黑果枸杞遗传多样性及可持续利用研究

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	黑果枸杞遗传多样性及可持续利用研究
	刘增根
学位类型	博士
导师	陶燕铎 ; 邵贇
	2014-05
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	植物学
关键词	黑果枸杞遗传多样性组织培养功能成分可持续利用研究
摘要	黑果枸杞（Lycium ruthenicum Murr.）为茄科枸杞属多年生灌木，是我国西北荒漠、盐碱地区特有的药食兼用植物资源。黑果枸杞成熟果实在我国的传统民族医药中占有重要地位，其味甘、性平、清心热，用于治疗心热病、心脏病、月经不调、停经等病症。现代药理学研究发现，黑果枸杞具有增强机体免疫、抗氧化、抗动脉粥样硬化等功效。本研究以我国西北地区野生黑果枸杞为研究对象，对其遗传多样性、组织培养、营养成分进行了较为系统的分析与评价，对有效成分多糖和花色苷的提取纯化及抗氧化作用进行了研究与探讨，为黑果枸杞资源保护及综合开发利用提供科学依据。主要研究结果如下： 1. 利用SRAP分子标记对我国14个野生黑果枸杞居群的遗传多样性和遗传结构进行了研究与分析。应用筛选出的31对SRAP引物组合对黑果枸杞基因组DNA进行PCR扩增，共检测到468个有效位点，其中多态性位点398个，多态位点百分率为85.04%。Nei基因多样性和AMOVA分析显示，总的遗传变异中15.55%存在于居群间，84.45%存在于居群内，表明遗传分化主要存在于种群内。居群间具有中等的遗传分化（Gst=0.2155）；基因流Nm=1.8199，表明各居群间有较高水平的基因流动，能有效防止基因空间异质性。Mantel检验结果表明黑果枸杞居群间的遗传距离和地理距离之间相关性显著（r = 0.303, P = 0.004）。通过聚类结果发现，地理分布区域相近的居群容易聚在一起，主成分分析结果与聚类分析基本一致。利用SPSS软件分析了遗传多样性指数（He，I和PPB）与生态地理因素间的Spearman秩相关性，结果显示，黑果枸杞遗传多样性与海拔和年均日照时数呈显著性正相关，而与年均湿度呈显著性负相关。以上研究可为黑果枸杞资源保护和遗传育种提供科学依据。 2. 以黑果枸杞种子萌发获得无菌苗，以其幼嫩叶片和茎段作为外植体，诱导愈伤、分化为无菌苗。种子需先经过200 mg/L赤霉素溶液处理，打破休眠。通过对不同类型的培养基进行筛选，得到最佳的诱导分化培养基为（a）MS0+6-BA 1.0mg•L-1(单位下同)+NAA 0.2、芽增殖培养基（b）MS0+6-BA 0.5+ NAA 0.1和（c）MS0+6-BA 0.5+NAA 0.2、生根培养基（d）1/2MS0+NAA 0.1，以上诱导分化及增殖培养基均加2.5% 蔗糖和0.8% 琼脂，生根培养基的蔗糖和琼脂量减半，pH 5.8～6.0。在黑果枸杞遗传转化实验中，农杆菌菌株为EHA105，含目的片段（Ps16F11）的质粒，构建到真核表达载体pBI121质粒上。筛选出的农杆菌转化系统是：OD600为0.5～0.8的EHA105菌液侵染转化受体15 min，在YEB共培养基上共培养3 d，延后2 d进行筛选培养为最佳技术参数，从而可提高愈伤组织遗传转化效率。 3. 黑果枸杞富含碳水化合物、还原性糖、蛋白质等营养成分。此外，矿质元素、氨基酸等营养成分含量较高，且种类较为丰富，鲜果含水量高（接近85%）。通过单因素实验及正交实验确定了亚临界流体萃取黑果枸杞籽油的最佳提取工艺并对籽油进行GC-MS分析，其主要成分为亚油酸和油酸，分别占脂肪酸总量的73.805%和19.599%。籽油还含有丰富的维生素E类物质（6.6501mg/100g）。 4. 通过响应面优化及多元回归分析，得到黑果枸杞多糖（LRP）微波提取的最佳工艺条件，多糖平均得率为8.25 ± 0.07%。LRP为糖蛋白复合物，糖含量为93.84%，蛋白含量为4.22%，其中酸性糖占22.9%，中性糖占69.6%；其单糖组成主要为阿拉伯糖和半乳糖醛酸，其次为半乳糖、木糖、鼠李糖、葡萄糖和甘露糖，它们的相对摩尔比为8.3：5.4：3.9：1.0：1.0：0.6：0.3。黑果枸杞多糖具有较强清除DPPH•、•OH、O2•-自由基的能力，IC50值分别为1.42 mg/mL、1.84 mg/mL、4.30 mg/mL。 5. 黑果枸杞经超声醇提、陶瓷膜过滤、AB-8大孔树脂纯化得到黑果枸杞花色苷（LRA）。LRA只有在酸性条件下才能保持稳定，在其提取、加工、贮藏过程中要注意密封、避光、低温，避免与强氧化剂和还原剂以及Al ³⁺ 、Cu ²⁺ 、Fe ³⁺等金属离子接触或共存。利用ORAC体系评价了黑果枸杞抗氧化能力。黑果枸杞干果的ORAC值为587 μmol TE/g，而从黑果枸杞干果和鲜果提取纯化得到的花色苷ORAC值分别为5219 μmol TE/g和5975 μmol TE/g，说明黑果枸杞及其花色苷具有较强的抗氧化能力。LRA能有效抑制H₂O₂引起的脂质代谢产物MDA堆积和抗氧化酶SOD活性的下降，从而抑制细胞凋亡，可能是其发挥保护作用的机制之一。而上述的调节作用很有可能是通过上调与激活HO-1活性，维持细胞氧化还原平衡状态，进而对抗H₂O₂诱导的H9C2心肌细胞氧化应激损伤。
其他摘要	Lycium ruthenicum belongs to the Solanaceae family and mainly distributes in salinized desert of Northwestern China. It is a dual-purpose plant resource of herb and food. The fruits have been widely used to cure illness among folks and it holds a highly position in the Minority Medicine and Chinese Traditional Medicine. L. ruthenicum has been recorded in Tibetan medical classic “Jing Zhu Ben Cao” as a traditional herb. Its ripe fruits had been used for treatment of heart disease, abnormal menstruation and menopause. It was found that L. ruthenicum has a large variety of pharmacological activities, such as enhancing body‘s immunity, antioxidant, anti-atherosclerosis, anti-aging and hypoglycemic effects. In this research, the wild L. ruthenicum in Northwestern China is used as research object, the genetic diversity, tissue culture and nutrient were analyzed and evaluated. The