甘蓝中硫氧还蛋白编码基因THL1的分子特性及表达研究

园艺学报 ›› 2007, Vol. 34 ›› Issue (4): 909-914.

甘蓝中硫氧还蛋白编码基因THL1的分子特性及表达研究

高启国¹ ; 宋　明¹; 牛　义¹ ;杨　昆² ; 朱利泉^2*;王小佳^1*

(¹ 西南大学重庆市蔬菜学重点实验室, 重庆400716; ² 西南大学植物生理生物化学实验室, 重庆400716)

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2007-08-25 发布日期:2007-08-25

Molecular Characterization and Expression of Thioredoxin-like Prote in THL1Gene from Brassica oleracea L.

GAO Qi-guo¹; SONG Ming¹; NIU Yi¹; YANG Kun² ; ZHU Li-quan^2*
;WANG Xiao-jia^1*

(¹Key Laboratory in Olericulture of Chongqing, Southwest University, Chongqing 400716, China; ² Plant Physiology and Biochemistry Laboratory, Southwest University, Chongqing 400716, China)

Received:1900-01-01 Revised:1900-01-01 Online:2007-08-25 Published:2007-08-25

摘要/Abstract

摘要： 采用PCR和RT2PCR技术, 以‘E1’甘蓝基因组DNA和柱头cDNA为模板对THL1基因进行扩增克隆, 得到的片段长度分别为732 bp和455 bp。序列分析表明, 克隆的DNA和cDNA序列与甘蓝‘西园四号’THL1的DNA和cDNA同源性分别为97.9%和98.3%, 两条序列内含子的大小不同; 同时, 前者第2内含子不符合典型的GT2AG规则: 即第2个内含子3′端碱基为AT。将THL1基因cDNA序列定向克隆到原核表达载体pET-43.1a ( + ) , 构建融合表达质粒pET43.1a ( + ) -THL1, 在大肠杆菌BL21中表达出分子量为74
kD的融合蛋白, 经胰岛素检测, THL1有氧化还原活性, 表明THL1在大肠杆菌中得到了正确表达。

关键词: 甘蓝, 类硫氧还蛋白, 基因克隆, 序列分析

Abstract: The DNA and cDNA fragments of the THL1 were amp lified from genomic DNA and stigma cDNA of Brassica oleracea ‘E1’by PCR and RT2PCR methods. Their lengths were 732 bp and 455 bp re
spectively. Sequence analysis indicated that the identities of DNA and cDNA with those cloned from Brassica oleracea ‘Xiyuan 4’ previously were 97.9% and 98.3% respectively, the introns of the two sequences were
different in size. Moreover, the second intron of THL1 from Brassica oleracea ‘E1’was not comp ly with the
typ ical GT-AG rule: AT existed in the 3′end of the second intron. The cDNA of THL1 was cloned into vector pET-43.1a ( + ) to be pET43.1a ( + ) -THL1, transferred into E1coli BL21 and exp ressed as a 74 kD fusion p rotein when induced with IPTG. Thioredoxin activitywasmeasured by ability of the THL1 to reduce insulin. The result showed the THL1 gene had been correctly exp ressed.

Key words: Brassica oleracea, Thioredoxin-like protein, Gene clone, Sequence analysis

高启国;宋　明;牛　义;杨　昆;朱利泉;王小佳. 甘蓝中硫氧还蛋白编码基因THL1的分子特性及表达研究[J]. 园艺学报, 2007, 34(4): 909-914.

GAO Qi-guo;SONG Ming;NIU Yi;YANG Kun;ZHU Li-quan;WANG Xiao-jia. Molecular Characterization and Expression of Thioredoxin-like Prote in THL1Gene from Brassica oleracea L.[J]. ACTA HORTICULTURAE SINICA, 2007, 34(4): 909-914.

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