长春花叶片三种生物碱含量及合成基因表达的初步研究
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摘要
文多灵(Vindoline)、长春质碱(Catharanthine)、长春碱(Vinblastine)三种生物碱产于药用植物长春花,具有抗肿瘤作用,但这些TIAs在天然长春花植株中含量较低并且分布具有组织和生长阶段的特异性,不能满足市场需求,使其价格较为昂贵。利用代谢工程在长春花中过量表达TIAs生物合成途径中的关键酶基因,为提高植物生物碱含量和改变生物碱组成成份开辟了新的途径。然而长春花(Catharanthus roseus L. G. Don)萜类吲哚生物碱(Terpenoid indole alkaloids, TIAs)的生物合成途径十分复杂,包含了多个关键酶和转录因子。目前已有30多个催化酶和转录因子基因被克隆。因此探明长春花叶片中生物碱的动态变化规律和生物碱合成途径中的关键酶和转录因子的表达量是十分必要的。
本研究以长春花叶片为研究材料,利用高效液相色谱技术研究了长春花叶片中生物碱的变化,利用荧光定量PCR技术检测了长春花叶片中生物碱合成途径中11个相关基因的相对表达量。得出了以下结果：1.探明了长春花叶片中三种生物碱的动态变化规律
长春花吲哚生物碱文多灵、长春碱、长春质碱的分布具有组织器官和生长发育的特异性。其中,不同时期长春花三种叶片形态中文多灵含量的差异较大,最高含量为1.18 mg/g,最低含量为0.02 mg/g,相差大约60倍左右。长春碱的最高含量为最低含量的四倍,为0.01 mg/g-0.04 mg/g。长春碱在长春花叶片中的含量比较低,最高含量只有万分之四。长春质碱的含量为0.4 mg/g-1.16 mg/g,大约相差2.5倍。
长春花吲哚生物碱文多灵、长春碱、长春质碱的组织特异性具有一定的规律性。三种叶片形态中的吲哚生物碱文多灵,长春质碱含量呈现规律性变化,在幼嫩叶片(L1)中最高,在成熟的叶片(L3)中的含量最低。两种生物碱,在前五个生长时期,L1和L2叶片状态的含量相差不到一倍,而L3状态叶片的两种生物碱含量和前两种状态叶片的生物碱含量相比差异比较大,最大到2.5倍。长春碱的含量大部分呈现L3>L2>L1的规律,叶片的L1和L2状态的叶片的长春碱含量相差不大,长春碱在长春花叶片中的含量比较低,最高含量只有万分之四。
长春花用于生物碱检测的叶片的最佳采集期在花蕾期到开花期。三种生长状态的长春花叶片的吲哚生物碱文多灵的含量为0.75 mg/g,长春质碱的含量为0.83 mg/g,成熟期2的三种生长状态的长春花叶片的吲哚生物碱长春碱的含量为0.027 mg/g,花蕾期,五对真叶生长期,开花期生物碱总含量较大,以长春花叶片的生物量最大为依据,从花蕾期到开花期为最佳的采收期。2.比较了长春花叶片中生物碱合成途径的11个合成酶基因的相对表达量
依照长春花悬浮细胞和毛状根中与生物碱含量相关性大的关键酶基因数据,选取长春花生物碱合成途径中的11个关键酶基因(ASA、CPR、D4H、TDC、GGPP、STR、ORCA3、G10H、PRX、SLS、DAT)。利用定量PCR检测了这些基因的相对表达量,发现环烯醚萜苷途径中的GGPP基因、G10H基因、SLS基因三个基因的表达可促进裂环马钱子苷的合成,STR基因的表达量增加可使生物碱长春质碱的含量提高,D4H基因,DAT基因的表达可促进文多灵的生成。文多灵和长春质碱在PRX基因编码的蛋白的催化下可促进长春碱的合成。而莽草酸途径中的ASA基因、TDC基因对叶片中生物碱的增加没有明显的促进作用。ORCA3基因的表达具有时空性,在嫩叶片中的表达量较高,在长春花成熟期的表达量较高。对D4H、DAT、SLS、TDC基因的表达具有正调控作用。
The medicinal plant Madagascar Periwinkle(Catharanthus roseus) possesses vindoline, catharanthine, vinblastine, and vincristine which play an important role in cancer. However, the contents of the three TIAs in C. roseus leaves are low and the diSTRibution has the specificity of tissue and growth stage. The use of metabolic engineering for improving the centents of the three TIAs in C. roseus leaves has opened up a new way by overexpression of TIAs biosynthesis of key genes. However, Biosynthetic pathway of the three TIAs is very complex, Currently more than 30 catalytic enzymes and transcription factor genes have been cloned. Therefore, Tt is essential to ensure the changes of alkaloids and the key enzymes and transcription factors of TIAs pathway in C. roseus leaves.
In this study, we analyzed the contents of the three TIAs by HPLC and the relative expression of the eleven catalytic enzymes and transcription factor genes in C. roseus leaves.The major results are as follows: 1.We discussed the rule of dynamic changes of three alkaloids in C. roseus leaves. The distribution of the three TIAs including vindoline, catharanthine and vinblastine in C. roseus leaves has the specificity of tissue and growth stage. The highest content of vindoline is 1.18 mg/g and the lowest is 0.02 mg/g, a differents of 60 times. The highest content of catharanthine is 2.5 times of the lowest one.
