of four troponin I isoforms was cloned by RT-PCR,named TnI-A,TnI-B,TnI-C and TnI-D(GenBank accession numbers:FJ785828,FJ785829,F3785830 and FJ890515 )respectively.Sequence homology comparison and functional prediction of the amino acids encoded by the four TnI gene isoforms in Bombyx mandarina indicate that they have characteristics of troponin families.The result of comparative analysis between the four TnI isoforms and TnI isoforms of Bombyx mori isolated by in silico clone showed that:the amino acid of TnI-B in silkworm is the same with that in wild silkworm;one amino acid of TnI-A and TnI-C in silkworm is different from that in wild silkworm,no TnI-D was isolated by in silico clone in Bombyx mori.3.Gene cloning and sequence analysis of Bombyx mandarina myosin light chain2The full length gene of myosin light chain2(MLC2)(GenBank accession No. EU332913) in IFM was cloned from wild silkworm by RT-PCR and RACE.The cDNA is 979 bp in full-length with three exons and two introns,containing a 5`URT of 63bp, an ORF of 603bp,termination codon TAA and a 3`URT of 310 bp.The MLC2 gene encodes 201 amino acids.The protein molecular weight is about 22 kD and its isoelectric point is 4.67.The result of functional protein predict analysis by SMART software suggest that MLC2 protein has two EFh domains and could bind Ca²⁺ which belongs to the member of troponin C superfamily.Meanwhile;the protein was thought to have the conservative amino acids that could be phosphorylated,so it is possible that the protein has the process of phosphorylation and participate in regulating the activity of actomyosin ATPase.4.Expression patterns of myosin light Chain 2 in moth period of Bombyx mandarina and Bombyx moriThe result of expression pattern of MLC2 gene in silkworm moth and wild silkworm moth by Real-Time Quantitative PCR showed that there was no significant difference in expression of MLC2 gene in silkworm and wild silkworm.They have the same expression trend:In the 5^th instar,the expression level is lowest,which increases great with eclosion and declines a little at 5 hours after eclosion,then increases again at 7 hours and 1 day after eclosion.This study compared the TnC,TnI,MLC2 gene in Bombyx mandarina with the corresponding gene in silkworm(or EST) in GenBank.The result shows that these genes in the two species are almost the same.No mutation affecting flight ability that have been identified in Drosophila melanogaster was found.The differences in moving behaviors between Bombyx mori and Bombyx mandarina may be caused by other mechanisms.