杉木ClKptA/Tpt1基因的克隆及其表达特性分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0177

摘要/Abstract

摘要： 杉木是我国南方重要的速生用材树种,磷是植物生长的重要元素,但在我国亚热带地区土壤中的磷含量相对较少。为了阐明杉木在磷限制条件下怎样充分利用磷来促进杉木生长,本研究克隆了杉木磷酸转移酶基因ClKptA/Tpt1,将该蛋白与其他物种的KptA/Tpt1亲缘关系进行了分析,同时预测了该蛋白的三级结构,并在大肠杆菌中表达了该蛋白。此外还检测了ClKptA/Tpt1基因在杉木不同部位和不同浓度磷处理下的表达模式。结果显示ClKptA/Tpt1的CDS全长为1 143 bp,编码380个氨基酸,与荷花的KptA/Tpt1亲缘关系最近,与番茄的亲缘关系最远;在大肠杆菌BL21（DE3）中表达了该蛋白,其分子量为55.5 kD,ClKptA/Tpt1的氨基酸序列不具有跨膜结构,其蛋白三级结构主要以α-螺旋和β-折叠的形式存在。表达模式研究发现ClKptA/Tpt1在杉木叶中的表达量最高,在茎中表达量最低;不同浓度磷处理过的杉木中,ClKptA/Tpt1的表达量和木质素含量在一定范围内随着磷浓度的增加而增多,该结果为阐明杉木充分利用磷促进木质素合成的分子机理研究奠定理论基础。

关键词: ClKptA/Tpt1, 亲缘关系, 蛋白表达, 磷处理, 表达模式

Abstract: Cunninghamia lanceolata（Lamb.）Hook is one of the most important fast-growing timber tree species in southern China. Phosphorus is an essential nutrition in plant growth and development. However,the concentration of phosphorus of soil is relatively low in the subtropical region of China. In order to elucidate how C.lanceolata fully uses phosphorus to promote its growth under phosphorus limitation,the phosphate transferase gene ClKptA/Tpt1 was cloned by RT-PCR from C. lanceolata,and genetic relationship between the protein and KptA/Tpt1 of other species was analyzed. Meanwhile,the tertiary structure of the protein was predicted,and the protein was expressed in Escherichia coli. Furthermore,the expression patterns of ClKptA/Tpt1 in the different parts of C. lanceolata treated with different concentrations of phosphorus were detected. The result showed that the total length of CDS（code sequence）of ClKptA/Tpt1 was 1 143 bp,and it encoded 380 amino acids,which had the closest relationship with the KptA/Tpt1 of Nelumbo nucifera and the furthest relationship with Solanum lycopersicum. The molecular weight of ClKptA/Tpt1 expressed in E. coli was 55.5 kD. The amino acid sequence of ClKptA/Tpt1 did not have a transmembrane structure,and tertiary structure of ClKptA/Tpt1 main constructed by α-heli and β-sheet. ClKptA/Tpt1 expressed in the leaves at the highest and the lowest in stem. The expression level of ClKptA/Tpt1 and lignin content of stem increased with phosphorus content increasingly added in the soil of planted C. lanceolata. These results lay a theoretical foundation for elucidating the molecular mechanism of fully utilizing phosphorus to promote lignin biosynthesis in C. lanceolata.

Key words: ClKptA/Tpt1, affiliation, protein expression, phosphorus treatment, expression pattern

杨世全, 彭丹, 费文杰, 杨丰, 屈高毅, 唐威威, 欧剑萍, 邓湘雯, 周波. 杉木ClKptA/Tpt1基因的克隆及其表达特性分析[J]. 生物技术通报, 2020, 36(8): 15-22.

YANG Shi-quan, PENG Dan, FEI Wen-jie, YANG Feng, QU Gao-yi, TANG Wei-wei, OU Jian-ping, DENG Xiang-wen, ZHOU Bo. Cloning and Expression of ClKptA/Tpt1 Gene from Cunninghamia lanceolata（Lamb.）Hook[J]. Biotechnology Bulletin, 2020, 36(8): 15-22.

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