虎杖白藜芦醇合酶基因的克隆与表达
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摘要
白藜芦醇(Resveratrol，3，4’5-trihydroxy-trans-stilbene，简称Res)是一种天然存在的多酚化合物。已经发现花生、葡萄、虎杖等植物中含有白藜芦醇。据报道，白藜芦醇有着广泛的生物学和约理学特性，如抗癌、抗炎症、心血管保护等作用。白藜芦醇在植物保护中也发挥着重要作用。
白藜芦醇合酶(resveratrol synthase，RS；EC 23.1.95)是芪类(stilbene)物质合成的关键酶，催化一分子coumaroyl-CoA(香豆酰-CoA)和二分子malonyl-CoA(丙二酰-CoA)合成白藜芦醇。RS的底物来自苯丙氨酸代谢途径，是控制植物次生代谢产物合成途径中重要的分支点的酶之一。苯丙酰代谢途径几乎存在于所有植物中，而Res只存在于少数有RS的植物中，大多数重要的作物都缺乏该基因，因此将RS基因转入无RS的植物，可以为该植物提供增强抗真菌侵染的防御体系或增强植物抗逆境胁迫的能力，还能提高一些作物的品质。
本研究利用RT-PCR和GeneRacer法，从药用植物虎杖(Polygonum cuspidatum)中克隆到了RS的全长cDNA，我们将它命名为PCRS。还从虎杖基因组DNA中克隆到了一个具有全长编码区的RS基因序列利一个比全长编码区少了72bp的RS基因(PCRS11)序列以及一个含有两个内含子的RS基因序列。将来自花生(Peanut)的RS基因(PNRS)与来自虎杖的PCRS及PCRS11进行了原核表达与植物表达。原核表达时发现，PNRS基冈与PCRS均能止常表达成蛋白，而PCRS11基因在诱导表达时，却未能检测到相应蛋白。植物表达时，用上述三个基因替换掉了pCAMBIA3301上面的GUS基因，从而构建了三个植物表达载体pCAMBIA3301-PNRS、pCAMBIA3301-PCRS、pCAMBIA3301-PCRS11，利用农杆菌介导法导入烟草品种Xanthi。经PCR和RT-PCR检测表明，目标基因已经转入烟草，且在mRNA的水平得剑了表达。但通过HPLC法没有检测到终产物白藜芦醇。
Resveratrol is a natural polyphenol compound found in various plants like peanuts, grapes, Polygonum cuspidatum, etc. It is reported that it has a variety of biological and pharmacological properties, such as anti-cancer, anti-inflammation, cardio-protective activities. It is assumed to be a healthy to take food containing high content of resveratrol like wine and peanuts. Resveratrol also plays important roles in defending stresses to plants.
Resveratrol synthase (RS; EC 2.3.1.95), the key enzyme in biosynthesis of stilbene-type phytoalexins, catalyzes the formation of resveratrol from one molecule of coumaroyl-CoA and three molecules of malony-CoA which come from phenylalanine metabolism pathway. Phenylalanine metabolism pathway is found in almost any plants, whereas resveratrol is only present in rare species which contain RS. Plants lacking of RS gene may be charged with the ability to synthesize resveratrol by transferring foreign RS gene into the plants, providing them with improved nutritional value and ability in defending microorganism infection.
In this paper, A RS cDNA (PCRS) was cloned from medicinal plants P. cuspidatum using RT-PCR and GeneRacer methods. A full-length encoding region of RS sequence, a sequence (PCRS11) lacking a 72 bp as compared with PCRS, and a sequence contains two introns were obtained from the genomic DNA of P.cuspidatum. PNRS gene (obtained from peanuts), PCRS and PCRS 11 from P.cuspidatum were expressed in prokaryotic cells and in plant cells. When expression was performed in prokaryotic cells, PNRS and PCRS could be successfully expressed and translated into proteins, whereas no proteins could be detected when PCRS11 was induced to express. In comparison, when expressed in plant cells, plant expression vectors pCAMBIA3301-PNRS、pCAMBIA3301-PCRS and pCAMBIA3301-PCRS11 were constructed by replacing the GUS gene in pCAMBIA3301 with PNRS, PCRS and PCRS11, respectively. The vectors were then used for Nicotiana tabacum transformation via Agrobacterium-mediated transfer methods. PCR and RT-PCR tests showed that the target genes had been successfully integrated into the tobacco genome in all the transformants.But no resveratrol could be detected by HPLC.
白藜芦醇合酶(resveratrol synthase，RS；EC 23.1.95)是芪类(stilbene)物质合成的关键酶，催化一分子coumaroyl-CoA(香豆酰-CoA)和二分子malonyl-CoA(丙二酰-CoA)合成白藜芦醇。RS的底物来自苯丙氨酸代谢途径，是控制植物次生代谢产物合成途径中重要的分支点的酶之一。苯丙酰代谢途径几乎存在于所有植物中，而Res只存在于少数有RS的植物中，大多数重要的作物都缺乏该基因，因此将RS基因转入无RS的植物，可以为该植物提供增强抗真菌侵染的防御体系或增强植物抗逆境胁迫的能力，还能提高一些作物的品质。
本研究利用RT-PCR和GeneRacer法，从药用植物虎杖(Polygonum cuspidatum)中克隆到了RS的全长cDNA，我们将它命名为PCRS。还从虎杖基因组DNA中克隆到了一个具有全长编码区的RS基因序列利一个比全长编码区少了72bp的RS基因(PCRS11)序列以及一个含有两个内含子的RS基因序列。将来自花生(Peanut)的RS基因(PNRS)与来自虎杖的PCRS及PCRS11进行了原核表达与植物表达。原核表达时发现，PNRS基冈与PCRS均能止常表达成蛋白，而PCRS11基因在诱导表达时，却未能检测到相应蛋白。植物表达时，用上述三个基因替换掉了pCAMBIA3301上面的GUS基因，从而构建了三个植物表达载体pCAMBIA3301-PNRS、pCAMBIA3301-PCRS、pCAMBIA3301-PCRS11，利用农杆菌介导法导入烟草品种Xanthi。经PCR和RT-PCR检测表明，目标基因已经转入烟草，且在mRNA的水平得剑了表达。但通过HPLC法没有检测到终产物白藜芦醇。
Resveratrol is a natural polyphenol compound found in various plants like peanuts, grapes, Polygonum cuspidatum, etc. It is reported that it has a variety of biological and pharmacological properties, such as anti-cancer, anti-inflammation, cardio-protective activities. It is assumed to be a healthy to take food containing high content of resveratrol like wine and peanuts. Resveratrol also plays important roles in defending stresses to plants.
