家蚕空泡型ATP酶（V-ATPase）基因的基本信息及表达特征

doi:10.3864/j.issn.0578-1752.2014.08.017

摘要/Abstract

摘要： 【目的】鉴定家蚕（Bombyx mori）中的空泡型ATP酶（vacuolar-type ATPase, V-ATP酶）A、B亚基的编码基因，并调查其在家蚕幼虫5龄第3天不同组织和上蔟时期丝腺不同区段的表达特征。【方法】利用生物信息学的方法鉴定家蚕的V-ATP酶A、B亚基的编码基因并在线预测V-ATP酶两个亚基所具有的结构域，采用软件将其分别与其他物种中V-ATP酶A、B亚基的氨基酸序列进行同源序列比对和进化树的构建，通过荧光定量PCR技术分析家蚕V-ATP酶A、B亚基的编码基因在5龄第3天家蚕各组织的表达情况。进一步利用向家蚕幼虫上蔟时期的气孔注射pH值指示剂溴酚蓝，对丝腺进行染色，通过颜色变化对丝腺不同区段的pH值进行调查。最后，采用荧光定量PCR对家蚕上蔟时期丝腺不同区段V-ATP酶A、B亚基编码基因的表达情况进行分析。【结果】获得了家蚕V-ATP酶A、B亚基的编码基因BGIBMGA008295(GenBank登录号NM_001098359.1)和BGIBMGA002241(GenBank登录号NM_001098358.1)。结构域预测发现这两个亚基均具有3个非常保守的结构域，分别为位于N端的β筒结构域、序列中部的核苷酸结合结构域和C端结构域。将这两个基因编码的V-ATP酶A、B亚基的氨基酸序列与其他物种V-ATP酶A、B亚基的氨基酸序列进行同源性比对和进化树的构建，同源比对结果发现不同物种中V-ATP酶A、B亚基的氨基酸相似度为90%，保守结构域间相似度高达95%以上，说明不同物种间V-ATP酶A、B亚基高度保守。进化树显示家蚕V-ATP酶A、B亚基与同属于鳞翅目的其他昆虫的V-ATP酶A、B亚基亲缘关系较近。5龄第3天家蚕各组织中V-ATP酶A、B亚基编码基因的荧光定量PCR结果显示，这两个基因主要在中肠、脂肪体、生殖腺和丝腺中表达。使用溴酚蓝染色的方法分析上蔟期丝腺不同区段的pH情况，结果显示家蚕后部丝腺、中部丝腺后区和中区为中性或碱性环境，中部丝腺前区pH急剧下降到酸性环境，前部丝腺也为酸性环境。进一步对V-ATP酶A、B亚基编码基因在上蔟期丝腺不同区段进行表达情况分析，发现V-ATP酶A、B亚基基因在前部丝腺和中部丝腺前区高量表达，推测V-ATP酶可能与这两个区域低pH环境的产生和维持相关。【结论】明确了V-ATP酶在家蚕各组织的表达情况，V-ATP酶可能与中部丝腺前区和前部丝腺腺腔的酸化相关，这为进一步研究V-ATP酶的生理功能提供了理论依据。

关键词: 家蚕 , V-ATP酶 , 系统进化 , 荧光定量PCR , 丝腺 , 组织特异性表达

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

王鑫, 李懿, 陈全梅, 衣启营, 谢康, 吴用, 赵萍. 家蚕空泡型ATP酶（V-ATPase）基因的基本信息及表达特征[J]. 中国农业科学, 2014, 47(8): 1611-1621.

WANG Xin, LI Yi, CHEN Quan-Mei, YI Qi-Ying, XIE Kang, WU Yong, ZHAO Ping. Bioinformational Analysis and Expression Pattern of V-ATPase in Silkworm (Bombyx mori)[J]. Scientia Agricultura Sinica, 2014, 47(8): 1611-1621.

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