黄鳝肠道cDNA文库构建及营养相关表达序列标签分析

Intestine cDNA Library Construction and Nutrition-related Expressed Sequence Tag Analysis of Monopterus albus

本研究旨在构建黄鳝肠道未剪切cDNA文库,对黄鳝肠道营养相关基因表达进行初步分析。以黄鳝全肠为试验材料,采用Invitrogen公司的Gateway技术构建了黄鳝肠道未剪切cDNA文库,并进行规模表达序列标签(expressed sequence tag,EST)测序和分析。结果表明:文库的库容量为1.46×107CFU,重组率达96.88%,平均插入片段长度2.11kb。随机挑取1056个cDNA阳性克隆进行测序,共获得906条有效的EST,其中886条的长度大于400bp,拼接组装得到762个单基因簇(unigene),包括61个重叠群(contig),701个单拷贝EST(singleton);与营养相关的EST共有5类59种,其中:蛋白质/氨基酸代谢类25种,碳水化合物代谢类13种,脂类代谢类5种,能量代谢类12种,无机离子代谢类4种。本文成功构建了黄鳝肠道未剪切cDNA文库,并进行了EST序列测定和分析,筛选出营养相关EST,为后续研究提供了基础数据。

In this study,we constructed an uncut intestine cDNA library,and examined nutrition-related gene expression in this library as a preliminary analysis.We used the whole intestinal tract tissue of M.albus and Invitrogen＇s Gateway technology to construct the cDNA library and to perform scale expressed sequence tag（EST）sequencing and analysis.Our results showed that the library capacity reached 1.46×107 CFU,and the percentage of recombination was as high as 96.88%.PCR results showed that the average insert size was 2.11 kb.A total of 1 056 randomly selected cDNA clones were sequenced,and 906 were used in high-quality EST.Of the 906 cDNA clones,886 were longer than 400 bp,with a splicing assembly of 762 unigenes,including 61 contigs and 701 singletons.We identified 5 categories within 59 species of putative nutrition-related genes based on their functional classification.These categories included 25 species for amino acid metabolism,13 species for carbohydrate metabolism,5 species for lipid metabolism,12 species for energy metabolism,and 4 species for inorganic ion metabolism.In this study,we successfully constructed an uncut intestine cDNA library,and performed EST sequencing and analysis,which identified nutrition-related EST.This study provided the basic data for follow-up researches related to nutrition in M.albus.