灯盏细辛血管保护作用及南方红豆杉等叶绿体基因组研究
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摘要
高脂血症(Hy percholesterolemia)是引起动脉粥样硬化(Atherosclerosis)等心血管疾病发生、发展的主要危险因素之一,与血管内皮细胞功能受损有关。灯盏细辛是菊科飞蓬属多年生植物短葶飞蓬Erigeron breviscapus (Vant.) Hand.-Mazz的干燥全草,其活性成分灯盏乙素(Scutellarin)在临床上多用于治疗脑血管意外引起的脑部缺血及其后遗症状。
本文预测了灯盏细辛的适宜生态产地,研究了灯盏乙素对高胆固醇饮食所致高脂血症大鼠的血管影响作用,为灯盏细辛的生产布局以及灯盏乙素调节异常血脂功能和延缓动脉粥样硬化、维持血管正常功能状态提供了实验依据。本实验喂食大鼠高胆固醇饮食(基础饲料添加1%胆酸、2%胆固醇和5.5%花生油),造成大鼠高脂血症模型,同时对各高胆固醇饮食组大鼠灌胃给予30、100和300mg/kg/day不同剂量的灯盏乙素,观察灯盏乙素对造模大鼠在调节血脂、抗氧化能力、乙酰胆碱所致的离体血管舒张、乙酰胆碱所致离体血管NO释放量、胸主动脉内皮一氧化氮合酶活性及其基因表达等方面的影响。结果表明：
(1)灯盏乙素能显著降低高胆固醇饮食所致大鼠的血清和肝脏组织总胆固醇升高,降低升高的肝脏总甘油三酯水平,恢复血清和肝脏组织中降低的高密度脂蛋白含量,减轻模型动物高脂血症症状,通过改善高脂血症大鼠的血脂代谢异常,阻断血管壁血脂沉积,达到延缓血管壁功能障碍,保护血管目的。
(2)乙酰胆碱引起的血管舒张作用是一个浓度依赖的过程,胆固醇饮食对血管内皮有不同程度的损伤作用。灯盏乙素各个给药组能够纠正血管内皮细胞功能异常,对高胆固醇饮食引发的高脂血症大鼠模型血管内皮依赖性舒张功能有一定的保护作用。
(3)灯盏乙素能上调模型组动物被抑制的eNOS基因表达,促进eNOS酶活力,增加NO释放量,从而改善血管内皮功能,延缓动脉粥样硬化,达到高脂血症模型动物血管保护作用。
(4)灯盏乙素具有大量清除DPPH自由基的能力,能显著改善血清和肝脏组织中谷胱甘肽过氧化物酶活性,降低血清和肝脏脂质过氧化物MDA含量。提示灯盏乙素是一种有效的抗氧化剂,其体内抗氧化作用可能通过直接作用于过氧化物实现,通过减轻脂质过氧化物对血管壁的刺激来达到保护血管功能的目的。
叶绿体基因组是叶绿体间质中所含的全部环状DNA分子,由于其高度的保守性和具备母系遗传的特性,可用于系统进化、基因工程和物种鉴定等领域。但目前可供研究的叶绿体基因组很少,在将近40万物种的植物界,截至2011年5月,只得到213条完整的叶绿体基因组序列(其中182条属于绿色维管植物),严重阻碍了植物叶绿体基因组学的进一步发展。
本实验在已有工作的基础上,优化出一种简便、快速、高效的叶绿体基因组DNA提取方法,通过454测序平台,采用多标签平行测序方法,进行De Novo和Reference Mapper方法拼接组装,得到了完整的南方红豆杉(Taxus chinensis var. mairei (Lemee et Levl.) Cheng et L. K. Fu)和苏铁(Cycas revoluta Thunb.)的叶绿体基因组序列。
对序列进行验证和注释分析后,得到南方红豆杉全长127,663bp,与其他陆地植物相比,缺少一个大的反向重复区域(Inverted Repeat, IR),导致无法区分其小单拷贝区(Small Single Copy Region, SSC)和大单拷贝区(Large Single Copy Region, LSC).南方红豆杉的GC含量为34.72%,是目前已测序裸子植物叶绿体基因组序列中GC含量最低的物种。南方红豆杉包含113个基因,其中109个是单拷贝基因,另外4个基因是各有2个拷贝的tRNA基因,分别是trnl-CAU和trnQ-UUG。它有4个核糖体RNA (rRNA)基因,29个转运RNA (tRNA)基因。在这113个基因中,有12个基因包含一个或一个以上的内含子,其中3个是tRNA基因,剩下的9个是蛋白编码基因。南方红豆杉拥有7个碱基以上(包括7个碱基)的Homopolymer结构199个；30个碱基以上(包括30个碱基)的重复序列411个,是目前已知裸子植物叶绿体基因组序列中重复序列个数最多的物种。
苏铁叶绿体基因组序列全长162,494bp,拥有一对长25,066bp的IRs区域,这对IRs区域被长23,379bp的SSC和长88,983bp的LSC隔离。苏铁的GC含量为39.38%,是已知裸子植物中GC含量第二高的物种,仅次于同属植物台东苏铁(Cycas taitungensis C. F.)。苏铁包含164个基因,其中142个是单拷贝基因,另外22个基因各有两个拷贝。在这164个基因中,8个为rRNA基因,38个为tRNA基因,22个基因包含一个或一个以上的内含子,其中8个是tRNA基因,剩下的14个为蛋白编码基因。苏铁具有7个碱基以上(包括7个碱基)的Homopolymer结构141个；30个碱基以上(包括30个碱基)的重复序列68个,远远少于台东苏铁(149个)。
对南方红豆杉、苏铁和其它已知裸子植物进行最大简约法(MP)和最大似然法(ML)分析,以烟草(Nicotiana tabacum Linn.)和问荆(Equisetum arvense L.)做为外类群,发现南方红豆杉与日本柳杉(Cryptomeria japonica (Linn, f.) D. Don)最相近,苏铁与台东苏铁最相近。
