【作者】 杨艳；

【导师】 王瑞辉；

【作者基本信息】 中南林业科技大学 ， 森林培育， 2015， 博士

【摘要】 白檀(Symplocos paniculata (Thunb.)Miq.)作为新型木本油料植物,其果实含油率高达36.6%,全果利用率接近100%。其籽油内含人体必需的两种不饱和脂肪酸,即油酸和亚油酸,含量高达70%以上,而硬脂酸含量低,是较为理想、有益健康的食用植物油,也是制备生物柴油的理想原料。白檀适生能力强,可垂直分布于海拔80-2500m山地及沟谷中,又可生长于贫瘠的荒山坡地以及废弃矿区,是退化环境修复理想的先锋树种。由此可见,白檀资源的开发与利用具有良好的生态,经济和社会效益。然而,白檀至今仍处于野生状态,相关研究报道甚少,为充分开发利用这种野生资源,本文对湖南分布比较集中的6个区域进行了白檀资源调查,利用形态学标记和ISSR分子标记相结合分析白檀资源的遗传多样性,开展白檀体细胞胚繁殖技术研究,观测研究白檀果实发育及脂肪酸积累规律,为白檀种质资源遗传多样性评价系统构建、挖掘白檀优质种质资源、建立优质种质保存体系和综合利用开发体系奠定基础,其主要研究结果如下：1、完善和规范了白檀种质资源主要表型性状观测记录方法及性状评价标准,并筛选出白檀资源集中且具有代表性的6个白檀居群。2、白檀表型性状遗传多样性丰富,居群间和居群内均存在不同程度的变异。不同居群间树高与枝下高、鲜果千粒重、种子千粒重、果穗长等表型性状差异极显著,具有较强的遗传分化；叶柄长、果形指数及果实紧密度等性状间遗传分化较小；居群内,除道县白檀单株表型性状遗传分化程度较低外,其余居群内均存在不同程度的差异性。3、白檀ISSR-PCR最优反应体系为：总体积20μL, DNA模板80ng(1.6pL), Mg2+3mmol/L(2.4μL), dNTPs0.2mmol/L (1.6μL),引物0.5umol/L (1μL) Taq DNA聚合酶1.25U (0.5μL), 10×PCR Buffer (Mg2+ free)2μL,灭菌的ddH2O10.9μL。反应程序为：94℃预变性4min；94℃变性30s,54℃退火45s,72℃延伸90s,30个循环；72℃延伸7min,4℃下保存。4、利用5条多态性较高的ISSR引物对6个不同居群的68份白檀样品进行DNA多样性分析,通过PCR扩增检测到74个扩增位点,电泳条带分布在200-2500bp之间,其中多态位点73个,多态性比率高达98.65%。居群内期望杂合度分布在0.10949～0.24805之间,最高的是大围山B区(0.24805),最低的是衡山(0.10949),表明大围山居群B群内遗传多样性丰富,而衡山居群群体内遗传同一性较高。5、居群间DNA多样性聚类分析,6个居群分为三大类,第一大类为大围山A区、大围山B区和衡山居群,第二大类为岳阳和道县居群,龙山居群单独成第三大类,整体上是地理上分布近的天然居群容易聚为一类,这与表型性状的聚类分析结果基本一致：对来自不同居群的68个样株聚类,被聚为11个类群,其中,来自龙山居群的单株被分成6个小类群,即居群内遗传分化丰富。6、以白檀种子未成熟胚为外植体进行离体培养,构建了体细胞胚快繁体系。以改良MS+0.25mg/L6-BA+0.15mg/LNAA为分化诱导培养基,胚性愈伤组织分化成体细胞胚及体细胞胚的增殖率分别高达71.99%和76.56%。将成熟体细胞胚在1/2改良MS+1.5%的蔗糖培养基上培养,成苗率可达85%。7、来自不同居群的白檀果实发育及油脂积累规律基本一致,但居群间脂肪酸含量差异显著。果实形态发育可划分为3个时期：快速生长期、缓慢生长期和果皮颜色变化期。油脂快速积累开始于果实缓慢生长期,果皮颜色变化期果实油脂总含量趋于稳定,饱和与不饱和脂肪酸的相对含量发生变化。白檀果实平均含油率为34.32%,不同居群间存在差异,大围山A区含油率最高,可达36.6%,大围山B区含油率最低,仅为31.32%。8、白檀果实油脂以油体和特化油细胞的形式贮存于全果中,以中果皮、胚乳和胚三个部位分布最丰富,其脂肪酸主要组成成分为油酸、亚油酸、亚麻酸、棕榈酸及硬脂酸,其中油酸和亚油酸含量与其果实生长发育阶段日均最高气温、年日照时数等气候因子呈显著正相关性。更多还原

