两个陆地棉过氧化物酶cDNA的克隆和鉴定  

Cloning and Characterization of Two Peroxidase cDNAs in Gossypium hirsutum L.

作　　者：郭媖[1] 郭旺珍[1] 张天真[1] 

机构地区：[1]南京农业大学作物遗传与种质创新国家重点实验室,江苏南京210095

出　　处：《作物学报》2007年第6期891-897,共7页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(30471104);国家重点基础研究发展计划(973计划)项目(2002CB111301);教育部新世纪优秀人才支持计划项目(NCET-04-0500);教育部长江学者和创新团队发展计划项目(IRT0432)

摘　　要：从陆地棉优质材料7235不同发育时期的棉纤维混合cDNA文库中分离出2个与过氧化物酶相关的cDNA克隆GhPOD1和GhPOD2。GhPOD1是利用5′RACE技术得到的长度为1 489 bp的完整cDNA序列,开放读码框长度为816 bp,编码271个氨基酸,BLAST结果表明,该基因和拟南芥中已报道的过氧化物酶基因同源性最高。GhPOD2是通过对cDNA克隆直接测序获得,其插入片段长度为1 355 bp,开放读码框长度为999 bp,编码332个氨基酸,BLAST结果表明,该cDNA序列与已报道的1个陆地棉纤维过氧化物酶基因(pod8,L08199)氨基酸相似性达99%。RT-PCR分析表明GhPOD1是一个组成性表达的基因,而GhPOD2在根中不表达,而在茎、叶、胚珠和纤维细胞中表达,并从开花后14 d起,在纤维细胞中表达量逐渐减弱。进化树分析显示GhPOD1与已知的11个过氧化物酶基因距离都较远,是1个新的陆地棉过氧化物酶基因;GhPOD2与相似性高的pod8在同一分支,但与其余的第3类过氧化物酶也有较大的差异。Southern杂交结果表明这两个基因在陆地棉基因组中都存在2个拷贝,推测A、D亚组中各有1个拷贝。GhPOD1和GhPOD2的棉花转基因功能验证正在进行。Pemxidases are iron haem-containing oxidoreductase （EC 1.11.1.7） that reduce peroxides, mainly hydrogen peroxide, to water and subsequently oxidize small molecules, often aromatic oxygen donors. The plant peroxidase superfamily can be divided into three classes. Of these classes, the class Ⅲ peroxidases have important function in plant metabolism processes such as cell wall organization, germination, hormonal, and stress responses etc. Cotton fibers are single-celled seed trichomes and fiber cells develop in four distinct but overlapping phases - initiation, cell elongation （primary wall synthesis）, cell wall deposition, and maturation. The objective of the study was to clone peroxidase genes, further put a foundation to illustrate these genes function in cotton fiber developmental stages. In this paper, two cDNA clones encoding class Ⅲ peroxidases were separated from developmentally different cotton fiber pools of elite material 7235 library. GhPOD1 was a 1 489 bp cDNA sequence obtained via 5＇RACE technique, its open reading frame was 816 bp, and encoded a polypeptide containing 271 amino acids. BLAST analysis indicated this gene had high homologies with the peroxidase reported in Arabidopsis thaliarta ; the insert fragment of GhPOD2 was 1 355 bp, its open reading frame was 999 bp, and encoded a polypeptide containing 332 amino acids. BLAST analysis indicated this gene had a 99% amino acid identity with the peroxidase（ pod8, 1.08199）reported in Gossypium hirsutum. GhPOD1 was expressed constitutively in every tissue, while GhPOD2 did not express in root, but express in stem, leaf, ovule, and fiber cells and its expression in fiber cells was decreased after 14 DPA in transcriptional level. The phylogenetic analysis suggested GhPOD1 was distinctly different from other peroxidases, indicating that it was a novel G. hirsutum peroxidase; GhPOD2 and pod8 were in the same branch group, but they were also distinctly different from the other class Ⅲ peroxidases. Southern blotting analyses showed that t

关 键 词：棉花 过氧化物酶 克隆 鉴定 

分 类 号：S562[农业科学—作物学]