阳春砂HMGR和DXR基因在烟草萜类化合物生物合成中的功能研究
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摘要
阳春砂Amomum villosum Lour.是中药砂仁来源植物之一,其性温,味辛,具有化湿开胃、温脾止泻、理气安胎之功效。砂仁化湿行气的主要药效物质为挥发油中的萜类化合物。药用植物中的萜类化合物具有广泛的药理作用和重要的生物活性。
植物萜类化合物的生物合成主要有两条途径,一条是甲羟戊酸途径(mevalonate pathway, MVA途径),另一条是2-甲基赤藓糖醇-4-磷酸途径(2-C-methyl-D-erythritol-4-phosphate pathway, MEP途径)。3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR)和1-去氧-D-木酮糖-5-磷酸还原异构酶(1-deoxy-D-xylulose-5-phos phate reductoisomerase, DXR)分别是MVA和MEP上游途径中的两个关键酶。通过调控HMGR和DXR基因的表达可以提高植物萜类化合物的含量。
1目的
利用阳春砂萜类化合物生物合成途径上游2个关键酶HMGR和DXR的基因——AvHMGR和AvDXR,进行萜类化合物生物合成调控研究,在模式植物烟草中分析AvHMGR和AvDXR的生物功能,为应用AvHMGR和AvDXR进行阳春砂萜类生物合成的基因工程调控,从根本上提高阳春砂的药效品质提供科学依据。
2方法
2.1AvHMGR、AvDXR转基因烟草T1代的筛选及形态观察
以卡那霉素(kanamycin, Kan)初步筛选AvHMGR、AvDXR转基因烟草T1代植株,对转基因植株进行以Kan抗性基因nptⅡ和目的基因AvHMGR、AvDXR为靶标的PCR验证。观察转基因烟草和野生型对照烟草在形态和抗虫性方面的差异。
2.2转基因烟草的AvHMGR、AvDXR表达量及酶活性分析
用半定量RT-PCR法,分别检测转基因烟草中的AvHMGR和AvDXR表达量。对AvHMGR和AvDXR基因超表达植株及其对照,采用专一底物,通过分光光度计法检测辅酶NADPH (nicotinamide adenine dinucleotide phosphate)的氧化速率来测定HMGR和DXR活性。
2.3超表达AvHMGR、AvDXR对烟草萜类生物合成的影响
对AvHMGR、AvDXR转基因烟草及其野生型对照中的萜类化合物进行分类提取和定性定量分析。对光合色素中的类胡萝卜素(四萜类)和叶绿素(二萜类),采用丙酮提取,分光光度法测定含量；对挥发性萜类,主要是单萜、倍半萜和二萜类,采用顶空固相微萃取(HS-SPME)提取,气质联用色谱(GC-MS)进行定性分析；对非挥发性萜类化合物,主要是三萜类植物甾醇、维生素E及二萜类植醇,采用二氯甲烷提取,GC-MS进行定量分析。
3结果
3.1AvHMGR、AvDXR转基因烟草T1代的筛选
通过Kan初步筛选和PCR验证,外源基因可能以单拷贝的形式遗传至AvHMGR转基因烟草的H1、H3、H4、H5株系及AvDXR转基因烟草的D1、D2株系基因组中。共筛选得到AvHMGR转基因烟草Tl代植株17株,AvDXR转基因烟草T1代植株12株。
对植物形态进行观察发现,AvHMGR转基因烟草的H5株系的株高极显著高于野生型。AvDXR转基因烟草表现出抗虫性且茎有横向生长趋势。
3.2转基因烟草的AvHMGR、AvDXR表达量及酶活性分析
经过检测,11株AvHMGR及8株AvDXR转基因烟草T1代植株外源基因获得超表达。酶活性测定结果显示,与野生型对照比较,AvHMGR超表达植株中有5株HMGR活性显著提高；AvDXR超表达植株中有2株DXR活性显著提高。
3.3超表达AvHMGR、AvDXR对烟草萜类生物合成的影响
AvHMGR和AvDXR转基因烟草与对照比较,能检测到更多的挥发性单萜(薄荷烯)、倍半萜(檀香醇,AvDXR转基因烟草D2-1)及二萜(黑松醇,AvHMGR转基因烟草H3-3),并促进了光合色素(叶绿素和类胡萝卜素)、甾醇(豆甾醇、菜油甾醇)及植醇的合成。其中,AvDXR对MEP途径的光合色素生物合成的促进作用更为明显,同样,AvHMGR对MVA途径的甾醇生物合成的促进也更为明显。而超表达烟草中的β-谷甾醇和维生素E的含量与野生型比较有所降低。
4结论
本研究证实,通过对萜类合成途径的上游不同关键酶基因HMGR和DXR的调控,均可促进下游萜类产物的合成,阳春砂AvHMGR和AvDXR基因在萜类生物合成途径中发挥重要作用,而AvHMGR和AvDXR分别超表达对不同萜类化合物的促进程度并不完全一致,与其在细胞内的不同定位有关,证明2条萜类合成途径之间既具有相对独立性,又存在着相互的关联和交流。本研究为应用AvHMGR和AvDXR进行阳春砂萜类生物合成的基因工程调控,从根本上提高阳春砂的药效品质提供了依据。
Fructus Amomi is mainly from Amomum villosum Lour.,which is a Chinese medicine with warm property and pungent taste and has such effects as resolving dampness to improve appetite, warming spleen to arrest diarrhea, regulating Qi to prevent miscarriage etc. The isoprenoids in its volatile oil are the main medicinal compounds of Fructus Amomi.
