家蚕FoxG转录因子的鉴定及其在精巢发育中的功能初探
Identification and functional analysis of FoxG transcription factors in the silkworm, Bombyx mori
胡启豪 裴梦圆 赵丹珲 戴玉玲 肖妍虹 莫东君 程心海 余小强 卢玉珍
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DOI：10.7679/j.issn.2095-1353.2021.063
作者单位：广东省昆虫发育调控与应用研究重点实验室，华南师范大学生命科学学院，广州 510631
中文关键词：家蚕；精巢；发育；转录因子；Fox家族蛋白
英文关键词：Bombyx mori; testis; development; transcription factor; Fox protein family
中文摘要：
【目的】 Fox家族蛋白是调控动物发育的关键转录因子。本文分析了鳞翅目昆虫家蚕Bombyx mori的BmFoxG亚家族蛋白在精巢发育中的作用，为基于生殖系统的家蚕性状优化或害虫不育技术的开发提供理论依据。【方法】 利用PCR克隆家蚕BmFoxG-1和BmFoxG-2基因；通过生物信息学工具对BmFoxG蛋白的结构及理化性质进行分析；采用qPCR技术检测BmFoxG基因在家蚕不同发育阶段的精巢中的表达变化；在家蚕Bm12细胞株中过表达BmFoxG-1，并通过qRT-PCR分析BmFoxG-1调控的靶基因。【结果】 本文克隆获得了BmFoxG-1（933 bp）和BmFoxG-2（702 bp）两个基因。BmFoxG-1和BmFoxG-2蛋白均包含保守的Forkhead结构域，但BmFoxG-1蛋白在C端多出约60个氨基酸。BmFoxG-2基因在家蚕不同发育时期的精巢中的表达量均较低；BmFoxG-1的表达均显着高于BmFoxG-2，且随着发育时期而变化，暗示BmFoxG-1参与精巢的发育。生殖细胞发育基因BmVasa、BmCyclinA等多个细胞周期基因以及精子鞭毛发育相关基因BMSK0009828在不同发育阶段的精巢中的表达趋势与BmFoxG-1类似，且BmFoxG-1在家蚕细胞中的过表达可以显著上调 BmVasa、BmCyclinA和BMSK0009828等精巢发育相关基因的表达。【结论】 我们推测BmFoxG-1蛋白可能通过调节精巢细胞周期或生殖细胞的功能来调节家蚕精巢的发育。
英文摘要：
[Objectives] The Fox transcription factor family has been
shown to play a critical role in the development of insects. In this study, we
investigate the function of Bombyx mori FoxG (BmFoxG) in the development of the testis, which may facilitate developing
a strategy to increase the production of silkworms, or the control of pests by
targeting their reproductive system. [Methods] The full-length cDNAs of BmFoxG-1 and BmFoxG-2 ORFs were cloned
from testis RNA samples by PCR and bioinformatics tools used to analyze the
characteristics of the two deduced amino acid sequences. The expression levels
of BmFoxG-1 and BmFoxG-2 in the testis of different developmental stages was determined by real-time
quantitative PCR (QRT-PCR). BmFoxG-1 was then overexpressed in B. mori Bm12 cells and the potential genes that might be regulated by
BmFoxG-1 detected by QRT-PCR. [Results]
The full length ORFs of BmFoxG-1 and BmFoxG-2 were 933 bp
and 702 bp, respectively, and both proteins contain the conserved Forkhead
domain with BmFoxG-1 having a longer C-terminal region. The mRNA expression
level of BmFoxG-2 remained low
in the testis of different developmental stages, but the transcript level of BmFoxG-1 changed during development and was always significantly higher than
that of BmFoxG-2, suggesting that BmFoxG-1 may play a major role
in testis development. Several cell cycle related genes, including BmVasa and BmCyclinA, were up-regulated in Bm12 cells overexpressing BmFoxG-1. [Conclusion] BmFoxG-1 may participate in testis
development in B. mori by regulating the function, or cell cycle
processes, of germline cells.