Hypercholesterolemia is a major risk factor for the development and progression of cardiovascular diseases including atherosclerosis, which is related with dysfunction of the vascular endothelial cells. Scutellarin is a major active ingredient from the plant Erigeron breviscapus. It is widely used for curing cerebral ischemia and other sequelaes caused by cerebrovascular accident in clinic.
Before this study, we predict the proper environments for Erigeron breviscapus, to find more producing areas for Erigeron breviscapus.
The aim of this study is to see whether scutellarin could adjust the abnormal lipid levels, delay the progress of atherosclerosis and maintain the normal function of thoracic aorta on the diet-induced hypercholesterolemia rats.
Animals of experimental groups are fed with a standard rat chow supplemented with 1%cholic acid,2%pure cholesterol and 5.5%oil to form hypercholesterolemia. Scutellarin is given by intragastric administration for 30,100 and 300 mg/kg/day. The lipid profile and oxidation resistance are monitored, and aortic functions in Sprague-Dawley rats are tested, including vasorelaxation, in vitro NO production, serum nitric oxide synthase and eNOS mRNA expression. The results indicate that:
(1) scutellarin can markedly attenuate the increased serum and liver total cholesterol induced by atherogenic diet, reduce the rised liver triglycerides, renew the high density lipoprotein cholesterol and alleviate hypercholesterolemia's symptoms. Its vascular protection may carry out by improving the lipid metabolism in the hypercholesterolemia rats, interrupting lipid aggrading and retarding dysfunction of vascular endothelial cells.
(2) scutellarin can ameliorate vascular endothelial cells'function and improve acetylcholine-induced endothelium-dependent vasorelaxation in hypercholesterolemia rats'isolated thoracic aortas.
(3) scutellarin administration can significantly enhance acetylcholine-induced NO production and increase the gene expression of endothelial nitric oxide synthase.
(4) scutellarin has the mightiness power of clarity the free radical of DPPH. It can also improve the activity of GSH-Px and reduce the amount of MDA in both serum and liver.
Chloroplast (cp) genome is the total circular DNA in the chloroplast. Because of its high conservation and maternal inheritance, cp genome can be used in phylogenetic analysis, genetic engineering, identification of plants and other fields as well. At present, there are only 213 complete cp genomes in publication. The lack of cp genome information may severely hamper the further progress of plant and cp genome studies.
