通过显微介导将鳜总DNA片段导入镜鲤基因组的分子验证  

Molecular Verification of Mirror Carp Genome Micro-injected With Total DNA of Mandarinfish Siniperca chuatsi

作　　者：闫学春[1] 栾培贤[1] 何立川 YAN Xuechun;LUAN Peixian;HE Lichuan(National Local Joint Engineering Laboratory of Freshwater Fish Breeding,Key Laboratory of Freshwater Aquatic Biotechnology and Genetic Breeding,Heilongjiang River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Harbin 150070,China)

机构地区：[1]中国水产科学研究院黑龙江水产研究所,淡水鱼类育种国家地方联合工程实验室,淡水水产生物技术与遗传育种重点实验室,黑龙江哈尔滨150070

出　　处：《水产学杂志》2020年第4期1-6,共6页Chinese Journal of Fisheries

基　　金：中央级公益性科研院所基本科研业务费专项(HSY201707M)。

摘　　要：利用外源基因导入技术,将1nL左右未经遗传修饰的鳜Siniperca chuatsi基因组DNA导入2500余粒镜鲤Cyprinus carpio受精卵核区附近,孵出仔鱼1500余尾,放入池塘300尾正常饲养。利用引物(E-AGG,M-CTA)对显微介导鳜基因镜鲤进行扩增片段长度多态性(AFLP)检测,研究显微介导外源总DNA转化当代的分子生物学结果。结果表明:在AFLP的15对引物中,显微介导鳜基因镜鲤中有3对引物扩增出了供体鳜基因片段,从DNA水平上验证了在受体鱼的基因组中整合了供体基因片段。将6尾阳性显微介导鳜基因镜鲤与供体鳜相同的AFLP条带进行切下、回收、克隆、测序,发现供体鳜片段序列与阳性显微介导鳜基因镜鲤片段序列完全一致,找到了精确的遗传证据。本研究结果为构建转全鳜基因镜鲤AFLP检测体系,从基因角度研究供体大片段在受体鱼中的导入情况提供了理论依据。About 1 nL of no genetic modification total genomic DNA derived from mandarinfish(Siniperca chuatsi)was micro-injected into the region near the nucleus of more than 2500 fertilized eggs of mirror carp(Cyprinus carpio), in which more than 1500 larvae were hatched and in which 300 postlarvae were reared in a pond. The microscopically mediated mandarinfish gene was detected in the micro-injected mirror carp individuals by amplified fragment length polymorphism(FLP) and by using E-AGG, M-CTA primers to explore the molecular biological results of microscopically mediated transformation of exogenous total DNA. In the 15 pairs of AFLP primers, three pairs of primers had amplified the gene fragments of donor, which verified that DNA fragments of the mandarinfish have already integrated into the genome of mirror carp parent individuals. The recovery and sequences of the AFLP bands from six positive micro-injected mirror carp individuals revealed that the gene fragment sequences of the mirror carp were the same as the donors.The finding provided theoretical basis for AFLP detection system construction of micro-injected mandarinfish gene mirror carp and verification of the integrated situation of macromolecular DNA fragments in receptor fish from molecular perspectives.

关 键 词：显微介导 鳜总DNA 镜鲤 AFLP 分子验证 

分 类 号：Q959[生物学—动物学] S917[农业科学—水产科学]