蚂蟥生长繁殖习性及其遗传多样性分子标记研究
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摘要
药材水蛭为蚂蟥Whitmania pigra Whitman、水蛭Hirudo nipponica Whitman和柳叶蚂蟥Whitmania acranulata Whitman的干燥品,是常用的活血化瘀药。目前心血管疾病给人类健康造成的危害已经越来越大,并已成为人类死亡的第一杀手,而以水蛭为主要原料的中成药产品有望成为这一顽症的克星,并且其抗炎作用可在饲料添加剂中进行大力开发应用。因此水蛭已成为中西药及饲料工业不可缺少的原料,且随着社会发展及其相关产品的开发,其需求量仍将快速增长,而野生资源却日益枯竭致使供需矛盾非常突出。近几年,水蛭已成为世界性的紧俏中药材之一。1984年水蛭被列入动物保护国际红皮书。保护现有的蚂蟥野生资源已成当务之急,为此蚂蟥种质资源亟待整理和评价,以便为蚂蟥核心种质资源库的建立提供有力保障。
本研究首先从蚂蟥内脏结构、皮肤表皮细胞类型的分布等形态学研究开始,进一步研究了蚂蟥的生理、生长、繁殖习性和药材中农残、重金属的积累,并对不同炮制品、不同种质的蚂蟥的内在质量进行系统的评价。在对其生理、生长、繁殖习性研究的基础上,收集了我国蚂蟥分布区部分种质资源,同时引入4个水蛭H.nipponica种群作为参照,在分子水平分析了蚂蟥遗传多样性,了解其群体遗传结构和多态性水平,并进行了类群的划分,为蚂蟥和水蛭的资源保护利用策略和人工选育提供有力的理论依据。其次,采用两种方法开发了蚂蟥的微卫星序列,并分析了其特点,为SSR分子标记在蚂蟥遗传多样性研究中的应用和蚂蟥种质资源的鉴定奠定了基础。具体研究内容和结果如下:
1.蚂蟥生长繁殖习性及药材质量评价研究
(1)应用组织石蜡切片和显微摄影方法,对蚂蟥内脏器官,包括口、咽、食管、嗉囊、肠、直肠、侧盲囊、精巢、储精囊、输精管、射精球、前列腺、皮肤等进行解剖观察分析。明确了蚂蟥的消化系统、生殖系统器官和皮肤的组织结构和作用,皮肤黏液细胞的类型与分布。为进一步研究蚂蟥的生长、繁殖习性和人工养殖及种质鉴别提供参考依据。
(2)应用呼吸室法研究了蚂蟥的耗氧率、耗氧量和窒息点。结果表明平均体重为10g的蚂蟥,15～35℃变温条件下耗氧率变化在0.049～0.094mg·g~(-1)·h~(-1)之间,耗氧量在0.44～0.67mg·p~(-1)·h~(-1)之间,窒息点为0.90～1.51mg·L~(-1),20℃条件下耗氧率为0.044～0.058mg·g~(-1)·h~(-1),耗氧量为0.19～0.77mg·p~(-1)·h~(-1),窒息点为1.40～1.57mg·L~(-1)。说明蚂蟥的耗氧率随温度的升高而上升,窒息点随温度的升高而降低,体重与耗氧率呈负相关,与耗氧量呈正相关,白昼和夜晚耗氧率的差异不大。
(3)研究了在不同温度条件下不同体重蚂蟥生长摄食情况,以及蚂蟥在24h内的摄食期律。结果表明蚂蟥生长的适宜温度是15～25℃,其饱食量随着体重增大和温度的升高而增加,摄食率随个体增大而降低,在24h之内蚂蟥有两个摄食高峰。
(4)在不同温度、体重条件下观察了蚂蟥产卵和孵化习性。结果表明,蚂蟥产卵和孵化的最适温度为25℃,产卵的最佳体重为20g左右;温度能影响蚂蟥产卵前后的体重,而体重大小对产卵前后体重变化影响不明显;当卵重在0.1～2.0g之间时与孵出量成正比,当卵重大于2.0g时孵出量反而有所下降。
(5)采用原子吸收分光光度计、原子荧光分光光度计和气相色谱仪对蚂蟥、基地土壤和养殖用水的重金属与农药残留进行了比较分析。结果表明除蚂蟥药材中铅超出国家现行有关标准外,蚂蟥药材、养殖基地土壤和养殖用水六六六、DDT和重金属残留各项指标均符合国家规定标准。建议今后在制订国家有关标准时,将类似蚂蟥类等动物的安全限量单独列出。
(6)采用《药典》一部(2005版)的方法测定了野生和人工养殖蚂蟥不同炮制品的水分、醇溶性浸出物、总灰分、酸不溶性灰分、抗凝血酶活性等指标,用电感耦合等离子体发射光谱仪测定了铁、锌、铜、铬、铅、镉、汞等元素,比较了野生和人工养殖蚂蟥不同炮制品的内在质量。结果表明,水分:人工养殖与野生炮制品两者之间差异不显著(P＞0.05);总灰分:滑石粉烫炮制品最高,与其它炮制品差异显著(P＜0.05);酸不溶性灰分:滑石粉烫炮制品最高,与其他炮制品差异不显著(P＞0.05);醇溶性浸出物:野生炮制品与人工养殖炮制品差异显著(P＜0.05);抗凝血酶活性:野生和人工养殖生晒品均达到药典标准,且二者之间差异不显著(P＞0.05);人工养殖及野生蚂蟥不同炮制品中除铅超标外,其余金属元素含量均符合国家标准。
(7)参考《药典》一部(2005版)测定了不同种群蚂蟥的水分、醇溶性浸出物、总灰分、酸不溶性灰分、抗凝血酶活性等指标,采用高效液相色谱法测定了其黄嘌呤,次黄嘌呤。结果表明南京人工养殖种群在水分、醇溶性浸出物、总灰分、酸不溶性灰分、抗凝血酶活性、黄嘌呤、次黄嘌呤等成分上均相对高于其他种群。
2.利用分子标记技术分析我国蚂蟥种质资源遗传多样性
(1)以全国蚂蟥主要分布区的8个省15份种质225个样本进行遗传多样性分析。利用RAPD标记技术分析,结果表明在物种水平上,多态位点百分率(PPL)为99.66%,Nei'S基因多样性指数(He)为0.3599。种群水平上的多态位点百分率在30.30%～56.06%之间,平均为44.14%,最高为安徽马鞍山,其次为江苏溧阳,最低为南京人工养殖。群体间遗传分化系数(G_(ST))为0.5539,种群间基因流(Nm)为0.4028。数据显示种群间分化较大,种群内存在一定程度变异。聚类分析显示,当相似系数为0.80时,可将15份材料分为8个类群,其中广西桂林、广东广州、云南大理、安徽马鞍山四个医蛭种群与其它大部分蚂蟥种群相似性较低。
