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为了探究菲律宾蛤仔(Ruditopes philippinarum)GnRH基因沉默对其性激素合成酶表达的调控作用,本研究首先根据菲律宾蛤仔GnRH cDNA序列,优化设计并合成了2条dsRNA,进行了作用时间、作用浓度的筛选。研究显示,30μg/50μL的GnRH-ds1干扰链注射到实验动物血腔后48 h,菲律宾蛤仔脏神经节中GnRH基因表达量较PBS组下降了73.3%,较阴性对照组下降了71.4%,达到了对菲律宾蛤仔体内GnRH基因表达的抑制作用。对性腺发育增殖期的菲律宾蛤仔进行dsRNA注射后48 h,采用荧光定量PCR测定了对照组和GnRH表达沉默组菲律宾蛤仔性腺中性激素合成相关基因CYP17、3β-羟基类固醇脱氢酶(3β-HSD)和17β-HSD的mRNA表达量,发现在GnRH沉默组的菲律宾蛤仔性腺中3β-HSD的表达量与对照组相比无显著变化,而CYP17和17β-HSD的表达量与空白对照组和阴性对照组相比分别下降了86.3%和84.9%(p<0.05)。实验结果初步说明菲律宾蛤仔体内GnRH可能通过调控其性腺中CYP17、17β-HSD的基因表达而影响性激素的合成并调控生殖发育。
Abstract：In order to study the function of GnRH on steroidogenesis regulation in bivalves, two double strands RNA(dsRNA) synthesized by in vitro transcription method was hemocoel injected into clams Ruditapes philippinarum. Effective time and concentration of GnRH-dsRNA was firstly determined, which showed that the expression of GnRH in visceral ganglion of clams from the 30μg/50μL GnRH-ds1 injection group was decreased by 73.3%, 71.4% compared with blank and negative control after 48 h, indicating that the expression of GnRH was effectively inhibited. Then 30μg/50μL GnRH-ds1 were hemocoel injected into the clams at gonadal developing stage and gonads were sampled after 48 h of injection. The results of real time PCR showed that there was no significant difference of 3β-HSD expression in gonads between GnRH-ds1 injection group and control. While the mRNA expression of CYP 17 and 17β-HSD was significantly decreased by 86.3% and 84.9% respectively(p<0.05), indicating that GnRH gene silencing significantly deduced the mRNA expression of CYP 17 and 17β-HSD. These results showed that GnRH may regulate gonadal development of bivalves through affecting the gene expression of steroidogenesis-related enzymes which allow R. philippinarum to endogenously produce sex steroids.
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基本信息:
DOI：10.16441/j.cnki.hdxb.20180412
中图分类号:S917.4
引用信息:
[1]姜珊珊,苗晶晶,刘力铷,等.菲律宾蛤仔GnRH基因沉默对其性激素合成酶基因表达的影响[J],2020,50(05):46-52.DOI:10.16441/j.cnki.hdxb.20180412.
基金信息:
山东省自然科学基金面上项目(ZR2016CM31)资助~~
2018-12-14
2018
2019-03-27
2020-06-25
2020
2