【Abstract】 Symplocos paniculata is a new woody oil plant, with highly fruit oil yield, oil content of fruit can reach as high as 36.6%.Utilization rate of the whole fruit can reach 100%. Symplocos paniculataadaptability is strong and can be vertical distributed between 80-2500 m in the mountains and valleys.Symplocos paniculata can also grow well in the barren mountains and land, which is the ideal tree for bioremediation. The seed oil contains two kinds of unsaturated fatty acid which are the human body essential.The two kinds of unsaturated fatty are oleic acid and linoleic acid and their content up to more than 70%. At the same time,the stearic acid content is low, so the oil of Symplocos paniculatais healthy and can be as edible vegetable oil.Meanwhile,it also can be the ideal raw material for biodiesel production.Therefore, the development and utilization of resources has good ecological, economic and social benefits. However, for a long time, Symplocos paniculata germplasm resources has been in the wild state. System evaluation of genetic diversity, improved variety breeding, germplasm conservation, fruit growth and the rule of accumulation of fatty acids is basically blank. For the full development and utilization of this wild resources, this paper lays the foundation for building Symplocos paniculata germplasm genetic diversity evaluation system, mining quality germplasm resources and establishing high quality germplasm preservation system and comprehensive utilization development system by surveying the resources which are mainly distributed in Hunan province, analysising the genetic diversity of Symplocos paniculata by using morphological markers and ISSR molecular markers,carrying out the Symplocos paniculata somatic embryo breeding technology research and studying the rule of Symplocos paniculata fruit growth and fatty acid accumulation.The main research conclusions are as follows:1.The main phenotypic traits observation and record method and character evaluation standard of Symplocos paniculata germplasm resources were improved and standardized.6 representative populations of Symplocos paniculata were selected from the regions where the Symplocos paniculata resources was concentrated.2.Symplocos paniculata in the natural population manifested rich morphological diversity. There are significant differences among populations and among individuals within populations. Phenotypical characteristic of tree body properties, fresh fruit grain, seed grain, longitudinal diameter of fruit had extremely significant difference and strong genetic differentiation.Tree form, petiole length, fruit shape index and fruit dense degree had low genetic differentiation. There were different degrees of difference within the population in addition to Symplocos paniculata from Daoxian.3.The experiment of single factor and orthogonal design was used to establish ISSR-PCR amplification system on Symplocos paniculate. An optimal reaction system contained total volume 20μL including template DNA at 80ng, Mg2+ at 3mmol/L, dNTP at 0.2mmol/L, radom primers at 0.5μmol/L, TaqDNA polymerase at 1.25U, 10×PCR Buffer at 2uL and ddH2O at 10.9μL.Then followed by 30 cycles each with pre-degeneration at 94 ℃ for 4 min, denaturation at 94 ℃ for 30 s, anneal at 54 ℃ for 45 s, extended at 72 ℃ for 90 s, extended at 72 ℃ for 7 min and preserved at 4℃. It could provide reference for genetic diversity analysis of Symplocos paniculata or similar species.4.5 primers with rich polymorphism were screened out and DNA diversity analysis was conducted within 68 Symplocos paniculata of 6 populations.74 ISSR markers were amplified from these clones with 73 polymorphic loci and polymorphism ratio was 98.65%.The expected heterozygosity (He) of 6 populations was ranged from 0.10949 to 0.24805, the maximumwas region B of Daweishan, data showed was 0.24805, the minimum was Hengshan, data showed was 0.10949. Hence, region B of Daweishan was abundant in genetic diversity and the population of Hengshan was high genetic identity.5.The results of UPGMA cluster analysis show that 6 populations were divided into three categories, the first major categories was region A and B of Daweishan and Hengshan population, the second major categories was Yueyang and Daoxian population, Longshan was separated into the third categories. The similar geographical provenance were easily clustered into one group, which was basically identical with morphological characters. Individual clustering analysis,68 samples were divided into 11 groups, basicallyaccording to geographical origin to clustering. But Longshan population was divided into 6 groups and different from others.6.The modified MS was the best basic medium for the immature embryo of Symplocos paniculata.Callus could be induced efficiently on the medium of GMS+0.2mg/L6-BA+0.1mg/LNAA,which was suitable for somatic embryogenesis. Somatic embryogenesis differentiation rate and proliferation rate on the medium of modified MS+0.25 mg/L6-BA+0.15 mg/LNAA can reach 71.99% and 76.56% respectively.The seedling percentage can reach 85% on the half of modified MS plusing 1.5% sugar.7.The rule of fruit growth in each group was consistent.The morphological development progress of fruit can generally be divided into three periods:fast growing of fruit size, slow growing of fruit size and the period of color change of pericarp.The oil accumulation began from the slow growing period and in the fruit color change period, the color of pericarp changed from green to purple. Oil content of fruit increased with the fruit growing up. At the fruit maturation period,the average oil content was 34.32%, the highest oil content was up to 36.6%(regionA of Daweishan),the lowest oil content was only 31.32%(regionB of Daweishan).8. Oil cells and parenchyma cells who have oil bodies were presented in the whole fruits and the main tissues where conducting oil accumulation were mesocarp, endosperm and embryo respectively.Fatty acid of Symplocos paniculata fruit oil was mainly composed of oleic, linoleic, linolenic,palmitic and stearic acid.There was a high correlation among oleic acid,linoleic acid and highest temperature, sunshine duration.更多还原