高山红景天红景天甙生物合成相关基因的克隆及功能分析
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摘要
高山红景天是一种新兴适应原性珍稀药用植物。世界范围内对其特征性功能成分红景天甙和甙元酪醇的生物合成途径的研究尚属空白。本文采用cDNA末端快速克隆技术(RACE),以高山红景天为试材,分离得到与红景天甙和酪醇生物合成相关的两个基因,对其基因特性、基因功能进行了鉴定和分析。
植物次生代谢中产生的小分子糖甙类化合物是由尿苷二磷酸葡萄糖基转移酶(UDP-glucosyltransferase,UDPGT,UGTs)催化合成的。本文首次从高山红景天中克隆了UDP-葡萄糖基转移酶全长cDNA 序列,命名为UGT1。对UGT1 进行基因特性分析的基础上,构建了植物高效表达载体pBSUGT1 并利用农杆菌介导法转化回高山红景天,经过分子鉴定证明UGT1已经整合到高山红景天基因组DNA分子中并在转录水平大量表达。转UGT1 基因高山红景天愈伤组织红景天甙含量HPLC 测定结果表明,UGT1 的超表达使红景天甙含量大幅增加。
酪醇在生物体内的合成目前只能够通过其分子结构和化学性质判断,苯丙烷类代谢途径中的苯丙氨酸解氨酶(PAL)为酪醇的合成提供了碳架结构最为相似的前体化合物来源。本文首次从高山红景天中分离了在植物次生代谢中起关键作用的苯丙氨酸解氨酶全长cDNA 序列,命名为PALcl1。对其基因特性、表达模式以及PALcl1 基因差异表达组织中的红景天甙含量进行了分析,结果表明,PALcl1 在不同组织和有诱导条件存在的情况下的转录水平与红景天甙含量变化呈正相关。综上,通过对红景天甙生物合成途径上游、下游相关关键基因的分离、鉴定、分析起始了对红景天甙生物合成代谢途径分子机制分析,为最终阐明红景天甙的生物合成代谢通路奠定了基础。
The genus Rhodiola L consists of nearly 90 species and nearly 70 species inChina. The Rhodiola sachalinensis is found in the northern parts of China, which isa cherish plant for medicinal materials. Salidroside and tyrosol are functionalcomponents identi?ed in a number of plants Rhodiola L. such as R. crenulata, R.fastigiata, R. kirilowii and R. saccharinensis. As one of the active components of thearctic root (Rhodiola sachalinensis), salidroside is suggested to function as anadaptogen, i.e. a biologically-active compound that is supposed to increaseresistance in humans to different stress-related disorders and a variety of ailments ordiseases. For example, salidroside has been reported to act as an anti-in?ammatory,anti-cancer, anti-viral, anti-aging, anti-oxygen deficiency, anti-fatigue, anti-radiation,double-regulation and other new action mechanism compounds. But there are threepuzzles which should be solved in the process of research and utilization of theseplants. Characteristics of reproductive ecology produce endangered stages, thesystem research of the plant is poor and the over utilization lead to the destroy ofecology environment.
Uridine diphosphate (UDP) glycosyltransferases (UGTs) mediate the transferof glycosyl residues from activated nucleotide sugars to acceptor molecules(aglycones), thus regulating properties of the acceptors such as their bioactivity,solubility and transport within the cell and throughout the organism. AUDP-glucosyltransferase cDNA gene was successfully isolated from Rhodiolasachalinensis and named as UGT1 with 1598 bp (GenBank accessionnumber:AY547304), which encodes a predicted polypeptide of 480 amino acids with53.87 kDa and pI 5.40 and contains only one possible transmembrane domains atamino-terminus. Phylogenetic tree analysis of the UGTs showed that it sharesgreater similarity with the other plant UGTs (24.23-54.24%). Carboxy-terminus ofdeduced amino acid sequence includes a part of the glucosyltransferase signaturesequence and has 44%-72% identity to other plant UDP-glucosyltransfereses withhighly homologous similarity to UDP-glucose: salicylic acid-inducibleglucosyltransferases. Amino-terminal consensus sequence showed 62% to 68%identity with the known salicylate-induced and phenylpropanoid: glucosyltransferasehomologs. Thus, it was indicated that UGT1 might function as aUDP-glucosyltransferase. Southern blot analysis with a UGT1 specific fragment asprobe showed that one copy of UGT1 gene in Rhodiola sachalinensis. The RNA gelblot analysis showed a different UGT1 expression patterns among the differentorgans at mRNA level with abundant in callus but none in leaves. The determinationof salidroside in callus and leaves of Rhodiola sachalinensis by HPLC showed
