红豆杉紫杉烷13a-羟基化酶基因的克隆及表达调控的研究
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摘要
紫杉醇(Taxol)是从紫杉树皮中分离提取到的天然抗肿瘤物质,是迄今为止最具抗癌活性的天然化合物之一,紫杉酚能够抑制细胞在G2-M期增殖,阻断有丝分裂,从而有效抑制癌细胞增生。然而紫杉醇在植物体内含量相当低,加之其生长缓慢,这对紫杉醇的进一步开发利用造成了很大的困难。紫杉烷13α–羟基化酶(Taxane 13α-hydroxylase)属于依赖细胞色素P450的单加氧酶,为紫杉醇合成途径的关键羟化酶,是紫杉醇及其半合成前体生物合成调控的十分重要的靶标。本研究克隆得到我国东北红豆杉(Taxus cuspidata)紫杉烷13α-羟基化酶基因,构建了其植物表达载体并转化烟草。在此基础上克隆了几个可能与紫杉烷13α-羟基化酶基因相作用的microRNA(miRNA)基因,构建其植物表达载体并进行了其中miRNA基因与紫杉烷13α-羟基化酶基因共转化试验。主要研究内容和研究结果如下:
(1)本研究克隆得到东北红豆杉13OH的基因全长,将它与报道的13OH基因进行同源性比较后发现9个碱基有差异,同源性为99.38%。
(2)根据不同的报告基因要求,成功构建了13OH基因在Pcambia1305.1和pcambia1304及pEGAD上的三种植物表达载体,分别含有GUS基因、GUS和mGFP5、EGFP作为报告基因。
(3)将构建在Pcambia1305.1的13OH基因成功转化到烟草中,获得可在烟草中表达13α-羟基化酶基因的转基因烟草植株。
(4)从水稻及拟南芥中克隆出miR160e、miR164e、miR165a、miR166m、miR169d小调节子前体,测序结果表明除164e有一个碱基差异166m有两个碱基差异外,其余同源性为100%。
(5)分别构建了miR160e、miR164e、miR165a、miR166m、miR169d在Pcambia1305.1上和miR165a、miR169d在pEGAD上的七种植物表达载体,便于不同的筛选。
(6)瞬时转化GUS染色结果显示miR169d对13OH基因的表达有很强的抑制作用。
(7)荧光定量PCR结果显示miR165a可能对13OH基因有小幅度的影响。
The nature product Taxol, produced by Taxus species, is a highly effective anticancer drug;Taxol inhibits cell proliferation at the G2-M phase, thereby it can block mitosis effectively. FDA has approved Taxol in the treatment of refractory ovarian and breast cancer in 1992. Despite its outstanding curative effect,the content of Taxol is at quite low level. For example, the commercial isolation of 1 kg Taxol required about 6.7 t of T.brevifolia bark, equivalent to that of 2,000-3,000 trees. To resolve this contradiction, scholars at home and abroad have made efforts such as artificial cultivation, total syntheses and semisynthesis, cell culture etc. However low yields and high costs preclude them from industrialization produce .For the foreseeable future, the metabolic regulation of the Taxol biosynthetic pathway may provide a feasible solution to the sustainable yield problem.
A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. 13α-hydroxylase is an important P450-dependent monooxygenases in the synthesis of Taxol. Here we cloned its full-length gene and constructed expression vectors.
The main results are summarized as follows:
(1) Full-length sequence coding for Taxane 13α-hydroxylase(13OH) was cloned from Taxus cuspidata cDNA library.The DNA was verified by sequencing after it was inserted into pGM-T easy vector. Compared with the nucleotide sequence in the Genbank, the cloned DNA in our paper showed 99.38% identity with the reported taxane 13α–hydroxylase of Taxus cuspidata.
(2) The 13OH gene was cloned in to Pcambia1305.1, pcambia1304 and pEGAD to construct three different plant expression vectors with GUS gene, GUS gene and mgfp5, EGFP gene as their report gene respectively.
(3) The binary vector with 13OH in pCAMBIA1305.1 was transformed into Agrobacterium tumefaciens GV3101 by electroporation. Plants were regenerated from the infected leaf dics under the selection of hygromycin. PCR ananlysis with 13OH-specific primer and assay of the fusion GUS reporter gene showed that the 13OH gene was successfully intergrated into and expressed in the leaf cells of transgenic tobacco plants.
(4) The precurcor of miR160e, miR164e, miR165a, miR166m, miR169d were cloned from the genomic DNA of Arabidopsis thaliana and Oryza sativa, Compared with the nucleotide sequence in the Genbank, the cloned DNA in our paper showed 100%, 99.24%, 100%, 99%, 100% identities respectively.
(5) Plant expression vectors for seven miRNA genes were constructed to test the function of the regulators.