extraction and purification of polysaccharides and anthocyanins, and their antioxidation were studied. Also these results provided a basis for future comprehensive exploitation and utilization of L. ruthenicum. The investigation results are as follows: 1. Sequence-related amplified polymorphism (SRAP) markers were used to assess the genetic diversity and population genetic structure in fourteen wild populations of L. ruthenicum from Northwestern China. Thirty-one selected primer combinations produced 468 discernible bands, with 398 (85.04%) being polymorphic, indicating relatively high genetic diversity at the species level. Analysis of molecular variance showed that the genetic variation was found mainly within populations (84.45%), but variance among populations was only 15.55%. And there was a moderate genetic differentiation (Gst = 0.2155) among populations. The mean of gene flow (Nm) was 1.8199, which showed relatively steady gene flow among populations, reduce genetic variance. Mantel test revealed a significant correlation between genetic and geographic distances (r = 0.303, P = 0.004), and the unweighted pair-group method using arithmetic average clustering and principal coordinates analysis demonstrated similar results. A total of nine significant (P < 0.05) correlations were detected between three indices of genetic diversity (He, I and PPB) and seven ecogeographic factors. It was positive significant correlation between genetic diversity and altitude and annual mean sunshine hours, and it was negative significant correlation between genetic diversity and annual mean humidity. Also recommendations for conservation of the endangered species resources and breeding program are proposed. 2. The aseptic seedlings were cultured through seed germination of L. ruthenicum. In this paper, the propagation by tissue culture were studied, including 200 mg/L gibberellin pretreatment for seed, different explants (young leaves and stem segments) induction and proliferation, callus induction and differentiation, root induction of plantlets of L. ruthenicum. Based on the selection of different types of media, the best callus induction and differentiation medium was (a) MS0+6-BA 1.0mg•L-1(next unit was the same)+NAA 0.2, the bud proliferation medium was (b) MS0+6-BA 0.5+ NAA 0.1and (c) MS0+6-BA 0.5+NAA 0.2, and the rooting medium was (d) 1/2MS0+NAA 0.1. The callus induction and differentiation medium and proliferation medium were added 2.5% sucrose and 0.8% agar, and the rooting medium was added 1.25% sucrose and 0.4% agar. The pH was 5.8～6.0. In the establishment of the genetic transformation system of L. ruthenicum mediated by Agrobacterium tumefaciens (EHA105), the fragment (Ps16F11) was constructed to the pBI121 plasmid of eukaryotic expression vector. The optimal Agrobacterium (OD600=0.5～0.8) transformation system indicated that infection time of 15 min, co-culture time of 3 days in YEB medium, and the sub-culture time of 2 days could enhance the transformation frequency. 