采用多种策略提高长春花萜类吲哚生物碱含量的研究
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摘要
夹竹桃科药用和观赏植物长春花(Catharanthus roseus (L.) G. Don)体内含有长春质碱(Catharanthine)、文多灵(Vindoline)、阿玛碱(Ajmalicine)、长春碱(Vinblastine)和长春新碱(Vincristine)等130种以上具有抗各类肿瘤活性的萜类吲哚生物碱(TIAs, Terpenoid indole alkaloids)。野生长春花中由于这些生物碱含量普遍较低，长春花的毛状根和悬浮细胞中由于无文多灵合成的相关酶，导致不能合成最重要的具有抗肿瘤活性的二萜类吲哚生物碱长春碱和长春新碱。目前国内外大多通过化学半合成法合成长春碱和长春新碱这两种二萜类生物碱，但由于这些生物碱具有复杂的化学结构，导致人工合成方法难度大、成本高、产量低，使得长春碱等活性价值高的生物碱大规模商业化受到了极大的限制，为此获得高含量的TIAs的长春花株系显得十分迫切。
本文从多方面研究提高长春花体内TIAs的含量，包括利用代谢工程策略和染色体加倍技术获得生物碱高产量的长春花植株。同时研究了花期对长春花叶片施用植物生长调节剂，以获得提高长春花生物碱含量的最佳生长调节剂组合，用于指导长春花的产业化生产。研究取得的结果如下：
1)构建了含长春花TIAs生物合成途径关键酶基因CrSTR和CrPRX1的单基因过量表达载体(pCAMBIA2300-STR，pCAMBIA2300-PRX1)，采用课题组建立的长春花遗传转化方法转化长春花下胚轴，获得了11株转CrPRX1基因的长春花植株、23株转CrSTR基因的长春花植株；Southernblot分析表明这些过量表达的内源基因整合到转基因长春花植株基因组中；Real-time PCR分析表明这些内源基因在转基因长春花植株中表达量有所增加；生物碱含量的HPLC分析显示，这些转目的基因长春花植株中文多灵、长春质碱以及长春碱等重要生物碱含量得到了显著提高。其中，转CrSTR基因的长春花植株中文多灵和长春质碱含量最高分别较对照（非转基因植株）提高了1.94、1.56倍。单转CrPRX1基因的长春花植株中长春碱含量最高为对照的2.88倍。
2)通过秋水仙素诱导长春花种子获得的植株，经流式细胞仪检测7.66%为四倍体植株，秋水仙素诱导长春花四倍体的最佳方案是0.2%的秋水仙素处理长春花种子24小时，该处理四倍体诱导率达到30%；四倍体长春花植株的气孔等形态指标较野生长春花有一定的差异；Real-time PCR分析结果表明，长春花四倍体植株的TIAs代谢途径的部分基因表达量较对照（同样经秋水仙素处理的植株）有显著提高；HPLC分析表明四倍体植株的文多灵、长春质碱和长春碱含量较对照植株分别提高了1.31倍、1.89倍和1.23倍。
3)花期用植物生长调节剂组合处理长春花叶片，结果表明：水杨酸(0.1mM)+乙烯利(0.1mM)、乙烯利(0.1mM)+矮壮素(0.1mM)、水杨酸(0.1mM)+矮壮素(0.1mM)和水杨酸(0.1mM)+乙烯利(0.1mM)+矮壮素(0.1mM)处理导致文多灵、长春质碱和长春碱三种生物碱含量有一定的提高；水杨酸(0.1mM)+乙烯利(0.1mM)+矮壮素(0.01mM)和水杨酸(0.1mM)+乙烯利(0.1mM)+矮壮素(1.0mM)的使用文多灵和长春质碱的含量得到了显著的提高，对长春碱含量却没有影响；水杨酸(0.1mM)+乙烯利(0.1mM)、乙烯利(0.1mM)+矮壮素(0.1mM)和水杨酸(0.1mM)+乙烯利(0.1mM)+矮壮素(0.1mM)能显著提高长春花植株中长春碱的含量，分别达到对照处理的209%（处理后48h）、246%（处理后48h）和213%（处理后24h）；与单个生长调节物质处理相比，组合处理可更大幅度地提高长春花TIAs的含量。
The Apocynaceae Vinca plant Catharanthus roseus (L.) G. Don is amedicinal and ornamental plant. More than130kinds of terpenoid indolealkaloids with anti-tumor activity have been isolated and identified in it,such as catharanthine, vindoline, ajmalicine, vinblasitne and vincristine.However, the contents of most alkaloids are low and the most valuabledimeric indole alkaloids in C. roseus, vinblastine and vincristine areimpossible to be yielded by hairy root culture and suspend cell culturesystems, because of lacking some enzymes related to vindoline biosynthsisin them. At present, the chemical semi-synthetic method is normallyemployed to produce more vinblastine and vincristine based on theextraction of relatively higher abundance of vindoline and catharanthine innatural C. roseus. Commercial production of important TIAs is very difficult such as vinblastine and vincristine by chemical synthesis due to thecomplexity of their chemical structures, high-cost and low-output forsynthesis. Therefore, it is necessary and urgent to find more efficient waysto improve the contents of important TIAs in C. roseus.
