克氏原螯虾卵黄蛋白原受体cDNA部分序列的克隆及mRNA表达量的相对定量

Cloning and Relative Quantification Analysis of Expression of the Partial Vitellogenin Receptor cDNA/mRNA of the Crayfish Procambarus clarkii

中文关键词: 卵黄蛋白原受体  克氏原螯虾  克隆  表达量

英文关键词: Procambarus clarkii  Vitellogenin receptor  Cloning  Expression

中文摘要:

      从成熟的克氏原螯虾卵巢中提取总RNA，通过同源克隆得到了卵黄蛋白原受体cDNA部分序列，长度为506 bp，在NCBI网站上进行比对后发现，其与斑节对虾、短沟对虾、罗氏沼虾的卵黄蛋白原受体(VgR) cDNA序列有较高的相似性。采用实时荧光定量PCR方法，测定了卵巢不同发育时期卵巢和肝胰腺两个组织中卵黄蛋白原受体mRNA的相对表达水平。结果显示，卵巢是卵黄蛋白原受体基因表达的主要部位，而肝胰腺只能检测到少量表达，卵黄蛋白原受体基因在卵巢的卵原细胞增殖期相对表达量最高，随后下降，成熟期达到最低值，恢复期开始回升。结合卵巢不同发育阶段卵巢质量的变化计算表达总量，结果发现，卵黄蛋白原受体基因的表达总量在卵原细胞增殖期为最低，随后持续升高并至成熟期达到峰值，恢复期急剧下降,但仍略高于卵原细胞增殖期的表达量。此外，运用所构建的系统进化树比较了克氏原螯虾VgR mRNA与其他物种间的遗传距离。

英文摘要:

      In oviparous animals, vitellin (Vt) and vitellogenin (Vg) are of importance for early embryonic development. Vitellogenin molecules are transported into a developing egg cell through vitellogenin receptor-mediated endocytosis. At present most studies of vitellogenesis have emphasized on vitellogenin. Here we analyzed the expression of vitellogenin receptor (VgR) mRNA in order to elucidate the dynamic relationship between vitellogenin and its receptor. Total RNA was extracted from ovary and hepatopancreas of the crayfish Procambarus clarkii at different ovarian developmental stages. A partial cDNA fragment of 506 bp was cloned with RT-PCR using degenerated primers. Relative VgR expression levels were compared between ovary and hepatopancreas of P. clarkii at different ovarian developmental stages using real-time fluorescent quantitative RT-PCR. It was found that in P. clarkii, ovary was the major organ that expressed VtR gene and there was also VgR mRNA in hepatopancreas. During the development of ovary, the relative VgR expression reached its climax at the beginning (the oogonium proliferation stage) and then decreased along with the developmental process until it reached the minimum at the mature stage. Considering the quantity changes in ovary during the whole developmental stages, the total relative VgR expression level was calculated as the relative VgR expression level multiplied by the mean value of ovary quantity at each developmental stage. It showed an opposite trend that the minimum appeared at the beginning (the oogonium proliferation stage) and the expression level constantly increased along with the developmental process until it reached the maximum at the mature stage. The total relative VgR expression level was similar to the Vg expression level in ovary and hepatopancreas in P. clarkia, which was an indicator of the relationship of the receptor and its target molecule between VgR and Vg. The present study was the first report of the relationship between vitellogenin and its receptor in Decapoda crustaceans in terms of the mRNA expression. Furthermore, we constructed the phylogenetic tree based on VgR sequences to evaluate the evolutional relationship between P. clarkii and the other arthropod species.