C．elegans的学习记忆异常基因及其功能的研究  

The abnormal genes of learning and memory in C. elegans and their functions

作　　者：许锡振[1] 吴利标[2] 罗信国[2] 赵彦 濮瀑 朱旭新[2] 

机构地区：[1]汕头大学医学院第一附属医院神经内科,汕头515041 [2]汕头大学医学院第一附属医院分子生物学实验室,汕头515041 [3]中国科学院上海生命科学研究院 上海交通大学医学院健康科学研究所

出　　处：《中华行为医学与脑科学杂志》2009年第12期1067-1070,共4页Chinese Journal of Behavioral Medicine and Brain Science

基　　金：国家自然科学基金资助项目（30470552）

摘　　要：目的探索学习记忆异常基因hab—1（cn308）的功能。方法1．在hab-1领域有Y63D3A．6、Y63D3A．7和Y63D3A．8等三个YAC克隆。在基因库里检索Y63D3A．6、Y63D3A．7和Y63D3A．8的cDNA序列，用DNASIS—Mac v3．6软件处理并设计引物，进行PCR。把PCR产物插入pMD^TM18-TVector载体，并培养提取Y63D3A．6、Y63D3A．7和Y63D3A．8的目的DNA，纯化DNA。2．进行rescue实验：把三种YAC克隆（clone）Y63D3A．6、Y63D3A．7和Y63D3A．8的DNA分别与GFP蛋白连接配制融合蛋白。应用OLYMPUS Axiovert S100共聚焦倒置显微镜操作系统，在心大小的C．elegans生殖腺（gonad）内分别把三种GFP融合蛋白溶液显微注射，发育传代至F1。在F1代选择带有GFP荧光的C．elegans，观察基因的表达部位，并进行Tap测试，通过观察在哪个YAC克隆把hab—1（cn308）的表现型恢复为野生型来确定hab-1的等位基因。3．DNA测序和同源性检索：hah-1的等位基因确定以后对cn308进行DNA测序。根据cn308的测序结果与已知的等位基因DNA序列进行Homology search，分析cn308的基因结构，并进行BLSTP homologic search，和其他物种比较目的基因的功能来阐明hab—1的基因功能。结果对Y63D3A．6、Y63D3A．7和Y63D3A．8等三种基因表达率进行χ^2检验结果表示差异有显著性（χ^2=26．84，P〈0．05）。表明Y63D3A．6、Y63D3A．7、Y63D3A．8等三种基因虽然连锁于第一常染色体上邻近的位置，但在C．elegans的头部表达率无关联性，并有显著性差异.同时可见Y63D3A．7强表达于C．elegans的头部，Tap测试证明Y63D3A．7把hab-1的表现型恢复野生型。DNA测序结果表明hab-1（cn308）是GAG错义突变为GGG，即Glu→Gly的突变体。BLSTP homologic search结果表明Y63D3A．7是调节线粒体complexⅠ的活性，就是说明hab-1基因与线粒体complexⅠ的功能有关。结论Y63D3A．7是hab-1的等位基因，主要表达于中枢神经系统。C．elegans的学习�Objective To research into the function of abnormal gene of learning and memory hab-1 （en308）. Methods 1. There were three YAC clones in the hab-1 area, Y63D3A. 6, Y63D3A. 7 and Y63D3A. 8. The eDNA sequence of Y63D3A. 6, Y63D3A. 7 and Y63D3A. 8 were retrieved in the gene bank, processed by DNASIS-Mac v3.6 software and designed primers,PCR was processed. The PCR product was inserted into pMD ^18TVector ,culturing and recovering target DNA of Y63D3A. 6, Y63D3A. 7 and Y63D3A. 8, purifying DNA . 2. Carrying out rescue experiments： DNA of three YAC clones Y63D3A. 6,Y63D3A. 7 and Y63D3A. 8 were linked with the C, FP Protein respectively for preparing DNA ： GFP fusion protein. The three DNA ： GFP fusion protein solution was nicroinjected into 1.4 size C. elegans gonad respectively by OLYMPUS Axiovert S100 confocal inverted microscope operating system, growing and subcuhring to F1 generation, observing gene expression site at the selected C. elegans with GFP fluorescence in F1 generation, carrying out Tap test , observing which YAC clone rescued the hab-1 （cn308） phenotype to wild-type phenotype to identify the hab-1 allele. 3. DNA sequencing and Homolog search： DNA sequencing for cn308 after hab-1 allele was ascertained. According to the results of cn308 sequencing and known allele DNA sequences to Homolog search, the genetic structure of cn308 was analyzed and BLST homologie search was earned out, comparing target gene function with other species to clarified gene funetion of hab-1. Results The result of χ^2 test for the rate of gene expression of Y63D3A. 6, Y63D3A. 7 and Y63D3A. 8 χ^2 showed χ^2 〉0.05, P〈0.05, there was a significant difference. It showed that although Y63D3A. 6, Y63D3A. 7 and Y63D3 A. 8 have linkage with the adjacent location of the first autosomal, expressing rate on the head of C. elegans had no relatedness and had significant differences. It revealed that Y63D3A. 7 was expressed strongly on thehead of C. elegans, and Tap test proved that Y63D3A. 7 rescued hab-1 to wild-type

关 键 词：Tap刺激 hab-1 线粒体complex Ⅰ 学习记忆 

分 类 号：R741[医药卫生—神经病学与精神病学]