| 申本昌,江红,乔传令.2009.基因组步移法克隆小菜蛾CYP9G2基因的上游序列.动物学杂志,44(5):112-116. |
| 基因组步移法克隆小菜蛾CYP9G2基因的上游序列 |
| Cloning of the Upstream Untranslated Region of CYP9G2 from Plutella xylostella by Genomic Walking |
| 投稿时间：2008-11-26 修订日期：2009-06-26 |
| DOI： |
| 中文关键词: 小菜蛾 CYP9G2 顺式调控元件 基因组步移 聚合酶链反应 |
| 英文关键词:Plutella xylostella CYP9G2 Cis-acting elements Genome walking Polymerase chain reaction |
| 基金项目:中国科学院知识创新工程领域前沿项目(No.KSCX3-IOZ-05);国科金外资助字第30311120206号项目;国家高技术研究发展计划(863)项目(No.2002AA601160);广州医学院博士/留学归国人员启动基金项目(No.2006GD056) |
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| 中文摘要: |
| 利用4种产生平端切头的限制性内切酶消化小菜蛾(Plutellaxylostella)的基因组DNA,然后利用DNA连接酶的催化作用,在4种不同平端切头的小菜蛾基因组DNA上连接一个氨基化的基因组步移衔接头序列,针对衔接头及已克隆的CYP9G2基因的序列,设计两对PCR上、下游引物,进行PCR扩增、T-A克隆和阳性克隆的巢式PCR验证,通过测序克隆到了小菜蛾CYP9G2基因上游未知序列约1.8kb。通过对该基因的上游序列进行信息分析,发现1个可能的节肢动物动物转录起始子(Inr),3个CAAT样盒及1个抗氧化剂样反应因子,共5个可能的顺式调控元件。研究还表明,利用基因组步移方法可以快速地克隆已知序列的上游未知序列,实验操作经济、简便,对于已知cDNA序列或部分基因组序列的基因,其上游调控序列的克隆,基因组步移具有较高的实用价值。 |
| 英文摘要: |
| To clone the upstream untranslated region of CYP9G2 from diamondback moth,the genomic walking was performed according to the routine procedure based on PCR amplification using the outer adaptor primer(AP1) and CYP9G2 specific primer(GSP1).After T-A cloning,positive clones were confirmed by nested PCR using the outer adaptor primer(AP2) and CYP9G2 specific primer(GSP2) as forward and reverse primers.Analysis of the upstream DNA sequence of CYP9G2 showed that 5 cis-acting elements existed in the upstream sequence of CYP9G2.One of the elements was a putative initiator of arthropoda,three of them were CAAT-like boxes,and the other one was an antioxidant-response-like element.This kind of genome walking could be used in rapid cloning of the promoters and other upstream regulatory elements in genes that only cDNA sequence was available,mapping of intron/exon junctions,and walking bidirectionally from any sequence-tagged site or expressed sequence tag.The method is simple,reliable and efficient. |
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