植物次生代谢中产生的小分子糖甙类化合物是由尿苷二磷酸葡萄糖基转移酶(UDP-glucosyltransferase,UDPGT,UGTs)催化合成的。本文首次从高山红景天中克隆了UDP-葡萄糖基转移酶全长cDNA 序列,命名为UGT1。对UGT1 进行基因特性分析的基础上,构建了植物高效表达载体pBSUGT1 并利用农杆菌介导法转化回高山红景天,经过分子鉴定证明UGT1已经整合到高山红景天基因组DNA分子中并在转录水平大量表达。转UGT1 基因高山红景天愈伤组织红景天甙含量HPLC 测定结果表明,UGT1 的超表达使红景天甙含量大幅增加。
酪醇在生物体内的合成目前只能够通过其分子结构和化学性质判断,苯丙烷类代谢途径中的苯丙氨酸解氨酶(PAL)为酪醇的合成提供了碳架结构最为相似的前体化合物来源。本文首次从高山红景天中分离了在植物次生代谢中起关键作用的苯丙氨酸解氨酶全长cDNA 序列,命名为PALcl1。对其基因特性、表达模式以及PALcl1 基因差异表达组织中的红景天甙含量进行了分析,结果表明,PALcl1 在不同组织和有诱导条件存在的情况下的转录水平与红景天甙含量变化呈正相关。综上,通过对红景天甙生物合成途径上游、下游相关关键基因的分离、鉴定、分析起始了对红景天甙生物合成代谢途径分子机制分析,为最终阐明红景天甙的生物合成代谢通路奠定了基础。
The genus Rhodiola L consists of nearly 90 species and nearly 70 species inChina. The Rhodiola sachalinensis is found in the northern parts of China, which isa cherish plant for medicinal materials. Salidroside and tyrosol are functionalcomponents identi?ed in a number of plants Rhodiola L. such as R. crenulata, R.fastigiata, R. kirilowii and R. saccharinensis. As one of the active components of thearctic root (Rhodiola sachalinensis), salidroside is suggested to function as anadaptogen, i.e. a biologically-active compound that is supposed to increaseresistance in humans to different stress-related disorders and a variety of ailments ordiseases. For example, salidroside has been reported to act as an anti-in?ammatory,anti-cancer, anti-viral, anti-aging, anti-oxygen deficiency, anti-fatigue, anti-radiation,double-regulation and other new action mechanism compounds. But there are threepuzzles which should be solved in the process of research and utilization of theseplants. Characteristics of reproductive ecology produce endangered stages, thesystem research of the plant is poor and the over utilization lead to the destroy ofecology environment.
Uridine diphosphate (UDP) glycosyltransferases (UGTs) mediate the transferof glycosyl residues from activated nucleotide sugars to acceptor molecules(aglycones), thus regulating properties of the acceptors such as their bioactivity,solubility and transport within the cell and throughout the organism. AUDP-glucosyltransferase cDNA gene was successfully isolated from Rhodiolasachalinensis and named as UGT1 with 1598 bp (GenBank accessionnumber:AY547304), which encodes a predicted polypeptide of 480 amino acids with53.87 kDa and pI 5.40 and contains only one possible transmembrane domains atamino-terminus. Phylogenetic tree analysis of the UGTs showed that it sharesgreater similarity with the other plant UGTs (24.23-54.24%). Carboxy-terminus ofdeduced amino acid sequence includes a part of the glucosyltransferase signaturesequence and has 44%-72% identity to other plant UDP-glucosyltransfereses withhighly homologous similarity to UDP-glucose: salicylic acid-inducibleglucosyltransferases. Amino-terminal consensus sequence showed 62% to 68%identity with the known salicylate-induced and phenylpropanoid: glucosyltransferasehomologs. Thus, it was indicated that UGT1 might function as aUDP-glucosyltransferase. Southern blot analysis with a UGT1 specific fragment asprobe showed that one copy of UGT1 gene in Rhodiola sachalinensis. The RNA gelblot analysis showed a different UGT1 expression patterns among the differentorgans at mRNA level with abundant in callus but none in leaves. The determinationof salidroside in callus and leaves of Rhodiola sachalinensis by HPLC showed
引文
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