3. There are rich in carbohydrates, reducing sugars and protein in fruits of L. ruthenicum. Also a wide variety of mineral elements, amino acids and vitamin were researched. And water content of fruit reached 85%. A method based on subcritical fluid extraction (SFE) before detection and identification by gas chromatography-mass spectrometry (GC-MS), respectively, has been developed for the isolation and extraction of L. ruthenicum seeds oil. And NIST02 spectral database were used to analyze and identify these components of oil. The optimum conditions of SFE were determined by single factor experiment and orthogonal experiment. The main components of seeds oil were linoleic acid (73.805%) and oleic acid (19.599%). Also the seeds oil was rich in vitamin E (6.6501mg/100g). 4. Dynamic microwave-assisted extraction (DMAE) technique was employed for the extraction of polysaccharides from L. ruthenicum (LRP). The extracting parameters were optimized by using three-variable-three-level Box-Behnken design and response surface methodology (RSM) based on the single-factor experiments. And the average yield of LRP was 8.25 ± 0.07%. LRP was polysaccharide-protein complex, and the total carbohydrate content and protein were 93.84% and 4.22%, respectively. It included 22.9% acidic polysaccharide and 69.6% neutral polysaccharide. Monosaccharide composition test indicated that LRP was composed of arabinose, galacturonic acid, galactose, xylose, rhamnose, glucose and mannose, and the relative molar ratio was 8.3 : 5.4 : 3.9 : 1.0 : 1.0 : 0.6 : 0.3. The antioxidant activities of LRP were investigated including scavenging activity of 2,2- diphenyl -1- picrylhydrazyl (DPPH), hydrogen peroxide and free radicals of superoxide anion in vitro. The results of antioxidant activity exhibited LRP had very strong free radical scavenging capacity and the IC50 values were 1.42 mg/mL, 1.84 mg/mL and 4.30 mg/mL, respectively. 5. L. ruthenicum anthocyanin (LRA) was isolated from the fruit of L. ruthenicum with ultrasonic ethanol extraction, ceramic membrane filtration and AB-8 macroporous resin purification. LRA was stable under acidic conditions, and the extraction, processing and storage process must be kept in the environment of avoiding light and low temperature, and avoid to contact with some metal ions (Al ³⁺ , Cu ²⁺ , Fe ³⁺ ) and strong oxidizing and reducing agents. The antioxidant capacity of L. ruthenicum. was evaluated using ORAC system. The ORAC value of dry fruit was 587 μmol TE/g. LRA was reached 5219 μmol TE/g and 5975 μmol TE/g, which extracted from dry fruit and fresh fruit. This indicated that the fruit of L. ruthenicum and LRA had strong antioxidative capacity. LRA could reduce the MDA content and increase the total SOD activity significantly, and then apoptosis was suppressed. This might be one of the mechanisms which played a protective role in the body. It could be explained that LRA protected H9C2 cardiomyocytes from H2O2-induced cell injury probably through the HO-1 mediated incremental and activation of activity with regulation of cellular redox balance．
文献类型	学位论文
条目标识符	http://210.75.249.4/handle/363003/4015
专题	中国科学院西北高原生物研究所
推荐引用方式 GB/T 7714	刘增根. 黑果枸杞遗传多样性及可持续利用研究[D]. 北京. 中国科学院研究生院,2014.

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