阔叶薰衣草芳樟醇合酶基因的克隆与植物表达载体的构建
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摘要
阔叶薰衣草(Lanvandula spica)是唇形科(Lamiaceae)薰衣草属(Lanvandula)的多年生草本植物,原产于地中海,耐热、耐湿,全株都能散发出宜人的香味,具有醒目提神、舒缓神经等功效,是薰衣草类较能适应海南高温高湿环境的一种。芳樟醇合酶一般通过植物叶片、花、果实等提取获得,是合成芳樟醇的关键酶,能在亲电机制的条件下将单一底物GPP催化形成单一产物芳樟醇。芳樟醇作为一种天然功能性单萜,是植物精油的主要成分,具有极高的经济价值和药用价值,广泛应用于医药、食品、化妆品和日化等行业。本实验通过对阔叶薰衣草芳樟醇合酶基因的克隆和序列分析,并构建基因转化载体,为热带园林植物中无香味或者香味较淡的物种进行定向遗传改良、培育出色、香、形俱全的优良转基因植物品种奠定基础。主要研究结果如下：
1、根据芳樟醇合酶同源序列设计出特异性引物,通过RT-PCR技术,成功从阔叶薰衣草叶片中克隆出芳樟醇合酶基因,其cDNA序列全长为1809bp,编码602个氨基酸,具有完整的开放读码框。
2、将克隆得到的阔叶薰衣草芳樟醇合酶基因与其他芳樟醇合酶基因进行蛋白质序列比对,该序列具有单萜类合酶的所有保守氨基酸功能序列,包括DDXXD以及5端的RR功能基团和(N, D)D(L, I, V)X(S, T)XXXE等高度保守功能域,属于芳樟醇合酶家族。Lslis和Lllis的蛋白二级结构预测结果表明,两者在整体结构上基本保持一致,但Lslis相对于Lllis要少一个α螺旋结构。因此将阔叶薰衣草芳樟醇合酶基因命名为Lslis。
3、利用植物双元表达载体pCAMBIA1303的CaMV35S启动子和NOS终止子可构成完整表达框的原理,将Lslis的片段插入到pCAMBIA1303载体中,成功构建了植物表达载体。再通过电转法将重组表达载体导入农杆菌LBA4404中,为进一步的Lslis转化植物中的遗传表达研究奠定良好的基础。
Lavandula spica, a kind of perennial herbage originating in Mediterranean, belonging to the lavender from the Family Labiatae, is hot and Submergence tolerance, which is a kind of lavender better adapted to the high temperature and humidity in Hainan. The whole plant of Lavandula spica could emit the pleasant fragrance with the effectiveness of striking refreshing_and relieving nervous. Linalool synthase usually extracted from leaves, flowers, fruit, and so on.It is a key enzyme,which can accept a single substrate geranyl diphosphate and transform it to a single product—linalool,under the condition of electrophilic mechanism. Linalool as a natural functional monoterpenes, is the main component of plant essential oils. So it has very high economic value and medicinal value, which is widely used in medicine, food, cosmetics and daily chemicals industry._In this experiment, the linalool synthase gene was extracted from the leaves of Lavandula spica, then cloning and sequences analysis of linalool synthase gene were performed. Then a plant expression vector of linalool synthase gene was constructed uccessfully. This study was targeted to clone the linalool synthase gene and construct the gene transfer vector, so to lay a foundation for the genetic advance of tropical landscape plants with no fragrance or lighter fragrance species. The main research results were as follows:
1、According to the linalool synthase homologous sequences, specific primers were designed, then linalool synthase gene was successfully cloned from the leaves of lavandula spica through the RT-PCR technology, which cDNA sequence was1809bp, encoding602amino acids, with a complete open reading frame sequence.
