Xu Guanfeng, Zhang Jie, Lyu Hao, Liu Jia, Ding Yang, Feng Qili, Song Qisheng, Zheng Sichun
Guangzhou Key Laboratory of Insect Development Regulation and Applied Research, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, China.
College of Agriculture, Food and Natural Resources, University of Missouri-Columbia, Columbia, USA.
Insect Biochem Mol Biol. 2017 Aug;87:117-126. doi: 10.1016/j.ibmb.2017.06.017. Epub 2017 Jul 1.
Insect chitin synthase A (CHSA) is an epidermis-specific enzyme that plays an essential role in insect development. In this study, the function and regulation of CHSA-2b, an alternative splicing variant of Bombxy mori CHSA that is discovered only in Lepidopteran insects, were investigated. Analysis of mRNA level showed that BmCHSA-2b was responsive to 20-hydroxyecdysone (20E) in pupal wing unlike BmCHSA-2a, which shares almost the identical sequence as BmCHSA-2b except the first 31 amino acids, suggesting that the expression of these two alternative splicing variants is driven by different promoters of CHSA gene. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis showed that BmCHSA-2b was up-regulated in the wing of mid-pupa unlike BmCHSA-2a, which was up-regulated in epidermis and wing disc at the beginning and end of pupal stage. Further analysis reveals that the up-regulations of BmCHSA-2a and BmCHSA-2b in pupal wing were consistent with the increase of chitin content and wing area at the same stages, respectively. Furthermore, the higher transcription level of BmCHSA-2b in the mid-pupal wing of male than that in female was consistent with the chitin content of pupal wing between genders. Injection of double-stranded RNAs of BmCHSA-2b resulted in the decrease in the area and chitin content of the wing, and irregular and crimpled vein. All these results together suggest that B. mori evolves an extra promoter in CHSA gene to activate BmCHSA-2b expression in the wing of mid-pupal stage in response to 20E, and BmCHSA-2b is required for the wing development in the mid-pupa of B. mori.
昆虫几丁质合成酶A（CHSA）是一种表皮特异性酶，在昆虫发育中起关键作用。在本研究中，对家蚕CHSA的一个选择性剪接变体CHSA-2b的功能和调控进行了研究，该变体仅在鳞翅目昆虫中发现。mRNA水平分析表明，与BmCHSA-2a不同，蛹翅中的BmCHSA-2b对20-羟基蜕皮酮（20E）有反应，BmCHSA-2a除前31个氨基酸外与BmCHSA-2b序列几乎相同，这表明这两个选择性剪接变体的表达由CHSA基因的不同启动子驱动。逆转录定量实时聚合酶链反应（RT-qPCR）分析表明，与在蛹期开始和结束时在表皮和翅盘中上调的BmCHSA-2a不同，BmCHSA-2b在蛹中期的翅中上调。进一步分析表明，BmCHSA-2a和BmCHSA-2b在蛹翅中的上调分别与同一阶段几丁质含量和翅面积的增加一致。此外，雄性蛹中期翅中BmCHSA-2b的转录水平高于雌性，这与两性蛹翅的几丁质含量一致。注射BmCHSA-2b的双链RNA导致翅面积和几丁质含量减少，翅脉不规则且卷曲。所有这些结果共同表明，家蚕在CHSA基因中进化出一个额外的启动子，以响应20E激活蛹中期翅中BmCHSA-2b的表达，并且BmCHSA-2b是家蚕蛹中期翅发育所必需的。
