CRT Black Spiny Ant Gene And CDNA Sequencing And Research HSC70 MRNA Levels Of Genes Affected By The Proposed Development

Calreticulin (CRT) exsits in all cells of higher organisms except erythrocytes. Calreticulin is a ubiquitous and extremely conservative endoplasmic reticulum calcium-binding protein, which possesses a variety of biological functions. Calreticulin play important roles in the regulation of calcium balance, assistance of protein folding, processing and maintenance of cell function. At present, the protein had been studied in many animals and plants.Heat stress proteins (HSPs) also called heat shock proteins, which will be produced in the body because of a series of stimulus from environment. The exsitence of HSPs in the cells called heat shock cognate proteins under normal physiological conditions. In HSP70families, HSP70family is the most conservative and has been widely studied, HSC70is one of the members of the HSP70family, this protein expresses in circumstances, will also be produced after stress and paly important roles in cell differentiation and development.The polyrhachis vicina roger as a sociaty insect possesses complex behaviors and significant grade differentiation. This ant possesses medicinal value and some economic significance, P.vicina as the experimental materials may be significant to investigate insect growth and development.In this paper, the full-length cDNA sequences of P.vicina CRT and HSC70gene were obtained by using RT-PCR and RACE. Protein sequences and gene sequences were analyzed by using related bioinformatics software, in addition, from fluorescence real-time quantitative PCR technique, the mRNA of different developmental stages and different castes in P.vicina were quantitative researched. The results are as follows:1. The full-length cDNA sequence of P.vicina CRT gene is1584bp, the open reading frame (ORF) is1251bp, encoding416amino acid residues,5’untranslated region(5’-UTR) is87bp,3’untranslated region (3’-UTR) is246bp, the obtained cDNA sequence of P.vicina CRT gene is named PvCRT. At the same time, the full-length cDNA sequence has been successfully submitted to the GenBank of National Biotechnology information Center (NCBI) of USA, accession number:JQ783054.2. The protein of PvCRT is analyzed by Bioinformatics methods. Homology analysis shows that PvCRT protein has high similarity with other insects, similarity with Nasonia vitripennis, Plutella xylostella and Drosophila melanogaster are99%,88%and89%, respectively, and this is consistent with highly conserved CRT protein. Primary structure analysis shows that the molecular weight of PvCRT protein is48.5kDa, the theoretical isoelectric point is4.37. The functional motif of PvCRT protein analysis that this protein has some functional sites. In addition, PvCRT encoding the last four amino acid sequence, which is the endoplasmic reticulum retention signal HDEL, the result consistent with the relevant reports of CRT position, and then verify that the cloned sequence is PvCRT cDNA sequence. The secondary structure shows that this protein is mixed and the tertiary structure shows that this protein’s best match template is cljhnA.3. The mRNA expression levels are researched by using quantitative real-time PCR technology. The results show that CRT gene expresses at different developmental stages and different castes, but the expression levels are different. During the developmental stage, the lowest expression levels is embryo, the first instar begin to increase, the second instar decrease, the third instar also increase, the fourth instar decrease again, the expression of pupae increase; at adult period, the highest expression levels is worker, which may assiociated with complex behavioral activity of worker, but the lowest expression levels is male. So CRT gene may play important roles in growth and development of P.vicina.4. The full-length cDNA sequence of P.vicina HSC70gene is2171bp, the ORF is1968bp, encoding655amino acid residues,5’-UTR is90bp,3’-UTR is113bp, and the obtained cDNA sequence of P.vicina HSC70gene is named PvHSC70.At the same time, the full-length cDNA sequence has been successfully submitted to the GenBank of NCBI, accession number:KC184398.5. The protein of PvHSC70is analyzed by Bioinformatics methods. Homology analysis shows that PvHSC70protein has high similarity with other insects, the similarity is greater than85%, especially with Hymenoptera, similarity with Nasonia vitripennis, Plutella xylostella and Ostrinia furnacalis are93%,90%and90%, respectively. Primary structure structure analysis shows that the molecular weight of PvHSC70protein is71.4kDa, the theoretical isoelectric point is5.43. In addition, the functional motif of PvHSC70protein analysis that this protein has some functional sites, PvHSC70encoding the last four amino acid sequence is EEVD, this is cytoplasmic HSP70motifs, which can prove that HSC70is the member of HSP70family, and the cloned cDNA sequence is P.vicina HSC70. The secondary structure shows that this protein is mixed and the tertiary structure shows that this protein’s best match template is c3d2fC.6. The results of quantitative real-time PCR shows that HSC70gene expresses at different developmental stages and different castes, but the expression levels are different. The expression levels are high both embryo and larve stages, but the expression level of adults is lower than larve stages, during adults stages, the highest expression levels is male, which may associated with mating flight of male, there is no related report now, so the function of HSC70gene needs to further research.This research first successfully cloned the full-length cDNA of CRT and HSC70gene in P.vicina, the full-length cDNA sequence has been successfully submitted to the GenBank of NCBI, and obtained accession number. At the same time, the prediction and analysis of CRT as well as HSC70protein by using bioinformatics software, the results are consistent with cell position and strcture of other insects, which further validate that the obtained cDNA sequences are CRT and HSC70in P.vicina. In addition, the results of quantitative real-time shows that CRT and HSC70gene expresse at different developmental stages and different castes, but the expression levels are different, which can provide data basis for exploring biological function of P.vicina.