(2)利用ISSR分子标记技术分析了蚂蟥遗传多样性,结果表明,在物种水平上,PPL为99.56%,He为0.3490。种群水平上的PPL在25.00%～53.95%之间,平均为37.73%,最高为广西桂林,其次为广东广州,最低为江苏南京人工养殖。G_(ST)为0.5730,Nm为0.3727。结果也表明蚂蟥种群间存在较高水平遗传分化,种群内也存在一定的遗传变异。聚类分析表明,当相似系数为0.78时,可将供试的15个蚂蟥种群分为5个类群。
(3)SRAP标记是一种新型技术,本文分析SRAP标记在蚂蟥遗传多样性研究上的适用性。结果表明在物种水平上,PPL为99.02%,He为0.3687。种群水平上PPL在31.69%～58.86%之间,平均为50.59%,最高为江西乐安,其次为江苏溧阳,最低为江苏南京人工养殖。G_(ST)为0.4699,Nm为0.5641。聚类分析显示,当相似系数为0.82时,可将15个种群分为6个类群。将SRAP标记结果同RAPD和ISSR结果分析比较,认为该新型标记可以用于蚂蟥遗传多样性研究。
(4)通过测定蚂蟥核糖体ITS碱基序列,分析其特点,了解种内变异类型。结果得到我国蚂蟥主要的14个种群14个样本rDNA中的ITS完全序列。ITS长度为857～863bp。ITS碱基频率差异显著,ITS较为保守。根据两者序列以邻接法建立分子系统发生树,表明蚂蟥种内至少存在3个变异类型。结合RAPD、ISSR和SRAP三种分子标记的研究结果,可将供试材料分为三个等级加以保护和利用。
3.蚂蟥微卫星(SSR)的开发
SSR标记具有共显性特点,开发蚂蟥微卫星对于遗传多样性研究具有重要意义。通过分析评价两种有代表性的SSR标记开发方法,为微卫星开发方法的选择提供参考信息。在现有方法基础上提出蚂蟥微卫星序列开发的两种策略。
(1)根据链亲和素磁珠和生物素特异结合的特性,将微卫星探针5'端生物素化后与链亲和素磁珠特异结合,用磁珠和探针的结合物与两端连接已知序列人工接头的蚂蟥基因组DNA酶切片段杂交,洗脱未杂交DNA片断后,建立微卫星文库。以此为模板用人工接头序列为引物进行PCR扩增,产物直接克隆,经菌液PCR筛选后测序分析。
(2)将特异接头连接到限制性内切酶消化的基因组DNA上,以此为模板用简并引物抑制性PCR获得SSR文库,克隆后经过两次菌液PCR筛选进行测序分析。结果表明,这两种方法可以快速、高效地开发出微卫星序列。
(3)结合前面从基因组DNA中已开发的微卫星序列,分析了蚂蟥SSR特点。结果发现二核苷酸重复类型在蚂蟥SSR占绝对优势,出现频率近93%,二核苷酸重复中以(AC)_n重复基元为主,达53.33%。此外,对SSR位点总共设计了14对引物,其中11对经检测有效可用于蚂蟥遗传多样性分析。
Medicinal leech,including Whitmania pigra Whitman,Hirudo nipponica Whitman and Whitmania acranulata Whitman,has been used in clinical practice for blood circulation promoting and stasis relieving.At present,cardiovascular disease,which has been the first killer to the human health,is expected to be cured by the Chinese Patent Medicine using Leech as major raw materials.It can be carried out and deeply applied in animal feed additives on anti-inflammatory effect.Leech has become indispensable raw materials of Western medicine.and feed industry.With the development of society and research of its related products,leech will in great need.The contradiction between supply and demand appears because of the deteriorating of the wild resources,leading to a worldwide shortage. Leech was included in the list of 1983 IUCN Invertebrate Red Data Book.It is urgent to protect existing leech wildlife resources,so,location,collation and evaluation germplasm resources of Hirudo is becoming an important task.
