【作者】 侯大斌；

【导师】 任正隆；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 川乌是一种传统中药,原植物是毛茛科乌头属植物乌头Aconitum carmichaeli Debx.。乌头母根为川乌,子根为附子,具有回阳救逆、补火助阳、散寒除湿等功效,药理上具抗炎、镇痛、强心、抗心律失常、降血糖、抗癌、对心血管系统和对神经系统作用等,有二千多年的药用历史和一千多年的栽培历史,主产于四川和陕西。四川江油是其道地性产地,其产品在国内外享有很高的盛誉。但是,一直以来,对附子栽培用种频繁调种和换种缺乏统一规范管理,栽培品种多为不同遗传分化类型的混合群体,存在不同程度的混乱和混杂现象,严重影响了川乌(附子)的产品质量;同时,对川乌资源缺乏深入系统的研究,对其种质资源的遗传分化状况缺乏了解,影响了对川乌(附子)资源的合理利用和保护。本研究在调查四川、陕西等主要栽培区资源的基础上,研究了川乌(附子)生长发育特性,物质和成分累积规律,栽培措施对川乌生长的影响;开展了对川乌资源遗传多样性的研究,探讨了各群体间的遗传关系及其与近缘属种的遗传关系;运用形态学、遗传学和分子标记对川乌资源遗传分化类型进行了鉴定和分类,对优良和优势遗传分化类型进行了产量和有效成分鉴定和比较,综合评价出一批可直接运用于生产的优良品种(遗传分化类型)。本文有如下创新点: 1.川乌的发育生物学 (1) 系统研究了川乌(乌头)植株生长发育特性,生长周期内各主要器官的发生特点、生物量变化和物质累积动态规律,根、茎、叶、花、果发育建成的关键阶段,建立了主要性状生物量的生长曲线。川乌在四川12月中旬栽种后,须根首先生长,次年1～2月,须根生长量相对植株总量不断增加,是植株须根系统发育形成的关键阶段。2月中旬植株出苗,茎、叶开始生长,不定根开始发生并形成子根。3～4月植株茎、叶生长加快,子根大量形成,是植株营养器官生长的关键时期。5～6月,子根迅速膨大,根、茎、叶生长最快,生物量增长最多,是植株获得高产的关键阶段。7月中旬,叶面积、子根数、须根量等达生长周期最大值,植株开始抽苔、孕育花蕾,根、茎、叶生长和块根膨大减慢,进入生殖生长阶段;8月中旬,子根和植株生物总量达生长周期最大值;7～8月是花粉和子房发育的关键时期。9月中旬植株开花,形成果实;11月中旬后,果实成熟,蓇葖果裂开、种子脱落。根、茎、叶、花、果实等主要器官的生长和发育依次发更多还原

【Abstract】 Chuanwu(Radix Aconiti) is a Chinese traditional medical herb, whose original plant is Aconitum carmichaeli Debx. Its mother tubers are Chuanwu and child tubers are FUZI(Radix Aconiti Lateralis Preparata). Chuanwu and FUZI have such medical functions as reviving Yang for resuscitation and rescuing patients from collapse, BuHuoZhuYang, Shanhanchushi, etc. It has such pharmacological effects as anti-inflammation, easing pain, analgesic effects, cardiotonic action, anti-arrhythmia, hypoglycemic action, anti-tumor, cardiovascular system effects and nerve system effects. Aconitum carmichaeli Dexb. has more than 2000 years of medicinal history and more than 1000 years of cultivation history. Chuanwu is mainly cultivated in Sichuan and Shanxi provinces, China. Jiangyou, Sichuan is its genuineness growing place. Its medicinal products are well reputed in the world. However, it has been a long time there is not a systematic method to control the varieties adjustment and exchange of Chuanwu (FUZI) , which seriously affected the medical quality of Chuanwu. At the same time, there is not an in-depth systematic research of the genetic resource for the differentiation types of Aconitum carmichaeli Dexb., which seriously affected the utilization and protection regarding the resources of Aconitum carmichaeli Dexb. Based on these problems, this research investigated the main resources in the main planting areas in Sichuan and Shanxi, studied such characteristics as the growing processes, accumulative rule of matters and components and cultivation conditions of Aconitum carmichaeli Dexb., performed the studies on Biology and Genetic diversity of Aconitum carmichaeli, evaluated and categorized the genetic evolution of populations of Aconitum carmichaeli and its related species. Identified and categorized the Genetic resources’ differentiation typesof Aconitum carmichaeli Dexb based on Morphology and Genetics and DNA molecule markers, effectively conducted planting outputs and gradients evaluation and compared to the fine and preponderant Genetic differentiation types, and comprehensively selected several good varieties that could be directly cultivated.This dissertation has the following innovations:1. The developmental biology of Aconitum carmichaeli Dexb.(1)The paper systematically studies growing characteristics and the organ features, biological amount changes and mass accumulative principles of Aconitum carmichaeli DexbM growing and development key stages of root, stem, leaf, flower and fruit system within its life cycle and established the growing curves for the growing amounts of main organs. After planted at the middle-ten days of December in Sichuan, China, the fibrous roots grow first and the growth percent matches the plant gross growth amount continuous growth from January to February. This stage is the key stage to the fibrous roots system. After seedling, stem, leaf and adventitious root begin to develop, and the first monkshood-tuber begins to birth. The stage that the stem-leaf growth increases and lots of monkshood-tubers birth is the key stage of vegetative organ from March to April. From May to June, the monkshood-tubers are swelling rapidly, and root, stem-leaf and plant growth yield are growing very rapidly, which is the key stage of high tuber yields. In middle July, the leaf area, tuber number, fibrous root amount, etc. reach their maximum growing stage, meanwhile, the plant begins to grow flower-bud