菠菜性别相关 EST-SSR 标记的开发及应用

为了明确菠菜EST序列中SSR的总体特点,开发菠菜EST-SSR引物;为利用EST-SSR引物进行菠菜性别相关特异序列的克隆奠定基础,本文从NCBI上获得1093条EST,利用在线软件SSRIT检测所含SSR序列,并进行分析。共检索出68条SSR序列,分布于64条EST中,检出率为6.22%,包括22种重复基元。其中二核苷酸重复基元的EST-SSR占主导地位,占总SSR数目的32.3%。利用在线引物设计软件Primer3.0设计了7对EST-SSR引物,在适合的PCR反应体系下,分别以雌、雄菠菜DNA基因组为模板,对设计的EST-SSR引物进行筛选,结果显示以EST序列HS097148设计的一对引物从菠菜雌雄基因组中扩增出一条雄性特异的条带,表明通过菠菜EST-SSR引物获得菠菜性别相关特异序列是可行的。     

中间锦鸡儿转录组EST-SSR标记系统性识别与引物筛选

旨在对中间锦鸡儿转录组数据库EST信息进行SSR系统性识别和初步验证,为进一步SSR分子标记开发提供依据。对Hi Seq2000测序技术获得的中间锦鸡儿转录组Unigenes进行SSR位点搜索,共获得45 706个SSR位点,出现频率为10.38%,平均4.30kb出现一个SSR位点。SSR重复类型以单核苷酸重复序列基元为主,所占比例为56.47%;二核苷酸、三核苷酸重复序列基元的数量所占比例分别是20.56%和21.04%;其他数量的基元所占比例仅为1.9%。多核苷酸重复类型中最多的为2核苷酸重复AG/CT;其次为3核苷酸重复AAG/CTT。针对EST-SSR位点随机挑选了150对引物,通过琼脂糖凝胶电泳进行PCR验证,其中有79对能获得扩增条带,21对引物扩增出单一条带,比例为14.0%。     

亚麻EST-SSR信息分析与标记开发

与基因组SSR相比,以EST为基础的EST-SSR分子标记具有自身的优点。本研究从11240条亚麻（Linum sitatissmum L.）EST序列中检索出877条含有SSR的序列,其出现频率为7.8%。其中以三核苷酸重复出现的频率最高,占总SSR序列的60.1%;其次是二核苷酸重复,占21.9%;四、五和六核苷酸重复占18%。根据这些含SSR的EST序列共设计了73对SSR引物,在8份亚麻材料间通过PCR扩增检测,有63对引物扩增出清晰条带,引物可用率86.3%;有17对引物在8份亚麻材料间显现出多态性,占可扩增引物的26.3%。     

龙眼顶芽转录组简单重复序列(SSR)标记信息分析及分子标记开发

随着新一代测序技术的发展,大量的转录组数据和表达序列标签(EST)成为开发简单重复序列(SSR)标记的可利用资源。本研究利用MISA软件筛选龙眼(Dimocarpus longan)顶芽转录组数据库序列,从114 445条龙眼转录组unigene序列中发现11 546个SSR位点,SSR出现频率为10.09%。其中1 975条unigene含有两个或两个以上EST-SSR位点,占所有SSR位点的比例为17.10%,SSR出现的平均距离为7.52 kb。从龙眼转录组SSR核苷酸基序类型来看,二核苷酸(52.11%)和三核苷酸(46.15%)出现频率最高,占所有核苷酸出现频率的99.26%。在龙眼转录组SSR中二核苷酸重复基元出现频率最高的是AG/CT(4 250个,占36.81%),三核苷酸重复基元出现频率最高的是AAG/CTT(1 109个,占9.61%)。对含SSR位点的9 571条unigene序列进行引物设计,共设计出了8 347对SSR位点特异引物。随机挑选合成50对EST-SSR引物,以‘石硖’、‘储良’、‘古山2号’、‘立冬本’等四份龙眼材料的基因组DNA为模板对这批引物进行PCR扩增、筛选,结果表明,其中21对引物能产生理想的PCR产物,有效扩增率为42%;16对引物扩增条带具有多态性,占有效引物的76.2%;16对多态性引物共扩增获得50个条带,其中多态性片段21个,每对引物平均产生1.31个多态性片段。     

蝴蝶兰EST-SSRs分析

对蝴蝶兰属EST序列进行了SSR分析。蝴蝶兰属EST总长为4.5Mb,含有609个SSR。SSR出现频率7.65%,平均距离7.39kb,平均长度为22.17bp。单碱基、二碱基和三碱基重复是主要重复类型,分别占EST-SSR总数的21.67%、40.39%和33.50%。A、AG和CCG分别是单碱基、二碱基和三碱基重复中主导重复基元,分别占96.21%、58.54%和32.25%。设计引物及检测的结果表明,蝴蝶兰EST-SSR标记对兰科其他属植物具有一定的通用性。     