2、protein sequences of the linalool synthase gene cloned by lavandula spica was compared with others. The sequence has all conserved amino acid function sequences of the monoterpene synthase, including DDXXD and the5'RR functionalgroup and (N, D) D (L, I, V) X (S, T) XXXE and other highly conserved functional domains, belonging to the linalool synthase family. The results of predicted secondary structure for Lllis and Lslis showed that both the mostly structures kept consistent, but Lslis had less a a helix than Lllis. so linalool synthase gene from lavandula spica was named Lslis.
3、Using the binary expression vector (including CaMV35S promoter and NOS Terminator) of plant expression vector pCAMBIA1303, the sense Lslis plant expression vectors were constructed and transferred into Agrobacterium LBA4404by electroporation.
1、根据芳樟醇合酶同源序列设计出特异性引物,通过RT-PCR技术,成功从阔叶薰衣草叶片中克隆出芳樟醇合酶基因,其cDNA序列全长为1809bp,编码602个氨基酸,具有完整的开放读码框。
2、将克隆得到的阔叶薰衣草芳樟醇合酶基因与其他芳樟醇合酶基因进行蛋白质序列比对,该序列具有单萜类合酶的所有保守氨基酸功能序列,包括DDXXD以及5端的RR功能基团和(N, D)D(L, I, V)X(S, T)XXXE等高度保守功能域,属于芳樟醇合酶家族。Lslis和Lllis的蛋白二级结构预测结果表明,两者在整体结构上基本保持一致,但Lslis相对于Lllis要少一个α螺旋结构。因此将阔叶薰衣草芳樟醇合酶基因命名为Lslis。
3、利用植物双元表达载体pCAMBIA1303的CaMV35S启动子和NOS终止子可构成完整表达框的原理,将Lslis的片段插入到pCAMBIA1303载体中,成功构建了植物表达载体。再通过电转法将重组表达载体导入农杆菌LBA4404中,为进一步的Lslis转化植物中的遗传表达研究奠定良好的基础。
Lavandula spica, a kind of perennial herbage originating in Mediterranean, belonging to the lavender from the Family Labiatae, is hot and Submergence tolerance, which is a kind of lavender better adapted to the high temperature and humidity in Hainan. The whole plant of Lavandula spica could emit the pleasant fragrance with the effectiveness of striking refreshing_and relieving nervous. Linalool synthase usually extracted from leaves, flowers, fruit, and so on.It is a key enzyme,which can accept a single substrate geranyl diphosphate and transform it to a single product—linalool,under the condition of electrophilic mechanism. Linalool as a natural functional monoterpenes, is the main component of plant essential oils. So it has very high economic value and medicinal value, which is widely used in medicine, food, cosmetics and daily chemicals industry._In this experiment, the linalool synthase gene was extracted from the leaves of Lavandula spica, then cloning and sequences analysis of linalool synthase gene were performed. Then a plant expression vector of linalool synthase gene was constructed uccessfully. This study was targeted to clone the linalool synthase gene and construct the gene transfer vector, so to lay a foundation for the genetic advance of tropical landscape plants with no fragrance or lighter fragrance species. The main research results were as follows:
1、According to the linalool synthase homologous sequences, specific primers were designed, then linalool synthase gene was successfully cloned from the leaves of lavandula spica through the RT-PCR technology, which cDNA sequence was1809bp, encoding602amino acids, with a complete open reading frame sequence.
2、protein sequences of the linalool synthase gene cloned by lavandula spica was compared with others. The sequence has all conserved amino acid function sequences of the monoterpene synthase, including DDXXD and the5'RR functionalgroup and (N, D) D (L, I, V) X (S, T) XXXE and other highly conserved functional domains, belonging to the linalool synthase family. The results of predicted secondary structure for Lllis and Lslis showed that both the mostly structures kept consistent, but Lslis had less a a helix than Lllis. so linalool synthase gene from lavandula spica was named Lslis.
3、Using the binary expression vector (including CaMV35S promoter and NOS Terminator) of plant expression vector pCAMBIA1303, the sense Lslis plant expression vectors were constructed and transferred into Agrobacterium LBA4404by electroporation.