This dissertation bases on early studies, having optimized a simple, rapid and effective method for cp DNA extraction. Using 454 Genome Sequencer FLX System and multiplex identifier (MID) parallel sequencing method, we obtain the complete cp genomes of taxus (Taxus chinensis var. mairei (Lemee et Levl.) Cheng et L. K. Fu) and cycad (Cycas revoluta Thunb.) through de novo and reference guided assembly.
After validation and annotation, we present the complete cp genome of taxus, which is 127,663 bp in length. Compared to other land plant genomes, this cp genome has lost one of the large IR regions. So we are unable to define the large and small single copy regions in this genome. The GC content of this genome is 34.72%, which is the lowest GC content among the already existing gymnosperms. It contains 109 single copy genes and two duplicated (trnl-CAU and trnQ-UUG) genes that give a total of 113 genes. There are 4 rRNA genes and 29 tRNA genes in taxus. Twelve of all the genes include one or more introns. Three of them are tRNA genes while the left 9 are protein coding genes. Taxus contains 199 homopolymers (7≥bp) and 411 repeat sequences (≥30 bp). It has the most repeat sequences in the known gymnosperms.
Cycad is 162,494 bp in length, which include a pair of IRs of 25,066 bp separated by a small single copy region of 23.379 bp and a large single copy region of 88.983 bp. Its GC content is 39.38%. It is the second highest GC content among the existing gymnosperms, just lower than Cycas taitungensis C. F.. another kind of cycad. Cycad contains 142 unique genes and 22 duplicated genes that give a total of 164 genes. Thirty eight of them are tRNA genes and 8 are rRNA genes. Twenty two genes contain two or more exons,8 of them are tRNA genes and the left 14 are protein coding genes. Cycad contains 141 homopolymers (≥7 bp) and 68 repeat sequences (≥30 bp). Its repeat sequences are far less than Cycas taitungensis.
We also perform a simple phylogenetic analysis in the existing 20 gymnosperms through maximum parsimony (MP) and maximum likelihood (ML) analysis using tobacco and horse-tail as outgroups. Both MP and ML trees indicate that taxus is close to Cryptomeriajaponica (Linn, f.) D. Don while cycad is sister to Cycas taitungensis C.F.
本文预测了灯盏细辛的适宜生态产地,研究了灯盏乙素对高胆固醇饮食所致高脂血症大鼠的血管影响作用,为灯盏细辛的生产布局以及灯盏乙素调节异常血脂功能和延缓动脉粥样硬化、维持血管正常功能状态提供了实验依据。本实验喂食大鼠高胆固醇饮食(基础饲料添加1%胆酸、2%胆固醇和5.5%花生油),造成大鼠高脂血症模型,同时对各高胆固醇饮食组大鼠灌胃给予30、100和300mg/kg/day不同剂量的灯盏乙素,观察灯盏乙素对造模大鼠在调节血脂、抗氧化能力、乙酰胆碱所致的离体血管舒张、乙酰胆碱所致离体血管NO释放量、胸主动脉内皮一氧化氮合酶活性及其基因表达等方面的影响。结果表明：
