银杏雄株叶片萜内酯类化合物的HPLC检测分析

目的:银杏叶萜内酯类化合物具重要的药用与保健开发利用价值.方法:在银杏最佳采叶期随机采取银杏叶片,经超声波70%乙醇水溶液提取、石油醚与正己烷萃取,再经ADS-17大孔树脂柱与酸性氧化铝柱纯化,以水∶甲醇∶四氢呋喃=7∶2∶1(V/V)为流动相,采用Waters ODS C18 柱 (250mm ×416mm,5μm)进行HPLC分析:0.6ml/min流速、219nm检测波长、26℃检测温度、15μl进样量.结果: GA、GB、GC和BB分别在23～460μg/ml、21～420μg/ml、22～440μg/ml和27～540μg/ml范围内呈线性相关,最小检出量0.5μg,回收率90.74%～102.38%,RSD 1.63%～4.2%.GBE3得率1%,GA 、GB、 GC 、BB占GBE3的12 55%,超过GBE761的6% 国际标准.结论:该HPLC检测分析技术与方法适用于银杏叶片萜内酯类化合物的提取、纯化和检测.     

银杏雌雄株叶片氨基酸组分及其含量

银杏是雌雄异株植物,依靠种子繁殖,是我国特有的活化石植物,也是重要的观赏树种。作为行道树以雄株为宜。为了有计划地进行定性栽培,就需要对幼龄植株进行早期性别鉴定。许多生理指标都可能作为性别鉴定的依据。通过对银杏中氨基酸含量和组成的分析表明,不同性别植株叶片中均含有被分析的17种氨基酸,各种氨基酸的含量和总含量在单株间没有表现出性别差异,而平均含量却表现出雄株高于雌株,其中胱氨酸和酪氨酸较为突出,分别高出雌株28.24%和40.94%,这是否反映了银杏雌雄的性别差异,值得进一步探讨。尽管脯氨酸含量与花药的正常发育有关,并大大高于雌蕊中脯氨酸的含量,但这一生理指标不适用于营养器官。     

银杏LEAFY同源基因的时空表达

以银杏雄株、雌株成年树和还未开过花的幼树的根、茎、叶,雌株幼果和不同时期的雄花芽、雌花芽为材料,利用同位素标记,制备Ginlfy和GinNdly两个特异探针,进行Northern分子杂交,研究银杏LFY同源基因Ginlfy、GinNdly在银杏不同器官,花芽不同生长发育时期的时空表达情况。结果显示,无论是幼树,还是成年的雌株、雄株,Ginlfy基因在各个器官,如根、茎、叶、雌花芽、雄花芽、幼果以及雌花芽、雄花芽的不同发育时期都有表达,属组成型表达,而GinNdly基因只在叶和不同时期的雄花芽、雌花芽中表达,其他器官都不表达,属特异性表达。银杏双拷贝LFY同源基因中的GinNdly基因可能与开花关系更为密切。 Abstract: Expressions of Ginlfy and GinNdly gene were studied by northern blotting in different organs and stages of Ginkgo Biloba. Ginlfy gene was expressed in different organs such as root, stem, leaf of juvenile tree, male tree and female tree, and in different stages of male flower bud and female flower bud. It was inferred that Ginlfy gene could be expressed constitutionally. GinNdly gene was only expressed in leaf of juvenile tree, male tree and female tree and in different stages of male flower bud and female flower bud, while GinNdly gene was not expressed in the other organs. Therefore it was thought that GinNdly gene could be expressed differentially and be a close relation to development of flower.     

犬冠状病毒大熊猫株纤突蛋白全基因的克隆与序列分析

在国内首次对犬冠状病毒大熊猫野毒株(CCVDXMV)纤突蛋白基因进行了克隆和序列测定。该基因全长4362bp,编码1453个氨基酸,N端前18个氨基酸为推测的信号肽序列,后1435个氨基酸构成成熟蛋白。与GenBank中已发表的11个CCV毒株s基因相比,s基因核苷酸序列同源性在40．2％-99．5％之间;推导的氨基酸序列同源性在15．9％-99．O％之间。DXMV株s基因变异区主要集中在该基因前1／2处,其中350．370、439．478、1718．1818三个区域碱基变异较大,而1060．1700区却十分保守。基于s全基因及其蛋白的聚类分析表明,DXMV株与K378、NVSL和USpatent株亲源关系最近。推导的DXMV株s蛋白氨基酸序列潜在的N-联糖基化位点与CCV强毒V54相同,为34个,比Insavc．1弱毒多一个;其中第566．568位糖基化位点为多数强毒拥有而弱毒没有的。另外,DXMV株S蛋白疏水性及抗原表位与其它毒株有一定的差异,这些差异对DXMV株致病性和免疫原性等影响尚待进一步的研究。     

