草鱼肌肉生长抑制素2的克隆、功能分析以及Tgf2转座子介导的鱼类插入诱变分析-动物遗传育种与繁殖专业论文.docxVIP

上海海洋大 上海海洋大学硕士学位论文 II II 鱼和团头鲂的受精卵（1-2 细胞期）中，以获得反义 mstn1 表达的转基因后代， 从而实现内源性 mstn1 基因的敲降。初步的研究结果显示，转反义 mstn1 的阳性 鱼比非转基因的阴性鱼体重增加显著（P0.05），表明了草鱼 mstn 基因在草鱼肌 肉生长发育中的负调控作用。 本文还开展了 Tgf2 转座子介导的鱼类插入诱变研究。以 Tgf2 转座子为基础， 分别构建了带有 4 种不同启动子的供体质粒——pTgf2-EF1α-eGFP、pTgf2-Krt8- eGFP、pTgf2-βactin-eGFP 和 pTgf2-MyoD-eGFP，以及辅助质粒 pCS2-β-actin-gfTP， 将供体质粒和辅助质粒或转座酶 mRNA 共同注射到草鱼、鲫和团头鲂的受精卵 中，初步建立了一个转座子介导的插入诱变库。研究结果显示，在鲫和团头鲂的 早期胚胎中，荧光率达到 90%，在草鱼中则达到 95%。随着注射的供体质粒的 不同，表现出不同的荧光特征——肌肉荧光、表皮荧光及全身荧光，eGFP 在平 游期的整合率平均为 53%，在 1 龄阶段的整合率在 35%左右。共得到草鱼插入 诱变突变体阳性个体 162 尾，其中有明显表型差异的 45 尾；鲫鱼插入诱变突变 阳性个体 60 尾，其中有明显表型差异的 14 尾；团头鲂插入诱变阳性个体 51 尾， 其中有明显表型差异的 10 尾。这些鱼类转座子插入诱变突变体可为目标性状功 能基因的挖掘和功能机制研究积累一定的基础。 关键词：草鱼，myostatin 基因，转座子，基因敲降，插入诱变 PAGE PAGE IV Cloning and functional analysis of myostatin in grass carp (Ctenopharyngodon idellus) and insertional mutagenesis by Tgf2 transposon in fish ABSTRACT Grass carp is one of the four major Chinese carps. The total output of grass crap for the whole nation was nearly over 4 million tons every year, so its rearch is important and the development and regulation of grass carp muscle is the hot point in grass carp rearch. MSTN stands for myostatin, which is a potent negative regulator of skeletal muscle and growth. In the mammal, there are only one kind MSTN ，MSTN1. However, there are two kinds of MSTN in teleosts, MSTN1 and MSTN2. We cloned MSTN2 in grass carp. The whole length was 1976 bp and encode 364 amino acid. RT-PCR results demonstrated that there was MSTN2 mRNA in different stages of embryo development , but it was low in the early stage of embryo, and it rised in the late stages. At the same time, MSTN2 mRNA was expressed in brain, muscle, gland，eyes and liver. And the brain had the most cotent. There was no cotent in the other tissues.And The whole mount in situ hybridization result demonstrated that there was no signals in the early stage of embryo. In the middle stage of embryo, the signals were in the brain and notochord and it was more strong in the