摘要
为了克隆维生素E缺乏时皱纹盘鲍(Haliotis discus hannai)外套膜差异性表达的基因,本研究配制了维生素E缺乏水平(3.5 mg/kg)和正常添加水平(52.8 mg/kg)的人工饲料,喂养皱纹盘鲍幼鲍,初始体质量为(0.71±0.00)g,初始壳长为(15.49±0.04)mm,240 d后,采用抑制消减杂交法(Suppression Subtractive Hybridization,SSH)构建了维生素E缺乏组与正常组外套膜组织差异表达的cDNA消减文库。经检验,差异表达的基因均被富集了25-28倍,证明构建的cDNA消减文库具有很强的消减效率。从文库中随机挑取100个白色克隆摇菌液进行PCR鉴定,95%的克隆中均有100-900 bp的插入片段,这些片段可能是维生素E缺乏差异表达基因的cDNA片段。选取含插入片段大小不同的48个克隆测序,其中有16个新基因,10个谷胱甘肽转移酶的基因,5个谷胱甘肽过氧化物酶基因,2个过氧化氢酶基因,3个羟基类固醇脱氢酶基因,4个酪氨酸激酶基因,1个类甲状腺过氧化物酶蛋白基因,3个纤维素酶基因,1个cAMP效应元件结合蛋白基因,3个贝壳生物矿化相关蛋白的基因。总的来说,利用抑制消减杂交的方法构建差异表达cDNA文库,可以比较好地反映维生素E对皱纹盘鲍影响的基因信息,为研究其他营养素对皱纹盘鲍基因表达的影响提供了基础数据。
A study was conducted to clone the differentially expressed genes in the mantle of abalone Haliotis discus hannai during vitamin E deficiency. Juvenile abalone [ initial weight （0.71 ± 0.00） g; initial shell length （15.49 ± 0.04）mm] were fed 240 d with semi-purified diets containing 2 levels of vitamin E： 3.5 mg/kg and 52.8 mg/kg. The cDNA subtractive library of the mantle of abalone vitamin E deficiencytreated was constructed using the method of suppression subtractive hybridization （SSH）. The results showed that the constructed cDNA subtractive library was a highly efficient one： the cDNAs were significantly enriched for differentially expressed sequences. One hundred positive clones were randomly picked and identified by PCR method;95 % clones contained 100 -900 bp inserts, which might be the cDNA fragments of differentially expressed genes in vitamin E deficiency group. Forty eight clones were sequenced, including 16 new genes, 10 glutathione-s-transferase genes, 5 glutathione peroxidase genes, 2 catalase genes, 3 hydroxysteroid dehydrogenase genes, 4 tyrosine kinase genes, 1 thyroid peroxidase-like protein gene, 3 genes for cellulose, 1 cAMP responsive element binding protein gene and 3 shell biomineralization related genes. In conclusion, the method to construct cDNA subtractive library could well reflect the gene information of abalone affected by vitamin E. Results of the present study provide the basal data to research of relationship between nutrients and gene expression of abalone. [Journal of Fishery Sciences of China, 2007, 14 （3）：383 - 389]
出处
《中国水产科学》
CAS
CSCD
北大核心
2007年第3期383-389,共7页
Journal of Fishery Sciences of China
基金
国家自然科学基金项目(30200215)
关键词
皱纹盘鲍
外套膜
维生素E
抑制消减杂交
CDNA消减文库
Haliotis discus hannai
mantle
vita rain E
suppression subtraetive hybridization
eDNA subtractive library
