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植物研究 ›› 2013, Vol. 33 ›› Issue (3): 308-316.doi: 10.7525/j.issn.1673-5102.2013.03.010

• 论文 • 上一篇    下一篇

阔叶薰衣草芳樟醇合成酶基因的克隆与表达载体构建

赵钟鑫;王健*;李琴;尚啸   

  1. 海南大学热带作物种质资源保护与开发利用教育部重点实验室,海南大学园艺园林学院,海口 570228
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2013-05-20 发布日期:2013-05-20
  • 通讯作者: 王健
  • 基金资助:
     

Cloning a Homologous Linalool Synthase Gene of Lavandula latifolia and Construction of Plant Expression Vector

ZHAO Zhong-Xin;WANG Jian*;LI Qin;SHANG Xiao   

  1. Key Laboratory of Protection and Developmental Utilization of Tropical Crop Germplasm Resources (Hainan University),Ministry of Education; College of Horticulture and Landscape Architecture,Hainan University,Haikou 570228
  • Received:1900-01-01 Revised:1900-01-01 Online:2013-05-20 Published:2013-05-20
  • Contact: WANG Jian
  • Supported by:
     

摘要: 以阔叶薰衣草(Lavandula latifolia)的鲜叶为试材,通过RT-PCR技术,获得阔叶薰衣草芳樟醇合成酶基因的cDNA序列。该序列长度为1 809 bp,编码602个氨基酸,包含一个1 809 bp的开放阅读框,与NCBI上已公布的阔叶薰衣草的芳樟醇合成酶蛋白序列相似度为98%,有21个核苷酸差异,11个氨基酸差异,将其命名为Lslis。序列分析研究表明:Lslis具有多数单萜烯类合成酶基因典型的保守结构域,即DDXXD,RRX8W和(N,D)D(L,I,V)X(S,T)XXXE;具有芳樟醇合酶活性必需的DDXXD功能基团。Lslis推导氨基酸序列的预测分子质量和等电点分别为70.36和5.66 kD;疏水性分析结果表明其大部分氨基酸区域为亲水结构,该基因为亲水多肽。Lslis和已公布的阔叶薰衣草芳樟醇合酶Lllis的蛋白二级结构预测结果表明,两者在整体结构上基本保持一致,但Lslis相对于Lllis要少一个α螺旋结构。将其连接到pMD18-T的载体上,通过中间载体pCAMBIA1303,构建了Lslis基因的植物表达载体,并将其导入农杆菌,经菌落PCR鉴定和测序结果表明该片段已插入表达载体。

关键词: 阔叶薰衣草, 芳樟醇合成酶, 基因克隆, 序列分析, 植物表达载体构建

Abstract: In this study, a full length cDNA encoding linalool synthase was successfully cloned from the fresh leaves of Lavandula latifolia, with newly gene-specific primers by reverse transcription (RT)-PCR technology. The sequence was 1 809 bp and contained a complete open reading frame (ORF) of 1 809 bp encoding 602 amino acids, and the protein sequences exhibited the highest similarity (98%) with L.latifolia linalool synthase announced on NCBI, with the differences of 21 nucleotide and 11 amino acid, respectively. The gene was named as Lslis. The sequence analysis showed that: Lslis presented three typical conserved motifs of many terpene synthases. There was a peptide sequence DDXXD which is essential for the enzymatic activity of linalool synthase. The molecular weight and isoeletric point of Lslis were predicted to be 70.36 kD and 5.66, respectively. Most of the amino acid sequences of Lslis were hydrophilic regions, which was hydrophilic polypeptide. The results of predicted secondary structure for Lllis and Lslis showed that both the most structures kept consistent, but Lslis had less a α helix to Lllis, which has been promulgated on NCBI. The PCR product was cloned into pMD18-T vector. The Lslis gene expression vector was constructed successfully, having the aid of intermediate plant expression vector pCAMBIA1303. Reconbinant expression vector was identified by PCR analysis, PCR indicated plant vector was transferred into A.tumefaciens.

Key words: Lavandula latifolia, linalool synthase, gene cloning, sequence analysis, construction of plant expression vector

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