Firstly,we studied the morphological structure of offal and the distribution of skin epidermal cell types of W.pigra.Then,we investigated the physiology,growth, reproductive habit,and residues of pesticide and heavy metals,and also systemically evaluated the inherent quality of different processing drugs and germplasm of W.pigra. Based on the research of the physiology,growth and reproductive habits,we sampled parts of germplasm resources of W.pigra in the distributed places of China and made the four H. nipponica population as reference.We analyzed the genetic diversity,population genetic structure and polymorphism of W.pigra and established a system of classification,which provided a strong theoretical basis for the protection and use of the W.pigra and H. nipponica resource.Secondly,we successfully isolated microsatellite sequence of W.pigra using two methods and develop its SSR markers application in W.pigra.The detail contents and results are listed below:
1.Growth aquaculture habits and quality assessment research
(1) Using the methods of paraffin-embedded myocardial specimens and photomicrography,we dissected and analyzed the visceral organ of W.pigra,including of cavity,swallow,esophagus,crop,bowel,rectum,parasomal sac stomach,testicle,seminal vesicle,vasa deferentia duct,ejaculatory ball,prostate gland and skin.As a results,we definited the Digestive System and Reproductive System of W.pigra,we also obtained the knowledge of structure and function of the skin and the organ framework of the digestive and reproductive system,as well as the types and distribution of the mucous cells,which provided information for further study on the living behaviour,reproductive habit,artificial aquaculture environment and species identification of W.pigra.
(2) Study the oxygen consumption rate,oxygen demand and suffocation point of W. pigra by the method of respiratory chamber.The result showed that the oxygen consumption rate(OCR) ranged from 0.049 to 0.094mg·g~(-1)·h~(-1)、the oxygen consumption volume(OCV) from 0.44 to 0.67mg·p~(-1)·h~(-1),and suffocation point(AP) from 0.9 to 1.51 mg·L~(-1),when the average weight of W.Pigra was 10g and the water temperature varied from 15 to 35℃.OCR ranged from 0.044 to 0.058mg·g~(-1)·h~(-1)、OCV from 0.19 to 0.77 mg·p~(-1)·h~(-1) and AP from 1.4 to 1.57mg·L~(-1),when water temperature was 20℃.We make the conclusion that the OCR will rise as the water temperature increased,but the AP was opposite.The weight was negatively correlated with OCR and positively with OCV.OCR had little variance from day to night.
(3) Comparison of the growth of W.pigra in different weight and temperature conditions,and study on the intake habit of W.pigra in 24h.The results show that the appropriate temperature for W.pigra growth is 15～25℃.And with the increase of the weight and the rise of temperature,the intake rate increased.The intake demand reduced with the increase of weight.W.pigra has two intake peaks in 24 hours.
(4) Observation the spawning and hatch of W.pigra in different temperature and weight,and determination the best spawning temperature and spawning weight.As a result, we found the most appropriate temperature for W.pigra spawning and incubation is 25℃, and the best spawning weight is 20g.Temperature can influence the weight before and after spawning,but the weight have no obvious effect.When the spawning weight range from 0.1～2.0g,it has the positive correlation with hatch amount,but when the spawning weight exceeds 2.0g,hatch amount will reduce.