小鼠Sry基因和Sox基因的克隆分离与鉴定

为了深入探讨性分化分子机制,构建了小鼠基因库以ＳＲＹ探针筛选,分离到了两组阳性克隆,一组含ＥｃｏＲⅠ／３．５ｋｂ,此片段中载有小鼠Ｙ染色体上的Ｓｒｙ基因,另一组含ＥｃｏＲⅠ／３．０ｋｂ或ＳａｌⅠ／３．０ｋｂ,此片段有小鼠Ｙ染色体之外的Ｓｏｘ基因。     

犬冠状病毒大熊猫株纤突蛋白全基因的克隆与序列分析

在国内首次对犬冠状病毒大熊猫野毒株(CCV DXMV)纤突蛋白基因进行了克隆和序列测定.该基因全长4362bp,编码1453个氨基酸,N端前18个氨基酸为推测的信号肽序列,后1435个氨基酸构成成熟蛋白.与GenBank中已发表的11个CCV毒株S基因相比,S基因核苷酸序列同源性在40.2%～99.5%之间;推导的氨基酸序列同源性在15.9%～99.0%之间.DXMV株S基因变异区主要集中在该基因前1/2处,其中350-370、439-478、1718-1818三个区域碱基变异较大,而1060-1700区却十分保守.基于S全基因及其蛋白的聚类分析表明,DXMV株与K378、NVSL和US patent株亲源关系最近.推导的DXMV株S蛋白氨基酸序列潜在的N-联糖基化位点与CCV强毒V54相同,为34个,比Insavc-1弱毒多一个;其中第566-568位糖基化位点为多数强毒拥有而弱毒没有的.另外,DXMV株S蛋白疏水性及抗原表位与其它毒株有一定的差异,这些差异对DXMV株致病性和免疫原性等影响尚待进一步的研究.     

长雄野生稻有利基因的发掘与利用

人类将普通野生稻驯化为亚洲栽培稻,其农艺性状如株高、落粒性、穗型等发生了重要变化,产量也大幅提高,但许多优良性状如抗逆性等却丢失。长雄野生稻与亚洲栽培稻同属AA基因组,蕴藏了许多生物胁迫和非生物胁迫的抗性基因,被认为是亚洲栽培稻遗传改良的潜在基因库。本文总结了长雄野生稻生物及非生物胁迫抗性、地下茎性状以及其他潜在应用价值性状,包括白叶枯抗性、抗旱性、耐热性、自交不亲和性、氮高效利用以及高产等有利性状。基于长雄野生稻地下茎性状开展多年生稻育种实践的应用研究,对长雄野生稻进行从头驯化的策略进行了探讨,以期为长雄野生稻基础研究及栽培稻遗传改良提供理论参考。     

腮腺炎病毒分离株(SP株)SH基因及其旁侧序列的初步分析

目的 比较腮腺炎病毒分离株SP株与其他野毒株的序列差异,以确定其遗传特征。方法 将2005年在云南省石屏县收集到的腮腺炎患儿唾液标本,于Vero细胞培养7 d后观察病变并分离收获病毒,用血细胞吸附试验验证。同时提取病毒RNA,采用反转录-套式聚合酶链反应(RT-nPCR)法从病毒RNA中扩增出SH基因及其旁侧序列,测序并以VectorNT16．0软件分析。结果 SH基因旁侧区序列分析表明:SP株与国内已知野毒株具有明显的亲缘关系,在SH基因旁侧区范围内的序列一致性为93％～96％,差异均＜8％。氨基酸序列分析发现:该毒株与其他国内野毒株在此区域中的氨基酸序列一致性为93．3％～98．25％,与Wlz2株的遗传距离最近。结论 SP株属于F基因亚型。     