indicating the reproduction stage begins. In the middle August, the plant growing amount reaches the maximum, which is the key stage of pollen growing and germen growing from July to August. In middle September, plant begins to flower and bearing. When fruits are mature in middle November, they become cracked while the seeds break off. During Aconitum carmichaeli life cycle, the main organ growing amounts during each development stage can be effectively established as an S curve, which is cyclical pattern processes. All of these results facilitate the recognition and understanding of the basic biological characteristics, which sets up the theoretical bases for the further studies on Biology, Cultivation Science, Physiology, Genetics, Breeding Science ofA.carmichaeli Dexb..(2) Based on the biological growing features of the organs, Chuanwu’s life cycle is divided into six main phases: fibrous roots growing period, seedling growing period, stem-leaf growing and tuber birth growing period, stem-leaf rapid growing and tuber expanding period, flower-bud emerging period, and flowering and fruiting period. The six phases’ time-ordered intervals and development time of the growing life cycle was clarified and the growing features for each of the growing phases and growing-development center are analyzed in the paper.(3)The paper completely studies the main influence of such factors as mother tubers size, cutting tubers and topping sprout during Aconitum catmichaeli Dexb.’s growing phases and deeply analyzed the main factors that affect child tubers amounts. The growing amount of the main organs such as root, stem, leaf, etc. is influenced significantly by mother tuber weight. These increases of the growing amounts match the mother tuber weight increase through the establishment of a fine correlation equation. There is also a significantly positive correlation between each of the main organs. There is no significantly increase on tuber number, tuber weight, the first tuber, and the second tuber, etc., when the mother tuber weight more than 20g. The optimal tuber yield comes from the mother tubers weight (about 20g) when without top-picking and tuber-cutting, however, the mother tubers weight should be about 10g when top-picking and tuber-cutting methods were used. In order to increase FUZI weight, it is a good practice to conduct tender top-picking and to adjust the traditional from 6-8 to 12 leaves. The optimal monkshood tubers’ harvest period for FUZI is middle July if planted in plain field, and middle August to middle October if planted in a mountain area. The research findings enriched Biology, Physiology and Cultivation Science of Aconitum carmichaeli Dexb. and provided theoretical basis for Aconitum carmichaeli Dexb.’s cultivation.(4) The paper studies the formation factors and influence factors on child tubers weight of Aconitum carmichaeli Dexb., which could be applied as the theoretical basis for seeds selection and cultivation management. The effective regressionequation is as following: Y = -16.204 + 0.7Kbc, +3.505x2 + 1.134x3 -0.901x42. The Biochemistry of Aconitum carmichaeli Dexb.(5) The paper studies the dynamical changing rules of Aconitum’s effective gradients and amino acid amounts. The alkaloids amount (except aconitine) and the amino acid amount (except Arg) of child-tubers at the initial stages are higher than that at other stages, however, they are decreasing gradually in the later growing stages. The development trends of aconitine and Arg amount are reverse. The alkaloids amount and the amino acid amount of child-tubers are stable after 160 days growing. The internal synthesis of Aconitum alkaloid may start-up in meristematic cell and produce in tender and eugonic tissues. The alkaloid amount of mother tubers is decreasing in the later sequential growing stages due to the degradation and the transportation of the stored substances whereas the aconitine amount change little. The amino acids amount in leaves is decreasing in the follows growing stages and the Glu and Leu amounts are higher than other amino acids amounts. The results can provide as the basis for the quality control of FUZI plantation and also is a good reference for the continuous studies on biosynthesis mechanism of alkaloid of Aconitum within the plant.