(5) Analyse the content of heavy metals and pesticide residues for soil and aquaculture water in W.pigra with the help of atomic absorption spectrophotometers,atomic fluorescence spectrophotometers and gas chromatography.The results showed that exception Pb,other indexs including aquaculture base of soil and aquaculture water HCH, DDT and heavy metals residues indicators are consistent with national standards in W. pigra.And we suggested that the animals similar to W.pigra should be separately listed in the national interrelated standards.
(6) Determine the moisture,alcohol extract,total ash,acid-insoluble ash,antiplatelet aggregation enzyme of the wild and the artificial aquaculture W.pigra using the method referred in the book(2005 edition).Mineral elements including iron,zinc,copper,chrome,lead,cadmium and Hg etc.are identified by the method of ICP-MS(Inductive Coupling Plasma Launching).In the results,in the parts of water there is no obvious difference between wild and artifical processing drugs(P＜0.05). The talcum powder bums have the highest content of total ash,which has obvious difference with other processing drugs(P＜0.05).Acid-insoluble ash:The talcum powder bums＞lives exposes to the sun＞the liquor product;alcohol extract:The talcum powder burnsz＞the liquor product＞to live exposes to the sun;antiplatelet aggregation enzyme:Lives exposes to the sun＞the liquor product＞the talcum powder to burn; Apart from artificial aquaculture W.pigra moisture and antithrombin higher than which in the wild,but have no significant difference(P＜0.05),the rest were lower than in the wild. The activity of antiplatelet aggregation enzyme in domestic W.pigra is higher than those in wild ones and has no significant difference(P＜0.05).But total ash,acid-insoluble ash and alcohol extract axe less than the wild.
(7) Analysis the moisture,alcohol extract,total ash,acid-insoluble ash,antiplatelet aggregation enzyme of concocting wild and the artificial aquaculture W.pigra using the methods consulted with the(2005 edition),and analysis the Xanthine and Hypoxanthine by HPLC.The results showed that the moisture,alcohol extract,total ash,acid-insoluble ash,antiplatelet aggregation enzyme,Xanthine and Hypoxanthine of artificial aquaculture population are better than other population.
2.Analysis of the genetic diversity of W.pigra germplasm resources in China using Molecular marker technology
(1) 225 samples from 15 germplasm resources distributed in 8 provinces were used to analyze genetics deiversity.Based on analysis of RAPD technology,the results showed that at the level of species the polymorphic loci percentage(PPL) is 99.66%,Nei'S gene diversity index(He) is 0.3599.At the population level,PPL is 30.30%～56.06%,averaging at 44.14%,in which the highest population is in Maanshan,followed by the Liyang population,Nanjing cultivated,the lowest of population.Genetic differentiation factor(G_(ST)) within groups is 0.5487,population gene flow(Nm) is 0.4028.Data shows that inter-population has more diversity,and intra-population has a certain extent variation. Cluster analysis revealed that when the similar coefficient is 0.80,15 materials can be divided into eight groups,in which Guangzhou,Guilin,Dali and Maanshan four population have lower similar to most of the other population.
(2) Analysed genetic diversity of W.pigra using SSR molecule marker technology. The results showed that at the species level PPL is of 99.56%,He is 0.3490.At the population level PPL is in 25.00%～53.95%,averaging at 37.73%,highest is the Guilin population,followed by Guangzhou,Nanjing cultivated the lowest population.G_(ST) is to 0.5730,and Nm of 0.3727.The results also showed that intm-population has much differentiation,and the inter-population has a certain level of genetic differentiation.Cluster analysis showed that as the coefficient is 0.78,15 population of W.pigra were divided into five groups.
(3) SRAP marker is a new technology.In this paper,we analysed the SRAP marker applicability in research of the genetic diversity of W.pigra.The results showed that at the species level,PPL is 99.02%,He is 0.3687.While at the population level PPL is within 31.69%～58.86%,averaging at 50.59%.the highest population is in Le'an,followed by Liyang,Nanjing cultivated is the lowest population.G_(ST) is to 0.4699,Nm is 0.5641.Cluster analysis revealed that as coefficient of 0.82,15 population were divided into 6 groups.By comparing SRAP,RAPD,ISSR data,the new technology can be used in W.pigra genetic structure study.
(4) To understand variation type within species,we tested the ribosomal ITS base sequences and analysed the characteristics of them.We obtained rDNA ITS complete sequences from 14 samples of 14 main W.pigra and H.nipponica population.The length of ITS was 857～863bp.The base frequence has more variation.ITS has more conservative.It constructed the phylogenetic tree using Neighbor-adjacent method,showing that there are at least 3 variations in W.pigra.Combined with RAFD,ISSR,and SRAP molecular markers data,it was suggested that 14 population should be divided into four grades to be protected and utilized.
3.Microsatellite exploiture of W.pigra
The main strategies of microsatellites development were reviewed in the paper. Analysis and evaluation of several representative methods are helpful to develop Micro-satellite method.Two micro-satellites development strategies for W.pigra were provided based on the existing methods.