(6) Analysis of transient transgenic Taxus shots showed that the expression of 13α-hydroxylase could be suppressed by the technology of miRNA .Compared with miR164e and miR165a ,miR169d was more efficient in silencing target gene due to its high specificity.
(7) The result of real-time PCR showed that miR165a has a little function on the expression of 13α–hydroxylase.
(1)本研究克隆得到东北红豆杉13OH的基因全长,将它与报道的13OH基因进行同源性比较后发现9个碱基有差异,同源性为99.38%。
(2)根据不同的报告基因要求,成功构建了13OH基因在Pcambia1305.1和pcambia1304及pEGAD上的三种植物表达载体,分别含有GUS基因、GUS和mGFP5、EGFP作为报告基因。
(3)将构建在Pcambia1305.1的13OH基因成功转化到烟草中,获得可在烟草中表达13α-羟基化酶基因的转基因烟草植株。
(4)从水稻及拟南芥中克隆出miR160e、miR164e、miR165a、miR166m、miR169d小调节子前体,测序结果表明除164e有一个碱基差异166m有两个碱基差异外,其余同源性为100%。
(5)分别构建了miR160e、miR164e、miR165a、miR166m、miR169d在Pcambia1305.1上和miR165a、miR169d在pEGAD上的七种植物表达载体,便于不同的筛选。
(6)瞬时转化GUS染色结果显示miR169d对13OH基因的表达有很强的抑制作用。
(7)荧光定量PCR结果显示miR165a可能对13OH基因有小幅度的影响。
The nature product Taxol, produced by Taxus species, is a highly effective anticancer drug;Taxol inhibits cell proliferation at the G2-M phase, thereby it can block mitosis effectively. FDA has approved Taxol in the treatment of refractory ovarian and breast cancer in 1992. Despite its outstanding curative effect,the content of Taxol is at quite low level. For example, the commercial isolation of 1 kg Taxol required about 6.7 t of T.brevifolia bark, equivalent to that of 2,000-3,000 trees. To resolve this contradiction, scholars at home and abroad have made efforts such as artificial cultivation, total syntheses and semisynthesis, cell culture etc. However low yields and high costs preclude them from industrialization produce .For the foreseeable future, the metabolic regulation of the Taxol biosynthetic pathway may provide a feasible solution to the sustainable yield problem.
A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. 13α-hydroxylase is an important P450-dependent monooxygenases in the synthesis of Taxol. Here we cloned its full-length gene and constructed expression vectors.
The main results are summarized as follows:
(1) Full-length sequence coding for Taxane 13α-hydroxylase(13OH) was cloned from Taxus cuspidata cDNA library.The DNA was verified by sequencing after it was inserted into pGM-T easy vector. Compared with the nucleotide sequence in the Genbank, the cloned DNA in our paper showed 99.38% identity with the reported taxane 13α–hydroxylase of Taxus cuspidata.
(2) The 13OH gene was cloned in to Pcambia1305.1, pcambia1304 and pEGAD to construct three different plant expression vectors with GUS gene, GUS gene and mgfp5, EGFP gene as their report gene respectively.
(3) The binary vector with 13OH in pCAMBIA1305.1 was transformed into Agrobacterium tumefaciens GV3101 by electroporation. Plants were regenerated from the infected leaf dics under the selection of hygromycin. PCR ananlysis with 13OH-specific primer and assay of the fusion GUS reporter gene showed that the 13OH gene was successfully intergrated into and expressed in the leaf cells of transgenic tobacco plants.
(4) The precurcor of miR160e, miR164e, miR165a, miR166m, miR169d were cloned from the genomic DNA of Arabidopsis thaliana and Oryza sativa, Compared with the nucleotide sequence in the Genbank, the cloned DNA in our paper showed 100%, 99.24%, 100%, 99%, 100% identities respectively.
(5) Plant expression vectors for seven miRNA genes were constructed to test the function of the regulators.
(6) Analysis of transient transgenic Taxus shots showed that the expression of 13α-hydroxylase could be suppressed by the technology of miRNA .Compared with miR164e and miR165a ,miR169d was more efficient in silencing target gene due to its high specificity.
(7) The result of real-time PCR showed that miR165a has a little function on the expression of 13α–hydroxylase.
引文
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[2] Craag G. M., Scheparat S.A., Suffness M., et al.The taxol supply crisis. New NCI policies for handling the large-scale production of novel natural product anticancer and anti-HIV agents. J Nat. Prod., 1993, 56:1657-1668
[3] Nicolaou K.C, Yang Z., Liu J.J., et al.Total synthesis of taxol. Nature, 1994, 367:630-634
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[8] Mirjalili N., Linden J.C. Methyl jasmonate induced production of taxol suspension cultures of Taxus cuspidata: Ethylene interaction and induction models.Biotechnol. Prog., 1996, 12:110-118
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