In higher plants, isoprenoids are produced through two universal pathways:one is the mevalonate (MVA) pathway, the other is2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. The enzymes,3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and1-deoxy-D-xylulose5-phosphate reductase (DXR) are the upstream key-enzymes for MVA and MEP pathway, respectively. Overexpresstion of HMGR and DXR gene can enhance the biosynthesis of isoprenoids in plants.
1Objective
Using the upstream key-enzymes in isoprenoid biosynthesis pathway of HMGR and DXR gene from A villosum to analyze the biological function of AvHMGR and AvDXR in transgenic tobacco. Furthermore, using the metabolic engineering method, to manipulate the expresstion of AvHMGR and AvDXR in isoprenoid biosynthesis, can be of value to improve the medicinal quality of A. villosum.
2Methods
2.1Screening of T1transgenic tobacco plants and phenotypic comparation
T1transgenic tobacco seeds were first screened by kanamycin, and then all kanamycin-resistance plants were screened by PCR for the presence of neomycin phosphotransferase Ⅱ (npt Ⅱ) and target genes of AvHMGR and AvDXR. Phenotypic differences were compared between the transgenic plants and untransformed plants.
2.2Expression and enzymes activity analysis in AvHMGR and AvDXR transgenic tobacco plants
RT-PCR (Reverse Transcription-Polymerase Chain Reaction) was used to analyze the expression level of AvHMGR and AvDXR in transgenic tobacco plants. Using the substrate specific method, HMGR and DXR activity were determined in AvHMGR and AvDXR transgenic tobacco plants and wild-type tobacco plants by spectrometer.
2.3Overexpression of AvHMGR and AvDXR genes effect of isoprenoids producion in tobacco plants
Extraction and qualitative analysis of isoprenoids contents in transgenic plants and wild-type plants. Chlorophylls (diterpene) and carotenoids (tetaterpene) were extracted by acetone and determined by spectrometer. Volatile isoprenoids, mainly of monoterpene, sesquiterpene and diterpene, were determinated by Head Space-SolidPhase Microextraction (HS-SPME), qualitative analysis was accomplished by GC-MS. Non-volatile isoprenoids, such as triterterpene of sterol, a-tocopherol, and diterpene of phytol, were extracted by dichloromethane and qualitative analysis by GC-MS.