(1) First method:Because of the strong affinity of biotin for streptavidin,the 5' end biotin of microsatellite probe was combined with magnesphere paramagnetic particles,and then combinations hybridization with the digested W.pigra DNA fragments both ends of them connected with special adaptor.Elution of the other DNA fragments,the micro-satellite library was established.The adaptors as PCR primers clone the products direct,and screening the results by technology of bacteria PCR amplification in the end.
(2) Second method:Connected the special adaptors to digested genomic DNA fragments,then make it as a template.The microsatellite library was established by inhibition PCR with mergers primer.Then analyze results of clone sequence after two screening by bacteria PCR.The results show that the two methods can quickly and efficiently be used to develop micro-satellite method.
(3) Finally,combining the isolated micro-satellites sequences from genome DNA,we analysis the characteristics of W.pigra.As a result,we found the dinueleotide repeat type in W.pigra SSR has absolute advantage,frequency nearly reached 93%.Dinucleotide repeat motif(AC)_n is up to 53.33%.In addition,14 pairs of SSR primers were designed,11 pairs of which can be used to effectively detect the genetic diversity of W.pigra.
本研究首先从蚂蟥内脏结构、皮肤表皮细胞类型的分布等形态学研究开始,进一步研究了蚂蟥的生理、生长、繁殖习性和药材中农残、重金属的积累,并对不同炮制品、不同种质的蚂蟥的内在质量进行系统的评价。在对其生理、生长、繁殖习性研究的基础上,收集了我国蚂蟥分布区部分种质资源,同时引入4个水蛭H.nipponica种群作为参照,在分子水平分析了蚂蟥遗传多样性,了解其群体遗传结构和多态性水平,并进行了类群的划分,为蚂蟥和水蛭的资源保护利用策略和人工选育提供有力的理论依据。其次,采用两种方法开发了蚂蟥的微卫星序列,并分析了其特点,为SSR分子标记在蚂蟥遗传多样性研究中的应用和蚂蟥种质资源的鉴定奠定了基础。具体研究内容和结果如下:
1.蚂蟥生长繁殖习性及药材质量评价研究
(1)应用组织石蜡切片和显微摄影方法,对蚂蟥内脏器官,包括口、咽、食管、嗉囊、肠、直肠、侧盲囊、精巢、储精囊、输精管、射精球、前列腺、皮肤等进行解剖观察分析。明确了蚂蟥的消化系统、生殖系统器官和皮肤的组织结构和作用,皮肤黏液细胞的类型与分布。为进一步研究蚂蟥的生长、繁殖习性和人工养殖及种质鉴别提供参考依据。
(2)应用呼吸室法研究了蚂蟥的耗氧率、耗氧量和窒息点。结果表明平均体重为10g的蚂蟥,15～35℃变温条件下耗氧率变化在0.049～0.094mg·g~(-1)·h~(-1)之间,耗氧量在0.44～0.67mg·p~(-1)·h~(-1)之间,窒息点为0.90～1.51mg·L~(-1),20℃条件下耗氧率为0.044～0.058mg·g~(-1)·h~(-1),耗氧量为0.19～0.77mg·p~(-1)·h~(-1),窒息点为1.40～1.57mg·L~(-1)。说明蚂蟥的耗氧率随温度的升高而上升,窒息点随温度的升高而降低,体重与耗氧率呈负相关,与耗氧量呈正相关,白昼和夜晚耗氧率的差异不大。
(3)研究了在不同温度条件下不同体重蚂蟥生长摄食情况,以及蚂蟥在24h内的摄食期律。结果表明蚂蟥生长的适宜温度是15～25℃,其饱食量随着体重增大和温度的升高而增加,摄食率随个体增大而降低,在24h之内蚂蟥有两个摄食高峰。
(4)在不同温度、体重条件下观察了蚂蟥产卵和孵化习性。结果表明,蚂蟥产卵和孵化的最适温度为25℃,产卵的最佳体重为20g左右;温度能影响蚂蟥产卵前后的体重,而体重大小对产卵前后体重变化影响不明显;当卵重在0.1～2.0g之间时与孵出量成正比,当卵重大于2.0g时孵出量反而有所下降。
(5)采用原子吸收分光光度计、原子荧光分光光度计和气相色谱仪对蚂蟥、基地土壤和养殖用水的重金属与农药残留进行了比较分析。结果表明除蚂蟥药材中铅超出国家现行有关标准外,蚂蟥药材、养殖基地土壤和养殖用水六六六、DDT和重金属残留各项指标均符合国家规定标准。建议今后在制订国家有关标准时,将类似蚂蟥类等动物的安全限量单独列出。
(6)采用《药典》一部(2005版)的方法测定了野生和人工养殖蚂蟥不同炮制品的水分、醇溶性浸出物、总灰分、酸不溶性灰分、抗凝血酶活性等指标,用电感耦合等离子体发射光谱仪测定了铁、锌、铜、铬、铅、镉、汞等元素,比较了野生和人工养殖蚂蟥不同炮制品的内在质量。结果表明,水分:人工养殖与野生炮制品两者之间差异不显著(P＞0.05);总灰分:滑石粉烫炮制品最高,与其它炮制品差异显著(P＜0.05);酸不溶性灰分:滑石粉烫炮制品最高,与其他炮制品差异不显著(P＞0.05);醇溶性浸出物:野生炮制品与人工养殖炮制品差异显著(P＜0.05);抗凝血酶活性:野生和人工养殖生晒品均达到药典标准,且二者之间差异不显著(P＞0.05);人工养殖及野生蚂蟥不同炮制品中除铅超标外,其余金属元素含量均符合国家标准。
(7)参考《药典》一部(2005版)测定了不同种群蚂蟥的水分、醇溶性浸出物、总灰分、酸不溶性灰分、抗凝血酶活性等指标,采用高效液相色谱法测定了其黄嘌呤,次黄嘌呤。结果表明南京人工养殖种群在水分、醇溶性浸出物、总灰分、酸不溶性灰分、抗凝血酶活性、黄嘌呤、次黄嘌呤等成分上均相对高于其他种群。
2.利用分子标记技术分析我国蚂蟥种质资源遗传多样性