2.The expressions of eleven catalytic enzymes and transcription factor genes by the real-time PCR show that the genes containing GGPP、G10H、SLS、D4H、DAT、PRX are the key enzymes genes in TIAs biosynthetic pathway. ORCA3 gene upregulates the expressions of the genes(D4H、DAT、SLS、TDC).
本研究以长春花叶片为研究材料,利用高效液相色谱技术研究了长春花叶片中生物碱的变化,利用荧光定量PCR技术检测了长春花叶片中生物碱合成途径中11个相关基因的相对表达量。得出了以下结果：1.探明了长春花叶片中三种生物碱的动态变化规律
长春花吲哚生物碱文多灵、长春碱、长春质碱的分布具有组织器官和生长发育的特异性。其中,不同时期长春花三种叶片形态中文多灵含量的差异较大,最高含量为1.18 mg/g,最低含量为0.02 mg/g,相差大约60倍左右。长春碱的最高含量为最低含量的四倍,为0.01 mg/g-0.04 mg/g。长春碱在长春花叶片中的含量比较低,最高含量只有万分之四。长春质碱的含量为0.4 mg/g-1.16 mg/g,大约相差2.5倍。
长春花吲哚生物碱文多灵、长春碱、长春质碱的组织特异性具有一定的规律性。三种叶片形态中的吲哚生物碱文多灵,长春质碱含量呈现规律性变化,在幼嫩叶片(L1)中最高,在成熟的叶片(L3)中的含量最低。两种生物碱,在前五个生长时期,L1和L2叶片状态的含量相差不到一倍,而L3状态叶片的两种生物碱含量和前两种状态叶片的生物碱含量相比差异比较大,最大到2.5倍。长春碱的含量大部分呈现L3>L2>L1的规律,叶片的L1和L2状态的叶片的长春碱含量相差不大,长春碱在长春花叶片中的含量比较低,最高含量只有万分之四。
长春花用于生物碱检测的叶片的最佳采集期在花蕾期到开花期。三种生长状态的长春花叶片的吲哚生物碱文多灵的含量为0.75 mg/g,长春质碱的含量为0.83 mg/g,成熟期2的三种生长状态的长春花叶片的吲哚生物碱长春碱的含量为0.027 mg/g,花蕾期,五对真叶生长期,开花期生物碱总含量较大,以长春花叶片的生物量最大为依据,从花蕾期到开花期为最佳的采收期。2.比较了长春花叶片中生物碱合成途径的11个合成酶基因的相对表达量
依照长春花悬浮细胞和毛状根中与生物碱含量相关性大的关键酶基因数据,选取长春花生物碱合成途径中的11个关键酶基因(ASA、CPR、D4H、TDC、GGPP、STR、ORCA3、G10H、PRX、SLS、DAT)。利用定量PCR检测了这些基因的相对表达量,发现环烯醚萜苷途径中的GGPP基因、G10H基因、SLS基因三个基因的表达可促进裂环马钱子苷的合成,STR基因的表达量增加可使生物碱长春质碱的含量提高,D4H基因,DAT基因的表达可促进文多灵的生成。文多灵和长春质碱在PRX基因编码的蛋白的催化下可促进长春碱的合成。而莽草酸途径中的ASA基因、TDC基因对叶片中生物碱的增加没有明显的促进作用。ORCA3基因的表达具有时空性,在嫩叶片中的表达量较高,在长春花成熟期的表达量较高。对D4H、DAT、SLS、TDC基因的表达具有正调控作用。
The medicinal plant Madagascar Periwinkle(Catharanthus roseus) possesses vindoline, catharanthine, vinblastine, and vincristine which play an important role in cancer. However, the contents of the three TIAs in C. roseus leaves are low and the diSTRibution has the specificity of tissue and growth stage. The use of metabolic engineering for improving the centents of the three TIAs in C. roseus leaves has opened up a new way by overexpression of TIAs biosynthesis of key genes. However, Biosynthetic pathway of the three TIAs is very complex, Currently more than 30 catalytic enzymes and transcription factor genes have been cloned. Therefore, Tt is essential to ensure the changes of alkaloids and the key enzymes and transcription factors of TIAs pathway in C. roseus leaves.
In this study, we analyzed the contents of the three TIAs by HPLC and the relative expression of the eleven catalytic enzymes and transcription factor genes in C. roseus leaves.The major results are as follows: 1.We discussed the rule of dynamic changes of three alkaloids in C. roseus leaves. The distribution of the three TIAs including vindoline, catharanthine and vinblastine in C. roseus leaves has the specificity of tissue and growth stage. The highest content of vindoline is 1.18 mg/g and the lowest is 0.02 mg/g, a differents of 60 times. The highest content of catharanthine is 2.5 times of the lowest one.
2.The expressions of eleven catalytic enzymes and transcription factor genes by the real-time PCR show that the genes containing GGPP、G10H、SLS、D4H、DAT、PRX are the key enzymes genes in TIAs biosynthetic pathway. ORCA3 gene upregulates the expressions of the genes(D4H、DAT、SLS、TDC).
引文
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