3Results
3.1Screening of AvHMGR and AvDXR T1transgenic tobacco plants and phenotypic comparation
AvHMGR and AvDXR transgenic tobacco plants were screened by Kan and verified by PCR, transgenic lines (H1, H3, H4, H5and D1, D2) were obtained, showed single-copy insertions, probably. Seventeen AvHMGR and twelve AvDXR transgenic tobacco plants were obtained.
The height of AvHMGR transgenic plants (line H5) showed obvious phenotypic differences compared with wild-type plants. The stems of AvDXR transgenic plants presented horizontal growth and AvDXR transgenic tobacco suffered from less pests.
3.2Expression and enzymes activity analysis in AvHMGR and AvDXR transgenic plants
The results showed that eleven AvHMGR and eight AvDXR T1transgenic tobacco plants were overexpressed. Compared with wild-type tobacco plants, the HMGR and DXR activity of five AvHMGR and two AvDXR transgenic plants had been significant increased,
respectively.3.3Overexpression of AvHMGR and AvDXR genes effect of isoprenoids producion in tobacco plants
Compared with wild-type plants, AvHMGR and AvDXR overexpressing plants can detected more volatile terpenoids, such as monoterpene (m-Mentha-4,8-diene), sesquiterpene (D2-1of AvDXR transgenic tobacco plants showed the production of santalol), diterpene (H3-3of AvHMGR transgenic tobacco plants showed the production of thunbergol).Furthermore, the overexpression of AvHMGR and AvDXR, resulted in elevated levels of photosynthetic pigments (chlorophylls and carotenoids), phytol, stigmasterol and campesterol. However, the contents of β-sitosterol and α-tocopherol had decreased, compared with wild-type plants.
4Conclusion
The results verified that regulation of HMGR and DXR can enhance biosynthesis of isoprenoids. AvHMGR and AvDXR genes play important role in isoprenoid biosynthesis pathway. However, all isoprenoids did not increase equally, each isoprenoid biosynthetic pathway could have its own localization in cells. Indicating that the MVA pathway and MEP pathway did not independent, cross-talk between the two pathways had also been documented. The study documented that using metabolic engineering, manipulate upstream key-enzymes of AvHMGR and AvDXR in isoprenoid biosynthesis, can improve the medicinal quality of Amomum villosum Lour.
植物萜类化合物的生物合成主要有两条途径,一条是甲羟戊酸途径(mevalonate pathway, MVA途径),另一条是2-甲基赤藓糖醇-4-磷酸途径(2-C-methyl-D-erythritol-4-phosphate pathway, MEP途径)。3-羟基-3-甲基戊二酰辅酶A还原酶(3-hydroxy-3-methylglutaryl coenzyme A reductase, HMGR)和1-去氧-D-木酮糖-5-磷酸还原异构酶(1-deoxy-D-xylulose-5-phos phate reductoisomerase, DXR)分别是MVA和MEP上游途径中的两个关键酶。通过调控HMGR和DXR基因的表达可以提高植物萜类化合物的含量。
1目的
利用阳春砂萜类化合物生物合成途径上游2个关键酶HMGR和DXR的基因——AvHMGR和AvDXR,进行萜类化合物生物合成调控研究,在模式植物烟草中分析AvHMGR和AvDXR的生物功能,为应用AvHMGR和AvDXR进行阳春砂萜类生物合成的基因工程调控,从根本上提高阳春砂的药效品质提供科学依据。