(1)灯盏乙素能显著降低高胆固醇饮食所致大鼠的血清和肝脏组织总胆固醇升高,降低升高的肝脏总甘油三酯水平,恢复血清和肝脏组织中降低的高密度脂蛋白含量,减轻模型动物高脂血症症状,通过改善高脂血症大鼠的血脂代谢异常,阻断血管壁血脂沉积,达到延缓血管壁功能障碍,保护血管目的。
(2)乙酰胆碱引起的血管舒张作用是一个浓度依赖的过程,胆固醇饮食对血管内皮有不同程度的损伤作用。灯盏乙素各个给药组能够纠正血管内皮细胞功能异常,对高胆固醇饮食引发的高脂血症大鼠模型血管内皮依赖性舒张功能有一定的保护作用。
(3)灯盏乙素能上调模型组动物被抑制的eNOS基因表达,促进eNOS酶活力,增加NO释放量,从而改善血管内皮功能,延缓动脉粥样硬化,达到高脂血症模型动物血管保护作用。
(4)灯盏乙素具有大量清除DPPH自由基的能力,能显著改善血清和肝脏组织中谷胱甘肽过氧化物酶活性,降低血清和肝脏脂质过氧化物MDA含量。提示灯盏乙素是一种有效的抗氧化剂,其体内抗氧化作用可能通过直接作用于过氧化物实现,通过减轻脂质过氧化物对血管壁的刺激来达到保护血管功能的目的。
叶绿体基因组是叶绿体间质中所含的全部环状DNA分子,由于其高度的保守性和具备母系遗传的特性,可用于系统进化、基因工程和物种鉴定等领域。但目前可供研究的叶绿体基因组很少,在将近40万物种的植物界,截至2011年5月,只得到213条完整的叶绿体基因组序列(其中182条属于绿色维管植物),严重阻碍了植物叶绿体基因组学的进一步发展。
本实验在已有工作的基础上,优化出一种简便、快速、高效的叶绿体基因组DNA提取方法,通过454测序平台,采用多标签平行测序方法,进行De Novo和Reference Mapper方法拼接组装,得到了完整的南方红豆杉(Taxus chinensis var. mairei (Lemee et Levl.) Cheng et L. K. Fu)和苏铁(Cycas revoluta Thunb.)的叶绿体基因组序列。
对序列进行验证和注释分析后,得到南方红豆杉全长127,663bp,与其他陆地植物相比,缺少一个大的反向重复区域(Inverted Repeat, IR),导致无法区分其小单拷贝区(Small Single Copy Region, SSC)和大单拷贝区(Large Single Copy Region, LSC).南方红豆杉的GC含量为34.72%,是目前已测序裸子植物叶绿体基因组序列中GC含量最低的物种。南方红豆杉包含113个基因,其中109个是单拷贝基因,另外4个基因是各有2个拷贝的tRNA基因,分别是trnl-CAU和trnQ-UUG。它有4个核糖体RNA (rRNA)基因,29个转运RNA (tRNA)基因。在这113个基因中,有12个基因包含一个或一个以上的内含子,其中3个是tRNA基因,剩下的9个是蛋白编码基因。南方红豆杉拥有7个碱基以上(包括7个碱基)的Homopolymer结构199个；30个碱基以上(包括30个碱基)的重复序列411个,是目前已知裸子植物叶绿体基因组序列中重复序列个数最多的物种。
苏铁叶绿体基因组序列全长162,494bp,拥有一对长25,066bp的IRs区域,这对IRs区域被长23,379bp的SSC和长88,983bp的LSC隔离。苏铁的GC含量为39.38%,是已知裸子植物中GC含量第二高的物种,仅次于同属植物台东苏铁(Cycas taitungensis C. F.)。苏铁包含164个基因,其中142个是单拷贝基因,另外22个基因各有两个拷贝。在这164个基因中,8个为rRNA基因,38个为tRNA基因,22个基因包含一个或一个以上的内含子,其中8个是tRNA基因,剩下的14个为蛋白编码基因。苏铁具有7个碱基以上(包括7个碱基)的Homopolymer结构141个；30个碱基以上(包括30个碱基)的重复序列68个,远远少于台东苏铁(149个)。
对南方红豆杉、苏铁和其它已知裸子植物进行最大简约法(MP)和最大似然法(ML)分析,以烟草(Nicotiana tabacum Linn.)和问荆(Equisetum arvense L.)做为外类群,发现南方红豆杉与日本柳杉(Cryptomeria japonica (Linn, f.) D. Don)最相近,苏铁与台东苏铁最相近。
Hypercholesterolemia is a major risk factor for the development and progression of cardiovascular diseases including atherosclerosis, which is related with dysfunction of the vascular endothelial cells. Scutellarin is a major active ingredient from the plant Erigeron breviscapus. It is widely used for curing cerebral ischemia and other sequelaes caused by cerebrovascular accident in clinic.
Before this study, we predict the proper environments for Erigeron breviscapus, to find more producing areas for Erigeron breviscapus.
