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题名: 电脉冲-精子介导转基因与转pCAhLFc草鱼抗出血病的研究
作者: 钟家玉
答辩日期: 2001
导师: 朱作言
专业: 遗传学
授予单位: 中国科学院水生生物研究所
授予地点: 中国科学院水生生物研究所
学位: 硕士
关键词: 转基因 ; 电脉冲 ; 精子 ; 稀有鮈鲫 ; 人类乳铁蛋白 ; 草鱼出血病 ; 攻毒
摘要: 本文共五章,包括以下主要研究内容:用稀有鮈鲫和草鱼发展和优化一种简便易行的转基因方法,即电脉冲-精子介导转基因法;研制转人类乳铁收白基因草鱼,并在其鱼种阶段检测其抗病力:以稀有鮈鲫为实验材料,进一步研究电脉冲-精子介导转基因法的机制。第一章:综述近年在鱼类转基因方法上的研究进展,各种转基因方法的优势与不足,人类乳铁蛋白hLF的生理功能的研究进展,草鱼出血病的研究现状。第二章:重组质粒pCAhLFc的构建,将我国鲤科“全鱼”重组质粒,即草鱼生长激素cDNA与鲤鱼的β-肌动蛋白基因启动子的重组体(pCAgcGHc)的启动子部分pCA和人类乳铁蛋白cDNA(hLFc)连接起来,构建为重组质粒pCAhLFc。第三章:以稀有鮈鲫为实验材料,优化电脉冲-精子介导转基因法的参数,并对这种方法的机制进行探讨。将稀有鮈鲫精子与重组质粒pCAhLFc线性DNA混和温育,经不同条件的电脉冲处理后,与卵子受精,孵化出苗,PCR检测,结果表明25.5%~66.7%鱼苗带有外源基因。用显微镜观察精子的活力并统计受精率,发现不同的电脉冲条件对精子有不同的损害作用。用DNA外切酶消化电脉冲处理过的精子。证明电脉冲可以促使精子摄入外源基因。与显微注射相比较,在卵子质量不高的条件下,电脉冲-精子介导转基因法获得较高的出苗率。第四章:通过电脉冲-精子介导转基因获得转pCAhLFc草鱼,并设置不同的电脉冲参数组合对这种方法进行优化。将草鱼精子与重组质粒pCAhLFc线性DNA混和,经电脉冲处理,与卵子受精,得到若干草鱼苗。将草鱼苗匀浆,进行PCR检测,结果表明19.6~46.8%的鱼苗带有外源基因。本实验获得的最佳实验参数是:外源基因在精液中的浓度为200ng/μl,电脉冲参数设置为10kV、12*4个循环、2~(10)脉冲/循环、0.4sec,此时鱼苗的阳性率可高达46.8%。第五章:用草鱼出血病病毒GCHV对5月龄的转pCAhLFc草鱼和对照组草鱼进行攻击性毒性实验,发现5月龄的转pCAhLFc草鱼抵抗GCHV的能力比对照鱼高。本文通过对上述问题的研究,发展了一种更简单快捷易行的转基因方法,并探讨了这种方法的机制;探索了转pCAhLFc增强草鱼抗出血病病毒能力的可行性,为从另一个角度培养抗GCHV草鱼品系打下了基础。有待进一步研究的问题包括:①用电脉冲-精子转基因法转入的外源基因的行为(如复制、整合和遗传等)和别的途径(如显微注射法)的有无差别;②hLFc在草鱼体内的表达量和抗GCHV能力的相关性;③将电脉冲-精子转基因法和基因定点整合技术结合起来研制抗病转基因草鱼品系的培育。
英文摘要: This 5-chapter of dissertation is mainly on three topics of research work. The first one is to develop and optimize one method of gene transfer and make it practicable on grass carp and rare minnow. The second is to produce pCAhLFc transferred grass carp and measure it's resistance to GCHV in order to get a strain of GCHV-resistant grass carp. The third is to use rare minnow to study the mechanism of electroporated-sperm-mediated gene transfer. The following is the introduction to each chaper. Chapter 1: Review about the methods of gene transferring, the research on haemorrage of grass carp, and human lactoferrin. Chapter 2: Construction of a recombinant plasmid, pCAhLFc, which contains the plasmid skeleton and promoter, pCA, and the coding fragment of hLFc. The pCA comes from removal of gcGH from pCAgcGHc, and "all-fish" gene construct, by cleavage at the double NcoI sites. The hLFc comes from phLFc, a recombinant plasmid given by Prof. Hong Mengmin in Institute of Plant Physiology and Ecology, CAS. So the 8.8kb recombinant plasmid, pCAhLFc, contains the human gene hLCc under the control of the promoter of carp β-actin gene. The recombinant plasmid was amplified in E. coli and extracted and purified. In order to get linear DNA, pCAhLFc is digested with HindIII. Chapter 3: Gene transfer in rare minnow via electroporated sperm. Sperm of rare minnow mixed with linear DNA (pCAhLFc) was electroporated. The mature eggs were in-vitro fertilized with the treated sperm cells. The fries were sampled and analyzed by PCR. The ratio of fries containing foreign genes is between 25.5% and 66.7%. The electroporation does less harm to sperms than incubation. The sperm cells electroporated with foreign genes were washed and digested by DNaseI and analyzed by PCR. Evidence for that foreign gene can traverse the membrane of sperm in the pulse electric field was found out. Chapter 4: Gene transfer in grass carp via electroporated sperm. Sperm of grass carp were mixed with linear DNA (pCAhLFc) and electroporated with different sets of electroporation parameters. Then mature eggs were in-vitro fertilized with the treated sperm cells. The fries were sampled and were analyzed by PCR. The result indicates that the foreign gene had been transferred into the genome of 19.6~46.8% fries. The optimal parameters were list below: final concentration of foreign gene, 200ng/μl, the sperm electroporated at 10kV and 2~(10)pulses/cycle for 12*4 cycles, and the burst time 0.4 second. And the optimal gene-transferring ratio is 46.8%. Chapter 5: Artificial infection of grass carp by GCHV. The GCHV, as known to be the pathogeny of haemorrhage, was injected into pCAhLFc transferred and control grass carp fries. The results of artificial infections of these fries show that pCAhLFc transferred fry, in average, is more resistant to GCHV and haemorrhage-resistant grass carp might be produced in this way. Electroporated-sperm-mediated gene transfer, a simpler and faster gene transferring method, was developed and optimized. And its mechanism, specially speaking, how foreign genes enter eggs along with electroporated sperms, was studied. The primary research on the resistance to pCAhLFc transferred grass carp to GCHV set a solid groundwork for breeding of a GCHV-resistant grass carp. However, there are still several interesting questions remained to be studied. The first one is whether the behaviors such as amplication, integration and descendibility of foreign gene transferred by electroporated-sperm are different from those transferred by other methods, for example, microinjection. The second one is about the relationship between the quantity of expression of hLFc and the level of resistance to GCHV. The third one is how to harmoniously utilize the techniques of electroporated-sperm-mediated gene transfer and site-specific integration of foreign gene at the same time to accelerate the breeding of transgenic fish which is more resistant to diseases.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12610
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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电脉冲-精子介导转基因与转pCAhLFc草鱼抗出血病的研究.钟家玉[d].中国科学院水生生物研究所,2001.20-25
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