银杏雌雄基因组DNA间的差异性分析

本研究应用RAPD技术,应用300个10bp随机单引物及200对随机双引物组合,检测了雌雄异株银杏基因组DNA的多态性。结果表明:雌雄基因组间具有极高的相似性,在检测到的3450个标记中,仅获得1个与银杏雄性基因组相关的RAPD标记。以该标记为探针,与雌雄银杏基因组DNA的Southern杂交分析,其杂交信号在两性之间表现为限制性片段长度多态性,该结果为寻找银杏早期性别鉴定的探针以及在细胞和分子水平进一步研究其性别问题奠定了基础。     

银杏雌配子体发育及原生质体分离与培养的研究

经观察,银杏雌配子体在4月下旬-5月下旬为游离核时期,5月上旬采集的雌配子体在0.5%纤维素酶(Onzuka R—10)与0.5%果胶酶(Serva)混合酶液中酶解4—5h,原生质体密度为6×10~5—8×10~5/ml,活性87.3%。原生质体在去掉NH_4NO_3的MT培养基中,附加BA1.0mg/L,NAA3.0mg/L,谷氨酰胺1000mg/L,Vc5mg/L,采用液体浅层培养获得了肉眼可见的多细胞团。     

银杏外种皮中银杏酸的分离方法

本文简单介绍了一种从银杏外种皮中分离银杏酸的方法,使用了皂化和酯化反应为后的两次柱层析分离提供便利条件。     

DIVISION OF THE GENERATIVE CELL AND LATE DEVELOPMENT IN THE MALE GAMETOPHYTE OF GINKGO BILOBA

Division of the generative cell in the male gametophyte of Ginkgo biloba to yield the sterile cell and spermatogenous cell was examined in vivo and in vitro. Evidence is presented in support of a new interpretation of development in which the sterile cell and spermatogenous cell arise from an unusual anticlinal ringlike division of the generative cell. This type of cell division is only known to occur during antheridial development in leptosporangiate ferns and stomatal development among certain ferns in the Schizaeaceae and Polypodiaceae. The strong similarities in development and cell arrangement within the male gametophytes of cycads and Ginkgo suggest that division of the generative cell in cycads may be the same as in Ginkgo. Although the ringlike (conically annular) divisions in the antheridia of leptosporangiate ferns and the male gametophytes of Ginkgo (and probably cycads) are remarkably similar and result in the production of a central spermatogenous cell, it is conjectural as to whether these patterns represent a striking convergence or evolutionary homology.     

银杏外种皮研究进展

银杏(GinkgobilobaL.)为银杏科银杏属多年生落叶乔木,又名白果,是我国特有树种,也是世界上珍贵的药用植物资源。对银杏叶和果的研究,尤其是对银杏叶黄酮的研究,已取得了多项成果〔1,2〕,但对银杏外种皮的研究还不多。银杏外种皮是种子硬壳外面的肉质部分,长期以来,被视为废物丢弃,既浪费资源又污染环境。国内自90年代末,扬州大学、中国药科大学等单位开展了银杏外种皮研究工作,但目前还没有规模生产的产品投向市场〔3〕。笔者在综合检索的基础上对银杏外种皮研究工作作一综述,希望引起对银杏外种皮开发利用工作的广泛重视。1　资源状况目前…     

银杏叶精油的化学成分

采集３－５年银杏苗叶,经常压水蒸汽蒸馏制得浅黄色精油,通过ＧＣ和ＧＣ／ＭＳ鉴定出４４个化学成分,主要由３１．１４％烃类、５．６１％醇类、１．７８％醛类、４６．１７％酮类、５．４１％酸类和１．１％酯类组成。在酮类中以六氢法呢酮含量最高,占１１．１５％,其次为橙花基酮（８．６８％）和β－紫罗兰酮（５．２４％）。     