2方法
2.1AvHMGR、AvDXR转基因烟草T1代的筛选及形态观察
以卡那霉素(kanamycin, Kan)初步筛选AvHMGR、AvDXR转基因烟草T1代植株,对转基因植株进行以Kan抗性基因nptⅡ和目的基因AvHMGR、AvDXR为靶标的PCR验证。观察转基因烟草和野生型对照烟草在形态和抗虫性方面的差异。
2.2转基因烟草的AvHMGR、AvDXR表达量及酶活性分析
用半定量RT-PCR法,分别检测转基因烟草中的AvHMGR和AvDXR表达量。对AvHMGR和AvDXR基因超表达植株及其对照,采用专一底物,通过分光光度计法检测辅酶NADPH (nicotinamide adenine dinucleotide phosphate)的氧化速率来测定HMGR和DXR活性。
2.3超表达AvHMGR、AvDXR对烟草萜类生物合成的影响
对AvHMGR、AvDXR转基因烟草及其野生型对照中的萜类化合物进行分类提取和定性定量分析。对光合色素中的类胡萝卜素(四萜类)和叶绿素(二萜类),采用丙酮提取,分光光度法测定含量；对挥发性萜类,主要是单萜、倍半萜和二萜类,采用顶空固相微萃取(HS-SPME)提取,气质联用色谱(GC-MS)进行定性分析；对非挥发性萜类化合物,主要是三萜类植物甾醇、维生素E及二萜类植醇,采用二氯甲烷提取,GC-MS进行定量分析。
3结果
3.1AvHMGR、AvDXR转基因烟草T1代的筛选
通过Kan初步筛选和PCR验证,外源基因可能以单拷贝的形式遗传至AvHMGR转基因烟草的H1、H3、H4、H5株系及AvDXR转基因烟草的D1、D2株系基因组中。共筛选得到AvHMGR转基因烟草Tl代植株17株,AvDXR转基因烟草T1代植株12株。
对植物形态进行观察发现,AvHMGR转基因烟草的H5株系的株高极显著高于野生型。AvDXR转基因烟草表现出抗虫性且茎有横向生长趋势。
3.2转基因烟草的AvHMGR、AvDXR表达量及酶活性分析
经过检测,11株AvHMGR及8株AvDXR转基因烟草T1代植株外源基因获得超表达。酶活性测定结果显示,与野生型对照比较,AvHMGR超表达植株中有5株HMGR活性显著提高；AvDXR超表达植株中有2株DXR活性显著提高。
3.3超表达AvHMGR、AvDXR对烟草萜类生物合成的影响
AvHMGR和AvDXR转基因烟草与对照比较,能检测到更多的挥发性单萜(薄荷烯)、倍半萜(檀香醇,AvDXR转基因烟草D2-1)及二萜(黑松醇,AvHMGR转基因烟草H3-3),并促进了光合色素(叶绿素和类胡萝卜素)、甾醇(豆甾醇、菜油甾醇)及植醇的合成。其中,AvDXR对MEP途径的光合色素生物合成的促进作用更为明显,同样,AvHMGR对MVA途径的甾醇生物合成的促进也更为明显。而超表达烟草中的β-谷甾醇和维生素E的含量与野生型比较有所降低。
4结论
本研究证实,通过对萜类合成途径的上游不同关键酶基因HMGR和DXR的调控,均可促进下游萜类产物的合成,阳春砂AvHMGR和AvDXR基因在萜类生物合成途径中发挥重要作用,而AvHMGR和AvDXR分别超表达对不同萜类化合物的促进程度并不完全一致,与其在细胞内的不同定位有关,证明2条萜类合成途径之间既具有相对独立性,又存在着相互的关联和交流。本研究为应用AvHMGR和AvDXR进行阳春砂萜类生物合成的基因工程调控,从根本上提高阳春砂的药效品质提供了依据。
Fructus Amomi is mainly from Amomum villosum Lour.,which is a Chinese medicine with warm property and pungent taste and has such effects as resolving dampness to improve appetite, warming spleen to arrest diarrhea, regulating Qi to prevent miscarriage etc. The isoprenoids in its volatile oil are the main medicinal compounds of Fructus Amomi.
In higher plants, isoprenoids are produced through two universal pathways:one is the mevalonate (MVA) pathway, the other is2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. The enzymes,3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and1-deoxy-D-xylulose5-phosphate reductase (DXR) are the upstream key-enzymes for MVA and MEP pathway, respectively. Overexpresstion of HMGR and DXR gene can enhance the biosynthesis of isoprenoids in plants.
1Objective
Using the upstream key-enzymes in isoprenoid biosynthesis pathway of HMGR and DXR gene from A villosum to analyze the biological function of AvHMGR and AvDXR in transgenic tobacco. Furthermore, using the metabolic engineering method, to manipulate the expresstion of AvHMGR and AvDXR in isoprenoid biosynthesis, can be of value to improve the medicinal quality of A. villosum.
