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RNA干扰蚤状溞(Daphnia pulex)doublesex1基因的表达研究
童巧琼1, 林重远1, 朱晓静1, 周伟1, 徐善良1, 王丹丽1, 赵云龙2
1.宁波大学海洋学院 宁波 315211;2.华东师范大学生命科学学院 上海 200062
摘要:
为了探索doublesex1dsx1)基因是否在蚤状溞的生殖转换过程中发挥作用,利用蚤状溞滤食摄食的特点将体外合成的dsx1双链RNA(dsRNA)通过浸泡得方法分别摄入不同生殖状态的溞体中,实现dsx1基因的表达沉默。随后采用荧光定量PCR(qPCR)以及整体原位杂交分别检测不同生殖状态下蚤状溞在RNAi前后体内dsx1的表达水平变化,进而研究dsx1在蚤状溞生殖转换过程中的作用。结果表明:雄性溞、两性溞、孤雌溞在RNAi后均出现了dsx1 mRNA表达水平的显著下调,且下调量具有显著性差异(P<0.05)其中在孤雌溞中下调68%,在三种生殖状态中最为显著,其次是雄性溞(56%)和两性溞(20%)。同时发现,干扰前能够在溞的第一触角、第一胸肢和复眼上检测到明显的信号位点,而在RNAi后,仅在溞体的第一触角上发现少量dsx1基因表达位点,且第一胸肢及复眼上未检测到相关信号。结合未干扰前dsx1在不同生殖状态溞的表达定量以及定位结果,我们推测dsx1可能在蚤状溞的生殖转化基因调控过程中扮演关键角色,并且在维持雄性蚤状溞性别特征的机制中发挥重要作用。
关键词:  蚤状溞  doublesex1  RNA干扰  qPCR  整体原位杂交  生殖转化
DOI:10.11693/hyhz20170200041
分类号:Q346
基金项目:国家自然科学基金项目,31572221号;宁波市农业重大项目资助,2017C110007号;宁波大学研究生创新科研基金项目,G17108号。
ANALYSIS OF DOUBLESEX1 GENE EXPRESSION AFTER RNA INTERFERENCE IN DAPHNIA PULEX
TONG Qiao-Qiong1, LIN Chong-Yuan1, ZHU Xiao-Jing1, ZHOU Wei1, XU Shan-Liang1, WANG Dan-Li1, ZHAO Yun-Long2
1.School of Marine Science, Ningbo University, Ningbo 315211, China;2.School of Life Science, East China Normal University, Shanghai 200062, China
Abstract:
To explore the role of doublesesx1 (dsx1) of Daphnia pulex in reproductive conversion, D. pulex of different reproductive stages were soaked in double-strand RNA (dsRNA) of dsx1 that had been synthesized in vitro. We adopted a dsx1 gene silencing method for D. pulex according to the characters of cuticula. Real-time PCR and whole-mount in situ hybridization were applied to detect the overall variation in dsx1 mRNA expression level in different individuals after interference. Real-time PCR result shows that dsx1 expression decreased significantly (P<0.05) in different reproductive stages in descending order of parthenogenetic females (68%), males (56%), and ephippial females (20%). Whole-mount in situ hybridization revealed that dsx1 expressed in the first antennae in males and ephippial females, whereas in the corresponding sites of parthenogenetic females, no expression was detected. Moreover, the expression was not detected in the first thoracic limb and compound eyes in all individuals, which is different from the situation before interference. Therefore, considering the expression patterns of different reproductive states, we believe that dsx1 is involved in the reproductive transformation and the maintenance of male sexual characteristics of D. pulex.
Key words:  Daphnia pulex  doublesex1  RNAi  qPCR  whole-mount in situ hybridization  reproductive conversion
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