(1)以全国蚂蟥主要分布区的8个省15份种质225个样本进行遗传多样性分析。利用RAPD标记技术分析,结果表明在物种水平上,多态位点百分率(PPL)为99.66%,Nei'S基因多样性指数(He)为0.3599。种群水平上的多态位点百分率在30.30%～56.06%之间,平均为44.14%,最高为安徽马鞍山,其次为江苏溧阳,最低为南京人工养殖。群体间遗传分化系数(G_(ST))为0.5539,种群间基因流(Nm)为0.4028。数据显示种群间分化较大,种群内存在一定程度变异。聚类分析显示,当相似系数为0.80时,可将15份材料分为8个类群,其中广西桂林、广东广州、云南大理、安徽马鞍山四个医蛭种群与其它大部分蚂蟥种群相似性较低。
(2)利用ISSR分子标记技术分析了蚂蟥遗传多样性,结果表明,在物种水平上,PPL为99.56%,He为0.3490。种群水平上的PPL在25.00%～53.95%之间,平均为37.73%,最高为广西桂林,其次为广东广州,最低为江苏南京人工养殖。G_(ST)为0.5730,Nm为0.3727。结果也表明蚂蟥种群间存在较高水平遗传分化,种群内也存在一定的遗传变异。聚类分析表明,当相似系数为0.78时,可将供试的15个蚂蟥种群分为5个类群。
(3)SRAP标记是一种新型技术,本文分析SRAP标记在蚂蟥遗传多样性研究上的适用性。结果表明在物种水平上,PPL为99.02%,He为0.3687。种群水平上PPL在31.69%～58.86%之间,平均为50.59%,最高为江西乐安,其次为江苏溧阳,最低为江苏南京人工养殖。G_(ST)为0.4699,Nm为0.5641。聚类分析显示,当相似系数为0.82时,可将15个种群分为6个类群。将SRAP标记结果同RAPD和ISSR结果分析比较,认为该新型标记可以用于蚂蟥遗传多样性研究。
(4)通过测定蚂蟥核糖体ITS碱基序列,分析其特点,了解种内变异类型。结果得到我国蚂蟥主要的14个种群14个样本rDNA中的ITS完全序列。ITS长度为857～863bp。ITS碱基频率差异显著,ITS较为保守。根据两者序列以邻接法建立分子系统发生树,表明蚂蟥种内至少存在3个变异类型。结合RAPD、ISSR和SRAP三种分子标记的研究结果,可将供试材料分为三个等级加以保护和利用。
3.蚂蟥微卫星(SSR)的开发
SSR标记具有共显性特点,开发蚂蟥微卫星对于遗传多样性研究具有重要意义。通过分析评价两种有代表性的SSR标记开发方法,为微卫星开发方法的选择提供参考信息。在现有方法基础上提出蚂蟥微卫星序列开发的两种策略。
(1)根据链亲和素磁珠和生物素特异结合的特性,将微卫星探针5'端生物素化后与链亲和素磁珠特异结合,用磁珠和探针的结合物与两端连接已知序列人工接头的蚂蟥基因组DNA酶切片段杂交,洗脱未杂交DNA片断后,建立微卫星文库。以此为模板用人工接头序列为引物进行PCR扩增,产物直接克隆,经菌液PCR筛选后测序分析。
(2)将特异接头连接到限制性内切酶消化的基因组DNA上,以此为模板用简并引物抑制性PCR获得SSR文库,克隆后经过两次菌液PCR筛选进行测序分析。结果表明,这两种方法可以快速、高效地开发出微卫星序列。
(3)结合前面从基因组DNA中已开发的微卫星序列,分析了蚂蟥SSR特点。结果发现二核苷酸重复类型在蚂蟥SSR占绝对优势,出现频率近93%,二核苷酸重复中以(AC)_n重复基元为主,达53.33%。此外,对SSR位点总共设计了14对引物,其中11对经检测有效可用于蚂蟥遗传多样性分析。
Medicinal leech,including Whitmania pigra Whitman,Hirudo nipponica Whitman and Whitmania acranulata Whitman,has been used in clinical practice for blood circulation promoting and stasis relieving.At present,cardiovascular disease,which has been the first killer to the human health,is expected to be cured by the Chinese Patent Medicine using Leech as major raw materials.It can be carried out and deeply applied in animal feed additives on anti-inflammatory effect.Leech has become indispensable raw materials of Western medicine.and feed industry.With the development of society and research of its related products,leech will in great need.The contradiction between supply and demand appears because of the deteriorating of the wild resources,leading to a worldwide shortage. Leech was included in the list of 1983 IUCN Invertebrate Red Data Book.It is urgent to protect existing leech wildlife resources,so,location,collation and evaluation germplasm resources of Hirudo is becoming an important task.
Firstly,we studied the morphological structure of offal and the distribution of skin epidermal cell types of W.pigra.Then,we investigated the physiology,growth, reproductive habit,and residues of pesticide and heavy metals,and also systemically evaluated the inherent quality of different processing drugs and germplasm of W.pigra. Based on the research of the physiology,growth and reproductive habits,we sampled parts of germplasm resources of W.pigra in the distributed places of China and made the four H. nipponica