2Methods
2.1Screening of T1transgenic tobacco plants and phenotypic comparation
T1transgenic tobacco seeds were first screened by kanamycin, and then all kanamycin-resistance plants were screened by PCR for the presence of neomycin phosphotransferase Ⅱ (npt Ⅱ) and target genes of AvHMGR and AvDXR. Phenotypic differences were compared between the transgenic plants and untransformed plants.
2.2Expression and enzymes activity analysis in AvHMGR and AvDXR transgenic tobacco plants
RT-PCR (Reverse Transcription-Polymerase Chain Reaction) was used to analyze the expression level of AvHMGR and AvDXR in transgenic tobacco plants. Using the substrate specific method, HMGR and DXR activity were determined in AvHMGR and AvDXR transgenic tobacco plants and wild-type tobacco plants by spectrometer.
2.3Overexpression of AvHMGR and AvDXR genes effect of isoprenoids producion in tobacco plants
Extraction and qualitative analysis of isoprenoids contents in transgenic plants and wild-type plants. Chlorophylls (diterpene) and carotenoids (tetaterpene) were extracted by acetone and determined by spectrometer. Volatile isoprenoids, mainly of monoterpene, sesquiterpene and diterpene, were determinated by Head Space-SolidPhase Microextraction (HS-SPME), qualitative analysis was accomplished by GC-MS. Non-volatile isoprenoids, such as triterterpene of sterol, a-tocopherol, and diterpene of phytol, were extracted by dichloromethane and qualitative analysis by GC-MS.
3Results
3.1Screening of AvHMGR and AvDXR T1transgenic tobacco plants and phenotypic comparation
AvHMGR and AvDXR transgenic tobacco plants were screened by Kan and verified by PCR, transgenic lines (H1, H3, H4, H5and D1, D2) were obtained, showed single-copy insertions, probably. Seventeen AvHMGR and twelve AvDXR transgenic tobacco plants were obtained.
The height of AvHMGR transgenic plants (line H5) showed obvious phenotypic differences compared with wild-type plants. The stems of AvDXR transgenic plants presented horizontal growth and AvDXR transgenic tobacco suffered from less pests.
3.2Expression and enzymes activity analysis in AvHMGR and AvDXR transgenic plants
The results showed that eleven AvHMGR and eight AvDXR T1transgenic tobacco plants were overexpressed. Compared with wild-type tobacco plants, the HMGR and DXR activity of five AvHMGR and two AvDXR transgenic plants had been significant increased,
respectively.3.3Overexpression of AvHMGR and AvDXR genes effect of isoprenoids producion in tobacco plants
Compared with wild-type plants, AvHMGR and AvDXR overexpressing plants can detected more volatile terpenoids, such as monoterpene (m-Mentha-4,8-diene), sesquiterpene (D2-1of AvDXR transgenic tobacco plants showed the production of santalol), diterpene (H3-3of AvHMGR transgenic tobacco plants showed the production of thunbergol).Furthermore, the overexpression of AvHMGR and AvDXR, resulted in elevated levels of photosynthetic pigments (chlorophylls and carotenoids), phytol, stigmasterol and campesterol. However, the contents of β-sitosterol and α-tocopherol had decreased, compared with wild-type plants.
4Conclusion
The results verified that regulation of HMGR and DXR can enhance biosynthesis of isoprenoids. AvHMGR and AvDXR genes play important role in isoprenoid biosynthesis pathway. However, all isoprenoids did not increase equally, each isoprenoid biosynthetic pathway could have its own localization in cells. Indicating that the MVA pathway and MEP pathway did not independent, cross-talk between the two pathways had also been documented. The study documented that using metabolic engineering, manipulate upstream key-enzymes of AvHMGR and AvDXR in isoprenoid biosynthesis, can improve the medicinal quality of Amomum villosum Lour.
引文
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