(6)The paper studies the relationships between alkaloid amount and amino acid amount of different genetic differentiation types of Aconitum and the relationships between alkaloid amount and amino acid amount of different positions in plant body. The analysis of simple correlation indicates that there is significantly positive correlation among the amount of gross alkaloid and the amounts of other nine (9) amino acids such as Asp and Glu, and the amounts of gross amino acid, Neutral amino acid and acidic amino acid. There are also obviously positive correlations between the amount of mesaconitine and the amount of His, the amount of hypaconitine and the amount of Pro, and the amount of aconitine and the amount of both of Tyr and acidic amino acids, and a highly negative correlation between the amount of aconitine and the amount of Glu and Met. Since the abundance of alkaloid and amino acids in the organs, in addition to the tubers, the leaf and fibrous roots also could be exploited to synthesize alkaloid and amino acids. Thestudied results could be applied as the reference to the further studies on internal metabolism and synthesis of Aconitum alkaloid and as the basis of comprehensive utilization of the plant of Aconitum carmichaeli Dexb.(7)The paper studies the influences of the ratio of N-P-K on the effective gradients, amino acid amounts and several enzymes vigor of Aconitum carmichaeli Dexb. N-P-K application quantity significantly influences the amounts of diesterifiable alkaloids, mesaconitine, Glu, Arg, Leu, lie and the vigor of GOT, GPT and NRA. Between each of them and N-P-K application quantity, there is a quadratic regression orthogonal equation. N-P-K application quantity also can influence the amounts of gross alkaloid, aconitine, Ser, Phe and Pro, and there is a simple regression orthogonal equation between each of them and N-P-K application quantity. All the amino acids that are influenced by N-P-K application quantity are important precursors or intermediates of internal main organic compound synthesis and metabolism. These research results could be applied as the reference for internal metabolism and the synthesis of Aconitum alkaloid and amino acid and as the basis of the ratio of N-P-K in the cultivation processes of Aconitum carmichaeli Dexb.3. The Genetics of Aconitum carmichaeli Dexb.(8) The paper studies the genetic characteristics of Aconitum chromosomes and discussed the formation processes and the mechanisms of Aconitum polyploidy. The hextraploid Aconitum carmichaeli is first discovered in southwestern China. There are a large quantity of octoploid, a few of hextraploid, and few tetraploid among both of the main planting populations and wild populations of Aconitum carmichaeli Dexb. in Southwestern China. The polyploid of subgen of Aconitum is mainly rooted from gametogamy of unreduced gamete.(9) The paper studies the genetic diversity of resources of Aconitum carmichaeli Dexb. By using RAPD, the genetic relationships among the populations of Aconitum carmichaeli Dexb. were determined, and the necessity and the advantages for breeding the elite varieties of the Aconitum carmichaeli Dexb were10analyzed. In the main planting region of A.carmichaeli, there is much more genetic diversity, more abundant germplasms and some unique resources among the wild population of A.carmichaeli Dexb. Percentages of genetic diversity among population and within population determined by Shannon index and Nei’s index are about 60% and 40%, respectively. The genetic variation of within-population was found to be relatively higher than that of among-population. The genetic diversity of planting population is higher than that of wild population, whereas the unique resource of planting population is much higher than that of wild population. For the main planting population, the genetic similarity is high and genetic distance is short. Historically, the seeds between Sichuan FUZI and Shanxi FUZI have been exchanged. The RAPD molecular marker can be applied effectively to identify the resources and pharmacognosy of A.carmichaeli and other Chinese traditional medical herbs. The results provide the theoretical basis for resources protection, application, and also provide the basis and references for genetic breeding.(10)The paper discusses the features of genetic diversity formation and changes of Aconitum carmichaeli Dexb. during cultivation and plantation or the self-sowing and evolution processes. Due to the frequent exchange of tuber-seeds, the gene flow among the population of Aconitum carmichaeli is high, which leads to the hybrid varieties of FUZI. Planting population has abundant genetic differentiation types whose genetic diversity is higher than that of wild population. Wind pollination out-crossing and insect pollination out-crossing play important roles in the sexual reproduction of wild Aconitum carmichaeli population. There is a high level of out-crossing reproduction among wild population. The out-crossing reproduction and tuber reproduction are the main causes that result in higher genetic diversity and abundant genetic differentiation types of Aconitum carmichaeli resources.