丙型肝炎病毒非结构区NS4b基因片段的克隆和表达

采用聚合酶链反应（ＰＣＲ）技术和ＤＮＡ体外重组方法,克隆出５７９ｂｐ的丙型肝炎病毒（ＨＣＶ）ＮＳ４ｂ基因片段,插入到原核高效表达载体ｐＥＴ－２８ａ中,构建重组质粒ｐＥＴ／ＮＳ４ｂ,转化大肠杆菌ＢＬ２１（ＤＥ３）菌株,经ＩＰＴＧ诱导培养后,获得了目的蛋白的高效表达。ＳＤＳ－ＰＡＧＥ分析显示在３０ｋＤ处有一条表达的目的蛋白区带。通过固定化金属配体亲和层析（ＩＭＡＣ）纯化目的的蛋白,ＥＬＥＳＡ检测结果表     

利用RFLP,SSR,AFLP和RAPD标记分析玉米自效系遗传多样性的比较研究

利用ＲＦＬＰ、ＳＳＲ、ＡＦＬＰ和ＲＡＰＤ４种分子标记方法研究了１５个玉米（Ｚｅａ ｍａｙｓ Ｌ．）自交系的遗传多样性,同时对４种标记系统进行比较。在供试材料中筛选到具多态性的ＲＦＬＰ探针酶组合５６个,６７６对ＳＳＲ引物,２０个ＲＡＰＤ引物和９个ＡＦＬＰ引物组合,分别检测到多态性带１６７、２０１、８７和１０８条。ＳＳＲ标记位点的平均多态性检测效率（Ａｉ,３２．２）。４种分子标记所得遗传相似自交系划分     

利用RFLP、SSR、AFLP和RAPD标记分析玉米自交系遗传多样性的比较研究

利用 ＲＦＬＰ、ＳＳＲ．ＡＦＬＰ和ＲＡＰＤ ４种分子标记方法研究了 １５个玉米（Ｚｅａ ｍａｙｓ Ｌ．）自交系的遗传多样性,同时对４种标记系统进行比较。在供试材料中筛选到具多态性的ＲＦＬＰ探针酶组合５６个,６６对ＳＳＲ引物,２０个ＲＡＰＤ引物和９个ＡＦＬＰ引物组合,分别检测到多态性带１６７、２０１、８７和１０８条。ＳＳＲ标记位点的平均多态性信息量（ＰＩＣ）最大（０．５４）,ＡＦＬＰ标记位点最小（０．３６）,但ＡＦＬＰ标记具有最高的多态性检测效率（Ａｉ,３２．２）。４种分子标记所得遗传相似系数相关性显著,比较相关系数表明 ＲＡＰＤ可靠性较低。依据 ４种分子标记结果将 １５个供试自交系划分为塘四平头、旅大红骨、兰卡斯特、瑞德和ＰＮ共５个类群,与系谱分析基本一致。认为ＳＳＲ和ＲＦＬＰ两种分子标记方法适合进行玉米种质遗传多样性的研究。     

RAPD标记构建水稻分子连锁图

利用随机扩增多态性ＤＮＡ（ＲＡＰＤ）,在一个水稻（Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．）的双单倍体（ＤＨ）群体中发展分子标记,仅用５２ 个ＲＡＰＤ标记建成了一个水稻ＲＡＰＤ分子连锁图。该图覆盖基因组的总长度为８９８．４ ｃＭ （ｃｅｎｔｉｍ ｏｒｇａｎ）,标记间的平均间距为１７．３ ｃＭ,它能与用同一群体构成的ＲＦＬＰ图谱互相补充     

棉铃虫核型多角体病毒p35基因的PCR扩增和克隆及其在原核细胞中的表达

用苜蓿银纹夜蛾核型多角体病毒（ Ａｃ Ｎ Ｐ Ｖ） 凋亡抑制基因ｐ３５ 的合成引物,从棉铃虫核型多角体病毒（ Ｈａ Ｎ Ｐ Ｖ） 基因组中用 Ｐ Ｃ Ｒ 扩增到了１ｋｂ 片段。采用 Ｐ Ｃ Ｒ－ 双脱氧核苷酸链终止银染法,测定了所扩增到的１ ０１０ｂｐ 全序列,发现其开放阅读框完整,长８９７ｂｐ ,编码２９９ 个氨基酸。用 Ｄ Ｎ Ａ Ｓ Ｉ Ｓ 和 Ｐ Ｒ Ｏ Ｓ Ｉ Ｓ 软件分析发现,此片段与 Ａｃ Ｍ Ｎ Ｐ Ｖ ｐ３５ 基因同源区核苷酸同源性为９１ ％ ,氨基酸同源性也达８１ ％ ,证明了所扩增的片段是 Ｈａ Ｎ Ｐ Ｖ 的ｐ３５ 基因。为了研究此ｐ３５ 基因的功能及其效应机制,克隆了这一基因并在大肠杆菌细胞中实现了表达。     