The aim of this study is to see whether scutellarin could adjust the abnormal lipid levels, delay the progress of atherosclerosis and maintain the normal function of thoracic aorta on the diet-induced hypercholesterolemia rats.
Animals of experimental groups are fed with a standard rat chow supplemented with 1%cholic acid,2%pure cholesterol and 5.5%oil to form hypercholesterolemia. Scutellarin is given by intragastric administration for 30,100 and 300 mg/kg/day. The lipid profile and oxidation resistance are monitored, and aortic functions in Sprague-Dawley rats are tested, including vasorelaxation, in vitro NO production, serum nitric oxide synthase and eNOS mRNA expression. The results indicate that:
(1) scutellarin can markedly attenuate the increased serum and liver total cholesterol induced by atherogenic diet, reduce the rised liver triglycerides, renew the high density lipoprotein cholesterol and alleviate hypercholesterolemia's symptoms. Its vascular protection may carry out by improving the lipid metabolism in the hypercholesterolemia rats, interrupting lipid aggrading and retarding dysfunction of vascular endothelial cells.
(2) scutellarin can ameliorate vascular endothelial cells'function and improve acetylcholine-induced endothelium-dependent vasorelaxation in hypercholesterolemia rats'isolated thoracic aortas.
(3) scutellarin administration can significantly enhance acetylcholine-induced NO production and increase the gene expression of endothelial nitric oxide synthase.
(4) scutellarin has the mightiness power of clarity the free radical of DPPH. It can also improve the activity of GSH-Px and reduce the amount of MDA in both serum and liver.
Chloroplast (cp) genome is the total circular DNA in the chloroplast. Because of its high conservation and maternal inheritance, cp genome can be used in phylogenetic analysis, genetic engineering, identification of plants and other fields as well. At present, there are only 213 complete cp genomes in publication. The lack of cp genome information may severely hamper the further progress of plant and cp genome studies.
This dissertation bases on early studies, having optimized a simple, rapid and effective method for cp DNA extraction. Using 454 Genome Sequencer FLX System and multiplex identifier (MID) parallel sequencing method, we obtain the complete cp genomes of taxus (Taxus chinensis var. mairei (Lemee et Levl.) Cheng et L. K. Fu) and cycad (Cycas revoluta Thunb.) through de novo and reference guided assembly.
After validation and annotation, we present the complete cp genome of taxus, which is 127,663 bp in length. Compared to other land plant genomes, this cp genome has lost one of the large IR regions. So we are unable to define the large and small single copy regions in this genome. The GC content of this genome is 34.72%, which is the lowest GC content among the already existing gymnosperms. It contains 109 single copy genes and two duplicated (trnl-CAU and trnQ-UUG) genes that give a total of 113 genes. There are 4 rRNA genes and 29 tRNA genes in taxus. Twelve of all the genes include one or more introns. Three of them are tRNA genes while the left 9 are protein coding genes. Taxus contains 199 homopolymers (7≥bp) and 411 repeat sequences (≥30 bp). It has the most repeat sequences in the known gymnosperms.
Cycad is 162,494 bp in length, which include a pair of IRs of 25,066 bp separated by a small single copy region of 23.379 bp and a large single copy region of 88.983 bp. Its GC content is 39.38%. It is the second highest GC content among the existing gymnosperms, just lower than Cycas taitungensis C. F.. another kind of cycad. Cycad contains 142 unique genes and 22 duplicated genes that give a total of 164 genes. Thirty eight of them are tRNA genes and 8 are rRNA genes. Twenty two genes contain two or more exons,8 of them are tRNA genes and the left 14 are protein coding genes. Cycad contains 141 homopolymers (≥7 bp) and 68 repeat sequences (≥30 bp). Its repeat sequences are far less than Cycas taitungensis.
We also perform a simple phylogenetic analysis in the existing 20 gymnosperms through maximum parsimony (MP) and maximum likelihood (ML) analysis using tobacco and horse-tail as outgroups. Both MP and ML trees indicate that taxus is close to Cryptomeriajaponica (Linn, f.) D. Don while cycad is sister to Cycas taitungensis C.F.
引文
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