漆树EST-SSR引物开发

利用NCBI数据库进行漆树EST-SSR引物开发,从NCBI数据库中共下载漆树EST序列87 856条。利用MISA软件进行序列处理、拼接及聚类后,从87 856条漆树EST序列中拼接组装成3 979条非冗余序列,含SSR位点的EST序列出现频率占EST序列总数的4.5%,从3 979条非冗余序列中检测到487个SSRs微卫星位点,出现频率为12.2%。这些SSR位点中,三核苷酸和二核苷酸重复基元所占比例较高。采用Primer5.0软件,共成功设计50对EST-SSR引物,50对EST-SSR引物在25个漆树个体上均能扩增出清晰的电泳条带,其中18对引物检测出了多态性条带,扩增率达36%。     

梅EST-SSR标记的开发及利用

利用MISA软件对10 123条梅EST序列进行SSR位点查找,得到含SSR位点的序列935条,SSR位点1233个,平均每100条EST序列中含有12.18个SSR位点.2核苷酸、3核苷酸重复是最主要的重复类型,分别占35.52%和41.36%.设计了40对EST-SSR引物并进行扩增,有24对引物能扩增出理想的PCR 产物,其中17对引物具有较好的扩增多态性.测序后发现13对引物中有73.08%的片段具有相应的SSR位点,对杏DNA指纹中部分谱带的测序结果也证明了是梅扩增出的相应的SSR位点.根据本研究含有SSR位点的测序结果推算,从梅EST中开发真实SSR位点的数目为901.随机选择13对引物对杏和梅进行DNA指纹构建与遗传多样性分析,结果发现,来源于梅的EST-SSR引物在杏中有很高的通用性,这些引物把梅和杏分成了两大群体,说明他们是遗传差异明显的两种植物.     

银杏EST序列中微卫星的分布特征

本文利用从NCBI下载的21 590条银杏EST序列,分析了银杏(表达序列标签微卫星)EST-SSR在银杏EST序列的分布和比较了在不同长度EST序列中的SSR特性.在剔除冗余和低质量序列后,得到总长为5 708.385 kb的无冗余EST序列7 961条,发现了405个EST序列(5.09%)含有475个SSR,长度400-1000 bp的EST序列含SSR位点数为445个,占SSR总数的93.68%.二核苷酸和三核苷酸基元类型是银杏EST-SSR的主要类型,分别占SSR总数的73.89%和24.00%,最常见的SSR基元是:(AT)_n、(AG)_n、(AC)_n、(AAG)_n和(AAT)_n.通过对银杏EST序列中SSR位点信息的发掘分析,为有针对性地设计EST-SSR引物,开发银杏EST-SSR分子标记奠定基础.     

亚麻EST序列中SSR标记的筛选

利用亚麻NCBI数据库中的7 941条亚麻EST序列进行SSR的筛选,共发现222个SSR,占整个EST数据库的2.73%,其中三核苷酸重复单元的EST-SSR占总SSR的72.1%,二核苷酸和四核苷酸二者出现的频率基本相近,分别占总SSR的14.4%和13.5%.AGAA是四核苷酸中的优势重复类型,占四核苷酸重复类型的67.67%.设计的21对EST-SSR引物中有18对在10个亚麻材料中有扩增产物,占设计引物的85%,有14对产物条带比较清晰并具有多态性.基于SSR标记进行聚类分析,可将10个亚麻材料划分为3个组.本研究建立的亚麻SSR标记,为亚麻遗传多样性鉴定、分子作图等研究提供了一种有效的分子标记系统.     

砂梨EST-SSR引物开发及其应用

利用GenBank和GDR数据库中的995条梨EST序列开发砂梨SSR引物,并根据开发的EST-SSR引物对砂梨'西子绿'×'喜水'F1群体的遗传变异进行分析.结果发现:(1)60个SSR位点分布于54条EST序列中,占整个EST数据库的5.4%,其中二核苷酸重复基元出现频率最高,达51.7%,其次为三核苷酸重复基元占25%.23类重复基序中AT重复基序出现的频率最高,为32.3%.(2)利用开发的25对EST-SSR引物对'西子绿'×'喜水'F1群体的遗传变异分析结果表明,其中9对引物呈现多态性,多态性引物占设计引物的36%;多态性引物扩增产物在F1群体中的等位基因数(No)平均为2,有效等位基因数(Ne)平均为1.932 4,平均杂合度观测值(Ho)和期望杂合度(He)分别为1和0.480 9.     

三七环二肽成分和人参内酰胺成分

From the roots of Panax notoginseng fourteen cyclodipeptides 1-14 were isolated including one new compound (1),seven new natural compounds (4-10) and six known compounds (2-3,11-14) together with one known other compound 15.The chemical structure of 1 was elucidated as cyclo-(Leu-Thr) based on spectral methods.From the roots of Panax ginseng five known lactams (16-20) includingpyrng lutamic acid were isolated together with butyric diacid,daucosterol and sucrose.The primary binactivity test showed that pyroglutamic acid and its n-butyl derivative have weak Ca^2 antagonistic activity.     