E和St基因组特异RAPD片段在部分小麦族植物中的分布

两个Ｅ基因组（包括Ｅｅ和Ｅｂ）特异ＲＡＰＤ片段和两个Ｓｔ基因组特异ＲＡＰＤ片段的序列分析表明,４个片段均为新的ＤＮＡ克隆片段。染色体原位杂交显示ＯＰＤ１２４４４为区域化连续高度重复序列,而ＯＰＦ０３１２９６（Ｅｂ特异）、ＯＰＢ０８５２５（Ｓｔ特异）、ＯＰＮ０１８１７（Ｓｔ特异）为弥散性高度重复序列。研究还显示：大部分ＤＮＡ高度重复序列在亲缘关系较近的小麦族植物基因组间是共享的,差异可能主要是在重复次数及片段长度上,而能否用ＲＡＰＤ技术扩增主要决定于某一基因组的这些重复序列中有无与特定引物相匹配的区域。文中就这些重复序列在小麦远缘杂交后代外源遗传物质检测、多倍体物种染色体组组成研究中的潜在价值进行了讨论。     

辣椒疫霉产毒共分离RAPD标记的研究

辣椒疫霉（Ｐｈｙｔｏｐｈｔｈｏｒａ　ｃｏｐｓｉｃｉ）毒素的产生是由一个不完全显性基因所控制,通过ＲＡＰＤ／ＢＳＡ分析证明,用ＯＰＷ１２扩增得到一条约１３００ｂｐ的ＲＡＰＤ标记带,该带与辣椒疫霉产毒共分离。纯化回收ＯＰＷ１２　１３００ＤＮＡ,共转化感受态Ｅ．ｃｏｌｉ　ＤＨ５ａ,筛选出３个白色阳性克隆,序列分析发现该标记ＤＮＡ为１２９１ｂｐ。这一标记ＤＮＡ序列的阐明为进一分析产毒遗传机理提供了新的信息。     

辣椒疫霉产毒共分离RAPD标记的研究*

辣椒疫霉（Ｐｈｙｔｏｐｈｔｈｏｒａ　ｃｏｐｓｉｃｉ）毒素的产生是由一个不完全显性基因所控制,通过ＲＡＰＤ／ＢＳＡ分析证明,用ＯＰＷ１２扩增得到一条约１３００ｂｐ的ＲＡＰＤ标记带,该带与辣椒疫霉产毒共分离。纯化回收ＯＰＷ１２　１３００ＤＮＡ,共转化感受态Ｅ．ｃｏｌｉ　ＤＨ５ａ,筛选出３个白色阳性克隆,序列分析发现该标记ＤＮＡ为１２９１ｂｐ。这一标记ＤＮＡ序列的阐明为进一分析产毒遗传机理提供了新的信息。     

辣椒疫霉产毒共分离RAPD标记的研究

辣椒疫毒（Ｐｈｙｔｏｐｈｔｈｏｒａ ｃａｐｓｉｃｉ）毒素的产生是由一个不完全显性基因所控制,通过ＲＡＰＤ／ＢＳＡ分析证明,用ＯＰＷ１２扩增得到一条约１３００ｂｐ的ＲＡＰＤ标记带,该带与辣椒疫霉产毒共分离。纯化回收ＯＰＷ１２１３００ＤＮＡ,共转化感受态Ｅ．ｃｏｌｉ ＤＨ５α,筛选出３个白色阳性克隆,序列分析发现该标记ＤＮＡ为１２９１ｂｐ。这一标记ＤＮＡ序列的阐明为进一步分析产毒遗传机理提供了新的信息     

毛乌素沙地柠条群体分子生态学初步研究:RAPD证据

毛乌素沙地柠条群体是一个杂种带,为了进一步阐明分子变异和基因流与生境或生态过渡带的联系,应用ＲＡＰＤ标记开展了柠条群体的分子生态学研究。根据ＲＡＰＤ数据利用Ｓｈａｎｎｏｎ信息指数估计了６个柠条群体的遗传多样性,发现大部分的分子变异存在于柠条群体之内（８２．４％）,只有少部分的分子变异存在于群体之间（１７．６％）,又利用ｅｉ指数统计了ＲＡＰＤ数据,也证实了大部分的遗传变异存在于群体之间,柠条锦鸡儿群     