(11) The paper discusses the classification of Aconitum L. and Subgen. Aconitum, and studies the relationship of the relevant species. The results confirm the traditional classification of Aconitum L. and the classification of Subgen. Aconitum and Subgen. Paraconitum. The analysis of UPGMA indicates that the geneticrelationship between Aconitum L. and Paeonia L is the most close one and the next is Ranunculus L, Anemone L and Coptis Salisb. The RAPD molecular marker can be applied effectively to research the genus relationship, but it is necessary to combine RAPD with other DNA molecular markers.4. The Breeding of Aconitum carmichaeli Dexb.(12) By applying morphology, genetics and DNA molecule markers, the paper completely studies the genetic differentiation types of Aconitum carmichaeli Dexb. resources. The results set up the basis for the further studies on genetics and breeding of Aconitum carmichaeli Dexb. There have been 265 genetic differentiation types identified from 62 germ plasm resource populations with a genetic differentiation index of 4.27. Because of affects of artificial selection in the course of planting, Sichuan population and Shanxi population’s genetic differentiation index is less than that of Chongqing population and Hubei population, planting population’s genetic differentiation index is less than wild population’s. The stronger the artificial affects are, the less the genetic differentiation index is and the less the number of genetic differentiation types is. RAPD could used to differentiate the 265 genetic differentiation types and to distinguish hextraploid from octoploid, which indicated the RAPD molecule marker can be applied effectively to identify genetic differentiation types and chromosome numbers. All the genetic differentiation types could be classified as 24 groups at the genetic similarity coefficient of 0.802 and based on these groups, six (6) cluster groups are classified.(13) The paper analyzes the formation factors of child tubers outputs and their structures, the total child tubers weight and first and second child tubers weight are used as the evaluation criteria to comprehensively evaluates the weight output and several elite materials are found which can be used as varieties directly.(14)The paper analyzes and discusses the medical quality and characteristics of FUZI by using such indices as gross alkaloids, diesterifiable alkaloids, amino acid amount, and chlorophyll amount and enzyme vigor. The paper comprehensivelyevaluates the medical quality and characteristics of dominant genetic differentiation types of Aconitum carmichaeli Dexb., and a series of FUZI varieties are found which have high alkaloids amounts, amino acid amounts, chlorophyll amount and enzyme vigor.(15) Several Chuanwu materials are selected which could be directly applied to commercial use, and is of great social and economic importance. On the basis of complete evaluation the economic output and the medicinal quality of genetic differentiation types of Chuanwu, the author selected the tuber number, tuber weight, the first and second tuber weight, gross alkaloid amount, diesterifiable alkaloids amount, amino acid amount and chlorophyll amount as the evaluation indices for a variety of genetic differentiation types of Chuanwu. By using DTOPSIS method, such fine genetic differentiation types as HacO1104-6, HacO.2106-3, Hac02102-8, HacO2126-3, etc, are identified. DTOPSIS method can be applied effectively to identify and select varieties of better Chuanwu and other Chinese traditional medical herbs, but it is necessary to pay attention to the selection of evaluation index and coefficients.(16) Through the multiple applications of Developmental Biology, Biochemistry, Genetics, Breeding and Molecular Biology, this research establishes a comprehensive new means for Chuanwu resource research, cultivation research and breeding. It is an important technical guidance for Chuanwu (FUZI) GAP formation, seed resource protection and continuous plantation, and Chinese traditional medical herb studies.更多还原