In this study, many ways on how to improve the contents of TIAs in C.roseus had been studied. They included over-expressing the key enzymegenes which involved in the TIAs biosynthetic pathway, CrPRX1andCrSTR in C. roseus plants by Agrobacterium tumefaciens-mediatedtransformation system, inducing tetraploid plants which have high contentsof TIAs by colchicine, and the plant growth regulator combinations applyingon the leaves of C. roseus during flower period had also been studied. Theresults are as follows:
1) Monovalent expression vectors (pCAMBIA2300-STR, pCAMBIA2300--PRX1) containing the rate-limiting enzyme genes in the TIAs biosyntheticpathway in C. roseus, STR and PRX1, were constructed. Through sonicationassisted Agrobacterium tumefaciens-mediated genetic transformationsystem,11independent transgenic C. roseus plants containing PRX1geneand23independent transgenic C. roseus plants containing STR gene wereobtained. The genes which were integrated in the genomes of transgenic C.roseus plants were verified by southern blot. The tranformants were further analyzed by real-time PCR, and the results indicated that the genes wereover-expressed in transgenic C. roseus plants. The contents of three TIAs asvindoline, catharanthine and vinblastine in transgenic C. roseus plants weredetermined by HPLC, and high TIAs-yielding plants were screened out fromthese transgenic plants. Compared to the control (non-transgenic plants), thecontents of the TIAs such as vindoline, catharanthine and vinblastine intransgenic C. roseus plants were significantly enhanced, with the highestover-expressing STR gene containing more1.94-fold of vindoline,1.56-foldof catharanthine and the highest over-expressing PRX1gene containing2.88-fold of vinblastine as the control.
2) The tetraploid plants of Catharanthus roseus (L.) G. Don was obtained bycolchicine induction from seeds explants, and the ploidy of the plants wasidentified by flow cytometry. The optimal treatment is0.2%colchicinesolution treated for24hours and the induction rate reaches up to30%.Compared with morphological characteristics and growth habits betweentetraploids and the control, we found that tetraploids of C. roseus had largerstoma and more branches and leaves. Real-time PCR results showed that theexpression of enzymes involved in terpenoid indole alkaloids biosynthesispathway had increased in the tetraploid plants. HPLC analysis showedtetraploidization could increase the contents of terpenoid indole alkaloids in C. roseus. Compared to the control (diploid plants which also treated withcolchicine), the contents of the TIAs such as vindoline, catharanthine andvinblastine in tetraploid C. roseus plants were significantly enhanced. Theywere increased1.31-fold,1.89-fold and1.23-fold respectively.
3) The effects of plant growth regulator combinations on the contents ofvindoline, catharanthine and vinblastine were investigated by short-termspray on an ornamental and medicinal plant Catharanthus roseus during theblooming period for commercial use. The results showed the combinationgroups such as ethylene (0.1mM)+chlormequat chloride (0.1mM),salicylic acid (0.1mM)+chlormequat chloride (0.1mM) and salicylic acid(0.1mM)+ethylene (0.1mM)+chlormequat chloride (0.1mM) resulted ina significant increase of the three alkaloids contents. The combinationgroups salicylic acid (0.1mM)+ethylene (0.1mM)+chlormequat chloride(0.01mM) and salicylic acid (0.1mM)+ethylene (0.1mM)+chlormequatchloride (1.0mM), affected vindoline and catharanthine contents but had noeffect on vinblastine. Among the combination treatments, salicylic acid (0.1mM)+ethylene (0.1mM), ethylene (0.1mM)+chlormequat chloride (0.1mM) and salicylic acid (0.1mM)+ethylene (0.1mM)+chlormequatchloride (0.1mM) could significantly increase the vinblastine content by209%at48h,246%at48h and213%at24h respectively. Thus compared to the single PGRs treatments, the combination treatments increase alkaloidaccumulations more effectively.