引文
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[2]. Chappell J, Wolf F, Proulx J, Cuellar R, Saunders C. Is the reaction catalyzed by 3-hydroxy-3-methylglutarylcoenzyme A reductase a rate-limiting step for isoprenoid biosynthesis in plants[J].Plant Physiology,1995(109):1337-1343
[3]. Chris C. N. van Schie Michel A. Haring Robert C. Schuurink. Tomato linalool synthase is induced in trichomes by jasmonic acid[J]. Plant Mol Biol.2007,64:251-263.
[4]. Christian L, Barbara F, Maria F et al, Cloning and functional characterization of three terpene synthasesfrom lavender (Lavandula angustifolia) [J], Archives of Biochemistry and Biophysics.2007,465:417-29.
[5]. Chu C L.Liu W T.Zhou T. Fumigation of sweet cherries with thymol and acetic acid to reduce postharvest brown rot and blue mold rot [J].Fruits,2001,56:123-130.
[6]. Colby S M. Alonso W R,Katahira E J,et al.4S-limonene synthase from the oil glands of spearmint (Mentha spicata) cDNA isolation, characterization,and bacterial expression of the catalytically active monoterpene cyclase[J].Bio/Chem,1993.268(31):23016-23024.
[7]. Dixon RA, Natural products and plant disease resistance[J]. Nature,2001,411:843-847.
[8]. Dubey N K,Kishore N.Exploitation of higher plant products as natural fumigants[C]. Proceedings of the Fifth International Congress on Plant Pathology.Kyoto(Japan),1988.
[9]. Dudareva N, Cseke L, Blanc V M, Pichersky E. Evolution of floral scent in Clarkia: novel patterns of S-linalool synthase gene expression in the C. breweri flower[J]. The Plant Cell,1996,8:1137-1148.
[10]. Dudareva N.,Pichersky E.,and Gershenzon J., Biochemistry of plant volatiles[J],Plant Physiol.,2004,135(4):1893-1902.
[11]. Facehini P J,Chavpsll J. Gene family for an elicitor-induced sesqniterpene cyclase in tobacco[J].Ptoc Nail Acad Sci.1992.89:11088-11092.
[12]. Hyatt DC,Youn B, ZhaoY,et al. Structure of limonene synthase,a simple model for terpenoid cyclase catalysis[J]. Science. USA,2007,104(13):5360-5365.
[13]. Jun-Wei Jia, John Crock, Shan Lu et al. (3R)-Linalool Synthase from Artemisia annua L.:cDNA isolation, characterization, and wound induction[J].. Biochemistry and Biophysics,1999.372:143-149.
[14]. Krasnyanski S, May RA, Loskutov A, Ball TM and Sink KC. Transformation of the limonene synthase gene into peppermint(Mentha piperita L)and preliminary studies on the essential oil profiles of single transgenic plants[J].Theor.Appl.Genet,1999, 99:676-682.
[15]. Lavy M,Zuker A,Lewinsohn E,Larkov O,Ravid U,Vainstein A,Weiss D.Linalool oxide production in transgenic carnation flowers expressing the Clarkia breweri linalool synthase gene[J].Mol Breeding,2002,9:103-111.
[16]. Lewinsohn E, Schalechet F, Wilkinson J, Matsui J, Tadmor Y, Nam KH, Amar O, Lastochkin E, Larkov O, Ravid U, Hiatt W, Gepstein S, Pichersky E. Enhanced levels of the aroma and flavour compound S-linalool by metabolic engineering of the terpenoid pathway in tomato fruits[J]. Plant Physiol,2001,127:1256-1265.
[17]. Liu W T.Chu C L.Zhou T.Thymol and acetic acid vapors reduce post harvest brown rot of apricot and plums [J].Hort Science,2002,37:151-156.
[18]. Lucker J, Bouwmeester H J, Schwab W, Blass J, van der Plas LHW, Verhoeven HA. Expression of Clarkia S-linalool synthase in transgenic petunia plants results in the accumulation of S-linalyl-β-D-glucopyranoside[J]. Plant,2001,27(4):315-324.
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