population as reference.We analyzed the genetic diversity,population genetic structure and polymorphism of W.pigra and established a system of classification,which provided a strong theoretical basis for the protection and use of the W.pigra and H. nipponica resource.Secondly,we successfully isolated microsatellite sequence of W.pigra using two methods and develop its SSR markers application in W.pigra.The detail contents and results are listed below:
1.Growth aquaculture habits and quality assessment research
(1) Using the methods of paraffin-embedded myocardial specimens and photomicrography,we dissected and analyzed the visceral organ of W.pigra,including of cavity,swallow,esophagus,crop,bowel,rectum,parasomal sac stomach,testicle,seminal vesicle,vasa deferentia duct,ejaculatory ball,prostate gland and skin.As a results,we definited the Digestive System and Reproductive System of W.pigra,we also obtained the knowledge of structure and function of the skin and the organ framework of the digestive and reproductive system,as well as the types and distribution of the mucous cells,which provided information for further study on the living behaviour,reproductive habit,artificial aquaculture environment and species identification of W.pigra.
(2) Study the oxygen consumption rate,oxygen demand and suffocation point of W. pigra by the method of respiratory chamber.The result showed that the oxygen consumption rate(OCR) ranged from 0.049 to 0.094mg·g~(-1)·h~(-1)、the oxygen consumption volume(OCV) from 0.44 to 0.67mg·p~(-1)·h~(-1),and suffocation point(AP) from 0.9 to 1.51 mg·L~(-1),when the average weight of W.Pigra was 10g and the water temperature varied from 15 to 35℃.OCR ranged from 0.044 to 0.058mg·g~(-1)·h~(-1)、OCV from 0.19 to 0.77 mg·p~(-1)·h~(-1) and AP from 1.4 to 1.57mg·L~(-1),when water temperature was 20℃.We make the conclusion that the OCR will rise as the water temperature increased,but the AP was opposite.The weight was negatively correlated with OCR and positively with OCV.OCR had little variance from day to night.
(3) Comparison of the growth of W.pigra in different weight and temperature conditions,and study on the intake habit of W.pigra in 24h.The results show that the appropriate temperature for W.pigra growth is 15～25℃.And with the increase of the weight and the rise of temperature,the intake rate increased.The intake demand reduced with the increase of weight.W.pigra has two intake peaks in 24 hours.
(4) Observation the spawning and hatch of W.pigra in different temperature and weight,and determination the best spawning temperature and spawning weight.As a result, we found the most appropriate temperature for W.pigra spawning and incubation is 25℃, and the best spawning weight is 20g.Temperature can influence the weight before and after spawning,but the weight have no obvious effect.When the spawning weight range from 0.1～2.0g,it has the positive correlation with hatch amount,but when the spawning weight exceeds 2.0g,hatch amount will reduce.