云南丽江人参、西洋参的氨基酸营养

本试验对云南丽江人参、西洋参的氨基酸含量进行了分析,并与吉林人参及西洋参的氨基酸含量进行比较。结果表明,两地人参、西洋参的总氨基酸含量稍有差异,但碱性、中性、酸性氨基酸在总氨基酸中所占百分比的高低基本一致。     

Genetic identification of Panax ginseng and Panax quinquefolius by pyrosequencing methods

This study was performed to determine whether two ginseng species (Panax ginseng and Panax quinquefolius) can be identified by genetic analysis and to verify pyrosequencing analysis, which was used to assess genetic variation. The pyrosequencing results constituted clear data. Panax quinquefolius showed a very different pattern than Panax ginseng. Pyrosequencing analysis might be able to identify the Panax species.     

人参的遗传改良*

遗传改良是人参育种的重要手段之一,而遗传转化和再生体系的建立是开展人参遗传改良工作的前提和基础。人参植株再生可以通过器官发生和体细胞胚发生,间接体细胞胚发生是人参植株再生的主要途径,从不同外植体,不同碳源,体细胞胚优化和无激素再生等方面进行了综述。在人参遗传转化方面,发根农杆菌和根癌农杆菌对人参的遗传转化均已成功,人参皂苷合成途径中的关键酶基因和抗除草剂基因也已陆续导入人参,得到了遗传改良的转化人参。发根培养系统可用于大量生产人参皂苷,讨论了rolC基因对人参发根诱导的作用,发根植株再生能力及生物反应器培养,最后指出了人参基因工程研究中存在的问题。     

Explant preparation affects culture initiation and regeneration of Panax ginseng and Panax quinquefolius

The influence of explant preparation on culture initiation and regeneration was investigated. Explant preparation was defined as the application of surface disinfection and homogenization to ginseng roots and embryos. Surface disinfection significantly affected culture initiation and subsequent embryogenesis. A high success rate (80–97%) in culture initiation was associated with explants from non-surface disinfected root tissue. The cumulative contamination rate after 8 months was below 4%; in contrast, a contamination rate of 85% was observed in disinfected root explants. For non-disinfected explants, visible callus was induced in 1 week, adventitious root and somatic embryos were formed in 2 and 6 months, respectively. Explants from disinfected roots required 1 month to induce visible callus, 5–7 months for adventitious roots and 10 months for somatic embryogenesis. For germinating embryos, disinfected embryos required double the time for embryogenesis than non-disinfected ones. It was considered that surface disinfection imposed a stress and subsequently a carry-over effect on explants.     

Cryopreservation of Panax ginseng cells

The impacts of cryoprotectants (CP) and cell status during the growth cycle on Panax ginseng cell viability during cryopreservation were investigated. The ginseng cells used had a 5–7 times proliferation rate (compared with inoculum) in 2–3 weeks and were subcultured at 2- and 4-week intervals in liquid and on solid media, respectively. After testing various CP solutions of glycerol, dimethylsulphoxide, ethylene glycol and sucrose, a combination of 10% (v/v) glycerol and 4% (w/v) sucrose was selected for its least cytotoxicity and highest cell viability after thawing. With this CP solution, cells throughout the growth cycle exhibited a ‘U’-shaped fluctuation of post-thaw cell viability. The highest viability (86.5%) occurred during the lag phase from cells already maintained in suspension culture and then in the late exponential phase (61.7%); the lowest level of 15.4% was in the mid-exponential phase. Callus freshly transferred to liquid medium showed a less obvious fluctuation pattern. The recovered cells were brown-to-reddish at first and gradually returned to a light yellow colour after several subcultures. Received: 1 October 1998 / Revision received: 6 January 2000 / Accepted: 11 January 2000     

竹节参化学成分研究

从竹节参的乙醇提取物分离得到了12个化合物,通过MS和NMR等方法鉴定为:竹节参皂苷Ⅳa(1),楤木皂苷A(2),竹节参皂苷Ⅰb(3),竹节参皂苷Ⅴ(4),尿嘧啶(5),胸嘧啶(6),尿苷(7),鸟苷(8),胸苷(9),腺苷(10),肌苷(11),胞苷(12),其中5 ～ 12为首次从该植物中分离.     

Molecular authentication of Panax species

Using conserved plant sequences as primers, the DNA sequences in the ribosomal ITS1-5.8S-ITS2 region have been amplified and determined for six Panax species, P. ginseng C. A. Mey. (Oriental ginseng), P. quinquefolius L. (American ginseng), P. notoginseng (Burkill) F. H. Chen (Sanchi), P. japonicus C. A. Mey. (Japanese ginseng), P. trifolius L. and P. major Ting, as well as two common adulterants of ginseng, Mirabilis jalapa L. and Phytolacca acinosa Roxb. An authentication procedure based upon the restriction fragment length polymorphism (RFLP) in the region is able to differentiate between P. ginseng and P. quinquefolius, and to discriminate the ginsengs from the two common poisonous adulterants. Broader application of this approach to authenticate other morphologically similar Chinese medicinal materials is rationalised.