本文从多方面研究提高长春花体内TIAs的含量，包括利用代谢工程策略和染色体加倍技术获得生物碱高产量的长春花植株。同时研究了花期对长春花叶片施用植物生长调节剂，以获得提高长春花生物碱含量的最佳生长调节剂组合，用于指导长春花的产业化生产。研究取得的结果如下：
1)构建了含长春花TIAs生物合成途径关键酶基因CrSTR和CrPRX1的单基因过量表达载体(pCAMBIA2300-STR，pCAMBIA2300-PRX1)，采用课题组建立的长春花遗传转化方法转化长春花下胚轴，获得了11株转CrPRX1基因的长春花植株、23株转CrSTR基因的长春花植株；Southernblot分析表明这些过量表达的内源基因整合到转基因长春花植株基因组中；Real-time PCR分析表明这些内源基因在转基因长春花植株中表达量有所增加；生物碱含量的HPLC分析显示，这些转目的基因长春花植株中文多灵、长春质碱以及长春碱等重要生物碱含量得到了显著提高。其中，转CrSTR基因的长春花植株中文多灵和长春质碱含量最高分别较对照（非转基因植株）提高了1.94、1.56倍。单转CrPRX1基因的长春花植株中长春碱含量最高为对照的2.88倍。
2)通过秋水仙素诱导长春花种子获得的植株，经流式细胞仪检测7.66%为四倍体植株，秋水仙素诱导长春花四倍体的最佳方案是0.2%的秋水仙素处理长春花种子24小时，该处理四倍体诱导率达到30%；四倍体长春花植株的气孔等形态指标较野生长春花有一定的差异；Real-time PCR分析结果表明，长春花四倍体植株的TIAs代谢途径的部分基因表达量较对照（同样经秋水仙素处理的植株）有显著提高；HPLC分析表明四倍体植株的文多灵、长春质碱和长春碱含量较对照植株分别提高了1.31倍、1.89倍和1.23倍。
3)花期用植物生长调节剂组合处理长春花叶片，结果表明：水杨酸(0.1mM)+乙烯利(0.1mM)、乙烯利(0.1mM)+矮壮素(0.1mM)、水杨酸(0.1mM)+矮壮素(0.1mM)和水杨酸(0.1mM)+乙烯利(0.1mM)+矮壮素(0.1mM)处理导致文多灵、长春质碱和长春碱三种生物碱含量有一定的提高；水杨酸(0.1mM)+乙烯利(0.1mM)+矮壮素(0.01mM)和水杨酸(0.1mM)+乙烯利(0.1mM)+矮壮素(1.0mM)的使用文多灵和长春质碱的含量得到了显著的提高，对长春碱含量却没有影响；水杨酸(0.1mM)+乙烯利(0.1mM)、乙烯利(0.1mM)+矮壮素(0.1mM)和水杨酸(0.1mM)+乙烯利(0.1mM)+矮壮素(0.1mM)能显著提高长春花植株中长春碱的含量，分别达到对照处理的209%（处理后48h）、246%（处理后48h）和213%（处理后24h）；与单个生长调节物质处理相比，组合处理可更大幅度地提高长春花TIAs的含量。
The Apocynaceae Vinca plant Catharanthus roseus (L.) G. Don is amedicinal and ornamental plant. More than130kinds of terpenoid indolealkaloids with anti-tumor activity have been isolated and identified in it,such as catharanthine, vindoline, ajmalicine, vinblasitne and vincristine.However, the contents of most alkaloids are low and the most valuabledimeric indole alkaloids in C. roseus, vinblastine and vincristine areimpossible to be yielded by hairy root culture and suspend cell culturesystems, because of lacking some enzymes related to vindoline biosynthsisin them. At present, the chemical semi-synthetic method is normallyemployed to produce more vinblastine and vincristine based on theextraction of relatively higher abundance of vindoline and catharanthine innatural C. roseus. Commercial production of important TIAs is very difficult such as vinblastine and vincristine by chemical synthesis due to thecomplexity of their chemical structures, high-cost and low-output forsynthesis. Therefore, it is necessary and urgent to find more efficient waysto improve the contents of important TIAs in C. roseus.