(5) Analyse the content of heavy metals and pesticide residues for soil and aquaculture water in W.pigra with the help of atomic absorption spectrophotometers,atomic fluorescence spectrophotometers and gas chromatography.The results showed that exception Pb,other indexs including aquaculture base of soil and aquaculture water HCH, DDT and heavy metals residues indicators are consistent with national standards in W. pigra.And we suggested that the animals similar to W.pigra should be separately listed in the national interrelated standards.
(6) Determine the moisture,alcohol extract,total ash,acid-insoluble ash,antiplatelet aggregation enzyme of the wild and the artificial aquaculture W.pigra using the method referred in the book
(7) Analysis the moisture,alcohol extract,total ash,acid-insoluble ash,antiplatelet aggregation enzyme of concocting wild and the artificial aquaculture W.pigra using the methods consulted with the
2.Analysis of the genetic diversity of W.pigra germplasm resources in China using Molecular marker technology
(1) 225 samples from 15 germplasm resources distributed in 8 provinces were used to analyze genetics deiversity.Based on analysis of RAPD technology,the results showed that at the level of species the polymorphic loci percentage(PPL) is 99.66%,Nei'S gene diversity index(He) is 0.3599.At the population level,PPL is 30.30%～56.06%,averaging at 44.14%,in which the highest population is in Maanshan,followed by the Liyang population,Nanjing cultivated,the lowest of population.Genetic differentiation factor(G_(ST)) within groups is 0.5487,population gene flow(Nm) is 0.4028.Data shows that inter-population has more diversity,and intra-population has a certain extent variation. Cluster analysis revealed that when the similar coefficient is 0.80,15 materials can be divided into eight groups,in which Guangzhou,Guilin,Dali and Maanshan four population have lower similar to most of the other population.
(2) Analysed genetic diversity of W.pigra using SSR molecule marker technology. The results showed that at the species level PPL is of 99.56%,He is 0.3490.At the population level PPL is in 25.00%～53.95%,averaging at 37.73%,highest is the Guilin population,followed by Guangzhou,Nanjing cultivated the lowest population.G_(ST) is to 0.5730,and Nm of 0.3727.The results also showed that intm-population has much differentiation,and the inter-population has a certain level of genetic differentiation.Cluster analysis showed that as the coefficient is 0.78,15 population of W.pigra were divided into five groups.
(3) SRAP marker is a new technology.In this paper,we analysed the SRAP marker applicability in research of the genetic diversity of W.pigra.The results showed that at the species level,PPL is 99.02%,He is 0.3687.While at the population level PPL is within 31.69%～58.86%,averaging at 50.59%.the highest population is in Le'an,followed by Liyang,Nanjing cultivated is the lowest population.G_(ST) is to 0.4699,Nm is 0.5641.Cluster analysis revealed that as coefficient of 0.82,15 population were divided into 6 groups.By comparing SRAP,RAPD,ISSR data,the new technology can be used in W.pigra genetic structure study.
(4) To understand variation type within species,we tested the ribosomal ITS base sequences and analysed the characteristics of them.We obtained rDNA ITS complete sequences from 14 samples of 14 main W.pigra and H.nipponica population.The length of ITS was 857～863bp.The base frequence has more variation.ITS has more conservative.It constructed the phylogenetic tree using Neighbor-adjacent method,showing that there are at least 3 variations in W.pigra.Combined with RAFD,ISSR,and SRAP molecular markers data,it was suggested that 14 population should be divided into four grades to be protected and utilized.
3.Microsatellite exploiture of W.pigra
The main strategies of microsatellites development were reviewed in the paper. Analysis and evaluation of several representative methods are helpful to develop Micro-satellite method.Two micro-satellites development strategies for W.pigra were provided based on the existing methods.
(1) First method:Because of the strong affinity of biotin for streptavidin,the 5' end biotin of microsatellite probe was combined with magnesphere paramagnetic particles,and then combinations hybridization with the digested W.pigra DNA fragments both ends of them connected with special adaptor.Elution of the other DNA fragments,the micro-satellite library was established.The adaptors as PCR primers clone the products direct,and screening the results by technology of bacteria PCR amplification in the end.
(2) Second method:Connected the special adaptors to digested genomic DNA fragments,then make it as a template.The microsatellite library was established by inhibition PCR with mergers primer.Then analyze results of clone sequence after two screening by bacteria PCR.The results show that the two methods can quickly and efficiently be used to develop micro-satellite method.
(3) Finally,combining the isolated micro-satellites sequences from genome DNA,we analysis the characteristics of W.pigra.As a result,we found the dinueleotide repeat type in W.pigra SSR has absolute advantage,frequency nearly reached 93%.Dinucleotide repeat motif(AC)_n is up to 53.33%.In addition,14 pairs of SSR primers were designed,11 pairs of which can be used to effectively detect the genetic diversity of W.pigra.
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