PHOTOSYNTHESIS IN THE FEMALE GAMETOPHYTE OF GINKGO BILOBA

The female gametophyte of Ginkgo biloba is the only seed plant gametophyte known to contain chlorophyll. Measurements of photosynthetically active radiation (PAR) indicate that a gametophyte growing within an ovule can receive significant quantities of light (70 μmol photons m^–2 s^–1). Under the conditions of our experiments, whole gametophytes dissected free from ovules were capable of gross photosynthesis, but not net photosynthesis. On a dry wt basis, the maximum rate of carbon fixation under near saturating light intensities was 3.64 × 10^–3 μmol CO₂g^–1 s^–1. The unique ability of the female gametophyte of Ginkgo biloba to produce chlorophyll and engage in photosynthesis appears to result from its exposure to sufficient levels of light and a predisposition to react to this stimulus by the development of a functional photosynthetic apparatus.     

凹叶厚朴大、小孢子发生和雌、雄配子体发育的研究

凹叶厚朴花药四囊型,腺质绒毡层有1-2层细胞,小孢子形成时胞质分裂方式为修饰性同时型．小孢子四分体排列方式为左右对称型,成熟花粉粒为二细胞型。四分体和小孢子在发生时有不规则变形。子房单心皮。心皮腹面壁上着生2个胚珠,胚珠倒生型,厚珠心,双珠被;抱原细胞一个,并且自表皮下第2层细胞处分化。胚囊发育为单孢蓼型。凹叶厚朴的胚胎学特征与木兰科其它植物的胚胎学特征基本相同,属于较原始的被子植物胚胎类型。在凹叶厚朴大、小孢子发生和雌、雄配子体发育过程中存在部分败育现象。本文初步探讨了凹叶厚朴濒危的生殖生物学原因。     

凹叶厚朴大、小孢子发生和雌、雄配子体发育的研究

凹叶厚朴花药四囊型,腺质绒毡层有1-2层细胞,小孢子形成时胞质分裂方式为修饰性同时型,小孢子四分体排列方式为左右对称型．成熟花粉粒为二细胞型。四分体和小孢子在发生时有不规则变形。子房单心皮,心皮腹面壁上着生2个胚珠,胚珠倒生型,厚珠心,双珠被;孢原细胞一个,并且自表皮下第2层细胞处分化。胚囊发育为单孢蓼型。凹叶厚朴的胚胎学特征与木兰科其它植物的胚胎学特征基本相同,属于较原始的被子植物胚胎类型。在凹叶厚朴大、小孢子发生和雌、雄配子体发育过程中存在部分败育现象。本文初步探讨了凹叶厚朴濒危的生殖生物学原因。     

LIGHT MICROSCOPE AND ULTRASTRUCTURAL STUDIES OF THE MALE GAMETOPHYTE IN GINKGO BILOBA: THE SPERMATOGENOUS CELL

A light microscope and ultrastructural study was made of the pollen tube of Ginkgo biloba, with special emphasis given to the spermatogenous cell that gives rise to two motile sperms. Just prior to the mitotic division that results in the formation of two sperms, the spermatogenous cell consists of a large nucleus, two blepharoplasts, two large osmiophilic globules, and a conspicuous lipo-protein body. Other organelles in the cytoplasm include numerous electron-dense proplastids (with some lamellar development), mitochondria, small vacuoles, and lipid bodies. Ribosomes are present in abundance, but endoplasmic reticulum and dictyosomes are sparse. The nucleus, prior to mitosis, is relatively Feulgen-negative, due undoubtedly to the diffuse distribution of DNA. Each blepharoplast, the main organelle of interest, is nearly spherical, measures 3.5–4.5 μm in diam, and supports about 1,000 probasal bodies. The interior of a blepharoplast consists of an electron-dense matrix and of less dense regions which appear to be infiltrated by a network of microtubules. Each probasal body is composed of a cylinder of nine separate tubules (singlets) at the basal or proximal end. The cylinder becomes elaborated distally into nine pairs of subtubules (doublets) and then into nine sets of subtubules (triplets). A central tubule is present the entire length of the probasal body. Some of the subtubules, as well as microtubules from the interior of the blepharoplast, extend into the cytoplasm and probably constitute the “astral rays” as seen with the light microscope. Comparisons are made with other published accounts of the organization of blepharoplasts in plants and of centrosomes and centrioles in animals.