In this study, many ways on how to improve the contents of TIAs in C.roseus had been studied. They included over-expressing the key enzymegenes which involved in the TIAs biosynthetic pathway, CrPRX1andCrSTR in C. roseus plants by Agrobacterium tumefaciens-mediatedtransformation system, inducing tetraploid plants which have high contentsof TIAs by colchicine, and the plant growth regulator combinations applyingon the leaves of C. roseus during flower period had also been studied. Theresults are as follows:
1) Monovalent expression vectors (pCAMBIA2300-STR, pCAMBIA2300--PRX1) containing the rate-limiting enzyme genes in the TIAs biosyntheticpathway in C. roseus, STR and PRX1, were constructed. Through sonicationassisted Agrobacterium tumefaciens-mediated genetic transformationsystem,11independent transgenic C. roseus plants containing PRX1geneand23independent transgenic C. roseus plants containing STR gene wereobtained. The genes which were integrated in the genomes of transgenic C.roseus plants were verified by southern blot. The tranformants were further analyzed by real-time PCR, and the results indicated that the genes wereover-expressed in transgenic C. roseus plants. The contents of three TIAs asvindoline, catharanthine and vinblastine in transgenic C. roseus plants weredetermined by HPLC, and high TIAs-yielding plants were screened out fromthese transgenic plants. Compared to the control (non-transgenic plants), thecontents of the TIAs such as vindoline, catharanthine and vinblastine intransgenic C. roseus plants were significantly enhanced, with the highestover-expressing STR gene containing more1.94-fold of vindoline,1.56-foldof catharanthine and the highest over-expressing PRX1gene containing2.88-fold of vinblastine as the control.
2) The tetraploid plants of Catharanthus roseus (L.) G. Don was obtained bycolchicine induction from seeds explants, and the ploidy of the plants wasidentified by flow cytometry. The optimal treatment is0.2%colchicinesolution treated for24hours and the induction rate reaches up to30%.Compared with morphological characteristics and growth habits betweentetraploids and the control, we found that tetraploids of C. roseus had largerstoma and more branches and leaves. Real-time PCR results showed that theexpression of enzymes involved in terpenoid indole alkaloids biosynthesispathway had increased in the tetraploid plants. HPLC analysis showedtetraploidization could increase the contents of terpenoid indole alkaloids in C. roseus. Compared to the control (diploid plants which also treated withcolchicine), the contents of the TIAs such as vindoline, catharanthine andvinblastine in tetraploid C. roseus plants were significantly enhanced. Theywere increased1.31-fold,1.89-fold and1.23-fold respectively.
3) The effects of plant growth regulator combinations on the contents ofvindoline, catharanthine and vinblastine were investigated by short-termspray on an ornamental and medicinal plant Catharanthus roseus during theblooming period for commercial use. The results showed the combinationgroups such as ethylene (0.1mM)+chlormequat chloride (0.1mM),salicylic acid (0.1mM)+chlormequat chloride (0.1mM) and salicylic acid(0.1mM)+ethylene (0.1mM)+chlormequat chloride (0.1mM) resulted ina significant increase of the three alkaloids contents. The combinationgroups salicylic acid (0.1mM)+ethylene (0.1mM)+chlormequat chloride(0.01mM) and salicylic acid (0.1mM)+ethylene (0.1mM)+chlormequatchloride (1.0mM), affected vindoline and catharanthine contents but had noeffect on vinblastine. Among the combination treatments, salicylic acid (0.1mM)+ethylene (0.1mM), ethylene (0.1mM)+chlormequat chloride (0.1mM) and salicylic acid (0.1mM)+ethylene (0.1mM)+chlormequatchloride (0.1mM) could significantly increase the vinblastine content by209%at48h,246%at48h and213%at24h respectively. Thus compared to the single PGRs treatments, the combination treatments increase alkaloidaccumulations more effectively.
引文
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