应用SNPs技术确定C．elegans形成异常习惯化的基因位置  

A study of ascertaining the exact gene location where C. elegans form abnormal habituation using SNPs methods

作　　者：许锡振[1] 张桂芳[2] 赵彦 濮瀑 乐卫东 

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]深圳市宝安区松岗人民医院 [3]中国科学院上海生命科学院 上海交通大学医学院健康科学研究所 上海交通大学医学院生物医学研究院

出　　处：《中华行为医学与脑科学杂志》2009年第11期970-972,共3页Chinese Journal of Behavioral Medicine and Brain Science

基　　金：基金项目：国家自然科学基金资助项目（30470552）

摘　　要：目的应用SNPs技术确定影响C．elegans的习惯化形成异常基因hab-1（cn308）的准确基因位置。方法（1）在C．elegans基因库里，检索连锁于第一常染色体上CB4856的Y71A12B的SNP标记（22．330MU）和C37A5的SNP标记（23．410MU），其基因型设为YC／YC，hab-1（cn308）的基因型为h／h。（2）把虫子进行同步化处理，同一虫子的大小。（3）野生型的雄性CB4856与雌雄同体cn308进行三因子杂交。对F1进行Tap测试，同时记录虫子的反应距离，统计处理，筛选习惯化和维持正常的杂合子（YC／h），传代至F2。（4）对F2代再进行Tap测试，筛选习惯化形成缓慢、习惯化维持时间短的虫子，进行PCR，用限制性内切酶消化，2％琼脂糖凝胶电泳分离目的片段。结果（1）第1次Tap测试结束后野生型的习惯化形成明显快于cn308[对Tap刺激形成习惯化均数（移动距离均数，单位：mm），野生型为（0．414±0．049）mm，cn308为（0．777±0．062）mm，t=10．08，P〈0．01]，1h后习惯化维持测试中，发现野生型的习惯化维持时间明显长于cn308（习惯化维持均数，野生型为（0．307±0．041）mm，cn308为（0．815±0．054）mm，t=11．54，P〈0．001）。（2）分析SNPs片段，统计Y71A12B的SNP标记与hab-1、C37A5的SNP标记与hab-1之间的基因重组数，观察HnonC的表现型，即Ch／h：20，Yh／h：6，测算重组比例可知hab-1离Y71A12B的SNP标记距离是0．831MU，hab-1离C37A5的SNP标记距离是0．249MU，所以确定hab-1位于Y71A12B的SNP标记和C37A5的SNP标记之间。结论影响C．elegans对无伤害性Tap重复刺激形成异常习惯化的hab-1基因位置是23．161MU。Objective To determine the enact gene location hab-1 （ cn308 ） which effects C elagans ab- normal habituation using SNPs methods. Methods （ 1 ） To retrieve the SNP markers of Y71A12B which chained at the first autosome the CB4856 （22. 330MU） and SNP markers of C37A5 （22. 330MU） in C. elegans gene bank, its genotype was set to YC/YC, the genotyp, es of hab-1 （cn308） was set to h/h. （2） By means of Synchro- nized processing, the size of worms was unified. （3）Wild-type CIM856 male was three-factor hybridized with her- maphroditism cn308. F1 generation were tested by Tap test ,recording the distance of response of the worms, statistical processing, screening habituation and retaining the Normal Heterozygote （ YC/h ）, subcuhuring to F2. （ 4 ） Then F2 generation were tested by Tap test, C. elegans with slow habituation were selected, processed by PCR, digested by restriction enzyme , separated target fragment with 2% agarose gel electrophoresis. Results After the first Tap test,the formation of habituation of the wild-type was significantly faster than the cn308. （ the average of habituation for Tap stimulation（ Moving distance, unit ： mm）, the wild-type＇ s was （ 0. 414 ± 0.049 ） mm, cn308 was （0. 777 ± 0. 062）mm, t = 10.08, P 〈 0. 001 ） ,in the habituation retention test 1 hour later, the habituation of wildtype was found that it retained significantly longer than cn308 （the average of habituation retention, wild-type was （0.307 ± 0. 041 ） ram, cn308 was （0.815 ± 0. 054 ） mm, t = 11.54, P 〈 0. 001 ）. （ 2 ） SNP fragments were analyzed,the genetic recombination number between SNP marker of Y71A12B and hab-1 and the genetic recombination number between SNP marker of C37A5 and hab-1 were gathered,observing the phenotype of H non C-type, namely, Ch/h ：20, Yh/h ：6 , by reckoning the restructuring proportion it was determined that the distance between hab-1 and SNP marker of Y71A12B was 0. 831MU. The distance between hab-1 and SNP marker of C

关 键 词：Tap刺激 习惯化 SNPS hab-1 

分 类 号：R363[医药卫生—病理学]