| A thorough study of insect olfactory mechanism and a complete explanation of olfactory signal transduction pathway are the base of exploring the specific prevention and control of pest.Batocera horsfieldi(Hope) is one of the most important insect pest of forest,and olfaction play an important role in its behavior such as mating,host location. The researches of distinguishing outside environmental principle and chemosensory protein supply a new strategy for sustainable management of Hope.In this study,two full length cDNA libraries from antennae of B.horsfieldi were constructed by SMART (Switching Mechanism At 5'end of RNA Transcript) technology,and hundreds of expressed tags(ESTs) were sequenced and analyzed.On the basis of ESTs,one chemosensory protein(CSP) gene was cloned by RT-PCR(Reverse Transcription PCR) and analysis of distribution of chemosensory proteins of B.horsfieldi in the body by protein gel electrophoresis.The main results showed that:1.We constructed 0.3-0.6kb and 0.6-1kb cDNA fragments cDNA libraries using the CreatorTM SMARTTM cDNA library construction kit(Clontech) and the quality of the two cDNA libraries were identified.The cDNA library of 0.3-0.6kb cDNA fragments has a titer of about 6.4×105 cfu/ml,the total number of colony-forming units(cfu) was 8.9×105 cfu,the insert size were mainly 0.5-0.75kb,average insert size was longer than 0.5kb,and the recombinant rate was 91%.And The cDNA library of 0.6-1kb cDNA fragments has a titer of about 4.1×106 cfu/ml,the total number of colony-forming units(cfu) was 5.8×106, the insert size are mainly 0.5-0.75kb,average insert size is longer than 0.5kb,and the recombinant rate was 91%.The library titer and recombinant rate all matched quality requirements for cDNA library and this indicated two cDNA libraries from antennae of B. horsfieldi were successfully constructed,which will be a valuable resource for functional genomics studies of goat B.horsfieldi.2.Some clones from two cDNA libraries from antennae of B.horsfieldi were randomly collected through sequencing 649 sequences were gotten which were analyzed by NCBI/GenBank/Blast,DNAMan,and Vector NTI and so on.On the basis of analyzing the sequences,we have gotten three chemosensory protein genes.One cDNA fragment chemosensory protein genes in B.horsfieldi was amplified with two specific primers by RT-PCR and the lengths was 393bp named BhoCSP1,and encoded 131 amino acid residues.Sequencing and analysis showed that BhoCSP1 gene was characterized by four typical conservative cysteine residues and domains.The N-terminus hydrophobic region predicted containing of 19 amino acid residues within the Has-CSP displayed the characteristic features of a signal peptide.Deduced amino acid sequences were highly similar to 19 CSPs of Tribolium castaneum which was the only one insect reported from Coleoptera,with sequence identities of 57%for TcaCSP10 and 59%for TcaCSP12.The alignment of amino acid sequences of BhoCSP1 with other 30 species insects' CSPs from Endopterygota also showed high sequence identity,with sequence identities of 21%-59%. From above analysis,we firmly suggest that the 393bp cDNA fragment was the chemosensory protein cDNA from B.horsfieldi.3.Electrophoretic analysis under 15%native PAGE of soluble proteins from different tissues from female B.horsfieldi showed that female antenna and leg had a great amount of BhoCSP1 protein,and female antenna also had BhoOBP1,BhoOBP2 proteins compared with female head,thorax and abdomen from B.horsfieldi.
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