Resveratrol synthase (RS; EC 2.3.1.95), the key enzyme in biosynthesis of stilbene-type phytoalexins, catalyzes the formation of resveratrol from one molecule of coumaroyl-CoA and three molecules of malony-CoA which come from phenylalanine metabolism pathway. Phenylalanine metabolism pathway is found in almost any plants, whereas resveratrol is only present in rare species which contain RS. Plants lacking of RS gene may be charged with the ability to synthesize resveratrol by transferring foreign RS gene into the plants, providing them with improved nutritional value and ability in defending microorganism infection.
In this paper, A RS cDNA (PCRS) was cloned from medicinal plants P. cuspidatum using RT-PCR and GeneRacer methods. A full-length encoding region of RS sequence, a sequence (PCRS11) lacking a 72 bp as compared with PCRS, and a sequence contains two introns were obtained from the genomic DNA of P.cuspidatum. PNRS gene (obtained from peanuts), PCRS and PCRS 11 from P.cuspidatum were expressed in prokaryotic cells and in plant cells. When expression was performed in prokaryotic cells, PNRS and PCRS could be successfully expressed and translated into proteins, whereas no proteins could be detected when PCRS11 was induced to express. In comparison, when expressed in plant cells, plant expression vectors pCAMBIA3301-PNRS、pCAMBIA3301-PCRS and pCAMBIA3301-PCRS11 were constructed by replacing the GUS gene in pCAMBIA3301 with PNRS, PCRS and PCRS11, respectively. The vectors were then used for Nicotiana tabacum transformation via Agrobacterium-mediated transfer methods. PCR and RT-PCR tests showed that the target genes had been successfully integrated into the tobacco genome in all the transformants.But no resveratrol could be detected by HPLC.
引文
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2．胡赕，陶春妙，陶朝鹏．白藜芦醇研究进展．化工时刊，2005，9(7)：53-56．
3．钟瑾，刘树君，马世嵩，等．核基质结合区对转爬山虎芪合酶基因烟草中产生白藜芦醇的作用．植物学报，2004，46(8)：948-954．
4. Baeuerle P A, Baichwal V R. NF-κB as a frequent target for immunosuppressive and anti-inflammatory molecules. Adv Immunol, 1997, 65:111-37.
5. Banerjee S, Bueso-Ramos C, Aggarwal BB. Suppression of 7, 12-dimethylbenz (a) anthracene-induced mammary carcino- genesis in rats by resveratrol: role of nuclear factor-κB, cyclooxygenase 2, and matrix metalloprotease 9. Cancer Res, 2002, 62 (17):4945-54.
6. Bennet R C, Walls grove R M. Secondary metabolites in plant defence mechanisms. New Phytol, 1994, 127:617-33.
7. Chunzhi Zhang*, Dai Li, Hongshan Yu, Bo Zhang, Fengxie Jin. *Purification and characterization of piceid-b-D-glucosidase from Aspergillus oryzae. Process Biochemistry, 2006,42 (2007):83-88
8. Chung I M, Park M R, Rehman S, Yun S J. Tissue specific and inducible expression of resveratrol synthase gene in peanut plants.Mol Cells, 2001,12(3):353-359.
9. Culpitt S V, Rogers D F, Fenwick P S, Shah P, De Matos C, Russell REK, Barnes PJ, Donnelly LE. Inhibition by red wine extract, resveratrol, of cytokine release by alveolar macrophages in COPD. Thorax, 2003, 58(11):942-6.
10. Dae-Yeon SUH, Junichi KAGAMI, Kazuki FUKUMA,Naoko IWANAMI, Yasuyo YAMAZAKI et al. Chalcone and stilbene synthase expressed in eukaryotes exhibit reduced cross-reactivity in Vitro. Chem Pharm.Bull, 2000, 48(7), 1051-1054.
11. Davis SR, Dalais FS, Simpson ER, Murkies AL. Phytoestrogens in health and disease. Recent Prog Horm Res, 1999, 54:185-210[discussion 210-181].
12. Donato MT, Castell JV. Strategies and molecular probes to investigate the role of cytochrome P450 in drug metabolism: focus on in vitro studies. Clin Pharmacokinet 2003, 42:153-78.
13. Fauconneau B, Waffo-Teguo P, Huguet F, Barrier L, Decendit A, Merillon J-M. Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis Vinifera cell cultures using in vitro tests. Life Sci, 1997, 61:2103-2110.
14. Fettig S, Hess D. Expression of a chimeric stilbene synthase gene in transgenic wheat lines.Transgenic Research, 1999, 8(3): 179-189.
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