东北梅花鹿鹿茸cDNA文库的构建及初步鉴定
摘要
本试验为克隆出与鹿茸生长发育相关基因的全长序列,采用SMART技术构建了东北梅花鹿鹿茸尖端组织的全长cDNA文库。采取人工驯养的健康4岁东北梅花鹿为试验动物,取其生茸期鹿茸尖端组织为实验材料,利用SV Total RNA抽提试剂盒分离提取和纯化总RNA；反转录成单链cDNA, LD-PCR方法合成双链cDNA; PCR产物经蛋白酶K水解、纯化后,用Sfil酶切；将酶切产物通过CHROMA SPIN-400TM柱进行分级分离,回收片段长度在500bp以上的cDNA组分,与pDNR-LIB质粒载体连接,电转化入E.coli.DH5a高效感受态细胞中,建成原始文库；鉴定文库的滴度和重组效率后,进行文库扩增；随机挑取单菌落噬菌斑,用载体克隆位点两端的通用引物进行PCR扩增,以检测所构建的cDNA文库的质量。试验结果表明：总RNA电泳中18S、28S两条带清晰明显无降解,总RNA质量好、纯度高,符合建库要求。经鉴定,文库得到约2.56x106个重组子,重组效率接近100%,插入片段长度在0.5-2.0kb之间,平均长度为1.1kb。本试验文库各项指标均达到了标准cDNA文库的要求,可用于全长cDNA的筛选,成功的构建了东北梅花鹿鹿茸cDNA文库。
东北梅花鹿鹿茸生长中心组织cDNA文库的成功构建,不仅保存了珍贵的梅花鹿基因资源,也为以后EST测序分析,基因表达谱构建,分析功能基因在鹿茸组织生长发育不同时期的表达奠定了前期基础。
In order to separate specific functional genes of velvet, we extracted total RNA from velvet top tissue of Sika deer (C. n. hortulorum) under artificial breeding.Total RNA was extracted by SV Total RNA Isolation System kit.SMART technique and reverse transcription were used for first-strand cDNA synthesis. Subsequently, we used LD-PCR method to synthesis double strand cDNA that were then digested by Sfil and fractionated by CHROMA SPIN-400 columns.The cDNA component above 500bp were retrieved and connectied with the pDNR-LIB plasmid vector.Then the ligation mixture was electroporated into E.coli DH5a.The qualities of original and amplified cDNA libraries were strictly cheeked by conventional titer determination.Then, the library was amplified, and clones were picked randomly from the library for screening size of cDNA inserts by PCR.The library contains 2.56×106 clones and the rate of recombinant was nearly 100%.The insert size ranged from 0.5-2.Okb.These data show that this library meets the requirement of a standard cDNA library.
The full-length cDNA library of Northeast sika deer velvet tissues has been constucted successfully by SMART techonology, which was essential for the coming researches, such as EST sequencing, analyze functional genes of the deer antler growth tissues and so on.
东北梅花鹿鹿茸生长中心组织cDNA文库的成功构建,不仅保存了珍贵的梅花鹿基因资源,也为以后EST测序分析,基因表达谱构建,分析功能基因在鹿茸组织生长发育不同时期的表达奠定了前期基础。
In order to separate specific functional genes of velvet, we extracted total RNA from velvet top tissue of Sika deer (C. n. hortulorum) under artificial breeding.Total RNA was extracted by SV Total RNA Isolation System kit.SMART technique and reverse transcription were used for first-strand cDNA synthesis. Subsequently, we used LD-PCR method to synthesis double strand cDNA that were then digested by Sfil and fractionated by CHROMA SPIN-400 columns.The cDNA component above 500bp were retrieved and connectied with the pDNR-LIB plasmid vector.Then the ligation mixture was electroporated into E.coli DH5a.The qualities of original and amplified cDNA libraries were strictly cheeked by conventional titer determination.Then, the library was amplified, and clones were picked randomly from the library for screening size of cDNA inserts by PCR.The library contains 2.56×106 clones and the rate of recombinant was nearly 100%.The insert size ranged from 0.5-2.Okb.These data show that this library meets the requirement of a standard cDNA library.
The full-length cDNA library of Northeast sika deer velvet tissues has been constucted successfully by SMART techonology, which was essential for the coming researches, such as EST sequencing, analyze functional genes of the deer antler growth tissues and so on.
引文
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