Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (8): 1611-1621.doi: 10.3864/j.issn.0578-1752.2014.08.017

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Bioinformational Analysis and Expression Pattern of V-ATPase in Silkworm (Bombyx mori)

 WANG  Xin, LI  Yi, CHEN  Quan-Mei, YI  Qi-Ying, XIE  Kang, WU  Yong, ZHAO  Ping   

  1. State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716
  • Received:2013-09-11 Online:2014-04-15 Published:2013-11-22

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Abstract: 【Objective】 The objective of this study is to identify the vacuolar-type ATPase (V-ATPase) subunit A and subunit B’s coding genes in silkworm (Bombyx mori) and analyze its expression level in different tissues on the 3rd day of 5th instar larvae and different sections of silk gland on wandering stage. 【Method】 The silkworm V-ATPase subunit A and subunit B’s coding genes were identified by bioinformatics. The domains of the V-ATPase subunit A and subunit B were predicted by online tools. Sequence alignment among other species as well as phylogenetic analysis were done by software. The expression levels in different tissues of silkworm on the 3rd day of 5th instar larvae were analyzed by real-time quantitative polymerase chain reaction (PCR). To understand the pH of different sections of silk gland on wandering stage, a pH indicator, bromphenol blue was injected into the body of silkworm through the pore. After that the silk gland was stained in different colors and the pH was indicated by those colors. At last, real-time quantitative PCR was used to make sure the expression levels of different sections of silk gland on wandering stage.【Result】The coding genes of V-ATPase subunit A and B BGIBMGA008295 (GenBank accession number NM_001098359.1) and BGIBMGA002241 (GenBank accession number NM_001098358.1) were identified. There were 3 highly conserved domains in subunit A and B, which were β barrel domain in N terminal, nucleotide binding domain in the central region, and domain in C terminal. Sequences alignment revealed that the identities of V-ATPases subunit A and B among different species were more than 90%. It indicated that the V-ATPases subunit A and B among different species were highly conserved. The cladogram showed that silkworm V-ATPase subunit A and B were clustered with the other lepidopterous insects. Real-time quantitative PCR results showed that these two genes were expressed mainly in mid-gut, fat body, gonad and silk gland on the 3rd day of 5th instar larvae. The pH for different sections of silk gland was analyzed by bromophenol blue staining. The results indicated that the pH of posterior silk gland (PSG), posterior section (PMSG) and middle section (MMSG) of middle silk gland were larger than 7 while the anterior section of middle silk gland (AMSG) and anterior silk gland (ASG) were acidic. The expression level of V-ATPase subunit A and B in different sections of silk gland on wandering stage were analyzed and it was found that the two genes were highly expressed in ASG and AMSG. The V-ATPase may involve in the generation and maintenance of the acidic environment in these two sections.【Conclusion】This study made clear the expression level of V-ATPases in different tissues in silkworm and the V-ATPases may involve in the acidification of the lumens of ASG and AMSG on wandering stage. This further provides a basis for functional study of V-ATPases.

Key words: Bombyx mori , V-ATPase , phyletic evolution , real-time quantitative PCR , silk gland , tissue expression profile

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