CAJ | 학술논문

　　目的构建虎眼万年青 EST 文库，筛选相关基因并对其进行功能鉴定。方法 CTAB 法提取虎眼万年青鳞茎总 RNA，通过SMART 方法构建全长 cDNA 文库，获得库容大于3×106 cfu/ml 的均一化全长 cDNA 文库。随机挑取1440个单克隆测序，并对得到的 EST 序列进行 Blast 分析（NR、NT、Swiss-Prot 和 KEGG）以及 COG 功能分类。结果获得1398条有效 EST 序列，含有1146条单一基因，cDNA 文库平均插入片段长度在1.5 kb 以上，全长率54%，冗余率3.3%。其中两段基因都与 Syntaxin 43基因相似，同源性分别为76%和89%，根据 Blast 比对结果，推断这两个基因片段是同一个 Syntaxin 基因的5'端和3'端。据此设计引物，以虎眼万年青 cDNA 为模板，通过常规的 RT-PCR 扩增得到全长 Syntaxin 基因，将其克隆到大肠杆菌表达载体，接着导入大肠杆菌进行诱导表达，SDS-PAGE 和 Western blot 证实该基因在大肠杆菌获得表达。结论首次构建成功虎眼万年青 EST 文库；并从中筛选得到一条和 Syntaxin 具有同源性的基因，实现了 Syntaxin 蛋白在大肠杆菌中的表达，为 Syntaxin 基因功能的鉴定奠定基础。
　　목적구건호안만년청 EST 문고，사선상관기인병대기진행공능감정。방법 CTAB 법제취호안만년청린경총 RNA，통과SMART 방법구건전장 cDNA 문고，획득고용대우3×106 cfu/ml 적균일화전장 cDNA 문고。수궤도취1440개단극륭측서，병대득도적 EST 서렬진행 Blast 분석（NR、NT、Swiss-Prot 화 KEGG）이급 COG 공능분류。결과획득1398조유효 EST 서렬，함유1146조단일기인，cDNA 문고평균삽입편단장도재1.5 kb 이상，전장솔54%，용여솔3.3%。기중량단기인도여 Syntaxin 43기인상사，동원성분별위76%화89%，근거 Blast 비대결과，추단저량개기인편단시동일개 Syntaxin 기인적5'단화3'단。거차설계인물，이호안만년청 cDNA 위모판，통과상규적 RT-PCR 확증득도전장 Syntaxin 기인，장기극륭도대장간균표체재체，접착도입대장간균진행유도표체，SDS-PAGE 화 Western blot 증실해기인재대장간균획득표체。결론수차구건성공호안만년청 EST 문고；병종중사선득도일조화 Syntaxin 구유동원성적기인，실현료 Syntaxin 단백재대장간균중적표체，위 Syntaxin 기인공능적감정전정기출。
Objective Construction of Ornithogalum saundersiae EST library to facilitate the gene screening and functional characterization. Methods The total RNA of Ornithogalum saundersiae was isolated by CTAB method. A normalized full-length cDNA library, with titer of above 3 × 106 cfu/ml, was established by the method of SMART using the total RNA of young bulb of Ornithogalum saundersiae as the template. Sequencing analysis of 1440 clones, which were selected randomly from the constructed EST library of Ornithogalum saundersiae was performed. Blast analysis (NR, NT, Swiss-Prot and KEGG) and COG function classification were done using the acquired EST sequences. Results The average size of cDNA inserts was 1500 bp with a redundancy rate of 3.3%. Meanwhile a total of 1146 unigenes were attained by sequencing. These results indicated that the normalized full-length cDNA library was established successfully. Two cDNA sequences, similar to Syntaxin gene, were screened from cDNA library. Blast analysis showed the two sequences were probably the 5' and 3' sequence of the same Syntaxin gene. Two pairs of primers based on the specific Syntaxin gene were synthesized. The full-length Syntaxin gene was isolated from Ornithogalum saundersiae by standard RT-PCR. The Syntaxin gene was cloned into the E.coli expression vector pET-28a(+) and the recombinant E.coli containing Syntaxin gene was induced to express by IPTG addition. SDS-PAGE and Western blot results indicated that the Syntaxin gene was successfully expressed in E.coli. Conclusion The Ornithogalum saundersiae EST library was successfully constructed. A full long cDNA similar to Syntaxin gene was isolated from Ornithogalum saumdersiae and then heterologous expression in E.coli was performed successfully, which laid the foundation for further functional characterization of Syntaxin gene in the future.