Analysis of the Genetic Structure of Sclerotinia sclerotiorum (Lib.) de Bary Populations from Different Regions and Host Plants by Random Amplified Polymorphic DNA Markers

The genetic diversity and genetic structure of a population of isolates of Sclerotinia sclerotiorum (Lib.) de Bary from different regions and host plants were investigated using the random amplified polymorphic DNA (RAPD) method with 20 random decamer primer pairs in order to provide some information on the phylogenetic taxa and breeding for resistance to sclerotinia stem rot. A minimum of three and a maximum of 15 unambiguously amplified bands were generated, furnishing a total of 170 bands ranging in size from 100to 3 200 bp, corresponding to an average of 8.5 bands per primer pair. One hundred and four of these 170bands (61.2%) were polymorphic, the percentage of polymorphic bands for each primer pair ranging from 0.0% to 86.7%. The genetic relationships among the isolates, based on the results of RAPD analysis, were examined. The genetic similarity of all selected isolates was quite high. At the species level, the genetic diversity estimated by Nei's gene diversity (h) was 0.197 and S hannon's index of diversity (I) was 0.300. The unweighted pair-group mean analysis (UPGMA) cluster analysis showed that most isolates from the same regions were grouped in the same cluster or a close cluster. The population of isolates from Hefei (Anhui Province, China) was more uniform and relatively distant to other populations. The Canadian population collected from carrot (Daucus carota var. sativa DC.) was relatively close to the Polish population collected from oilseed rape (Brassica napus L.) plants. There was no relationship between isolates from the same host plants. An analysis of molecular variance (AMOVA) revealed that the percentage of variance attributable to variation among and within populations was 50.62% and 49.38%, respectively. When accessions from China, Europe, and Canada were treated as three separate groups, the variance components among groups,among populations within groups, and within populations were -0.96%, 51.48%, and 49.47%, respectively.The genetic differentiations among and within populations were highly significant (P ＜ 0.001). Similarly, the coefficient of gene differentiation (Gst) in total populations calculated by population genetic analysis was 0.229 4, which indicated that the genetic variation among populations was 22.94%. The gene flow (Nm)was 1.68, which indicated that the gene permutation and interaction among populations was relatively high.     

云南丽江山慈菇遗传多样性的DALP分析

采用DALP (Direct amplification of length polymorphism) 分子标记技术, 对产自云南的药用植物丽江山慈菇Iphigenia indica (L.) Kunth的9个居群进行DNA指纹检测。筛选出5个引物组合, 扩增共产生131条DNA片段, 其中104 条谱带具有遗传多态性, 约占79 39%, 平均每组引物扩增所得多态条带为20 8, 9个居群平均多态百分率为42 21%。9个居群平均观察等位基因数Na为1 4224, 总Na为1 7939; 平均有效等位基因数Ne 为1 3141, 总Ne 为1 4810; 平均遗传多样性指数H为0 1745, 总H为0 2831; 平均Shannon 多样性指数I 为0 2527, 总I为0 4231; 总基因多样性Ht为0 2831, 居群内多样性Hs 为0 1745, 居群间基因分化系数Gst为0 3834, 即丽江山慈菇有61 66%的遗传变异来自居群内, 38 34%来自居群间, 居群间存在较高水平的遗传分化。滇西北居群的遗传多样性明显高于滇中居群的遗传多样性, 这与滇中地区丽江山慈菇野生资源被大规模挖掘有着直接的关系。     

烟草SCAR标记技术的建立

目的:通过烟草随机扩增多态性DNA(RAPD)标记技术建立烟草特征序列扩增区域(SCAR)标记技术,用于烟草品种鉴定。方法:对12个烟草品种的复烤叶片DNA进行RAPD分析,得到2个RAPD特异片段S1和S2,通过切胶回收,连接pUCm-T载体克隆转化,片段测序,设计特异性引物S1-1/S1-2和S2-1/S2-2,对SCAR-PCR扩增退火温度进行优化。结果:2个RAPD标记成功地转化为稳定快捷的SCAR标记,可将红花大金元和NC102等2个品种从12个烟草品种中快捷准确地鉴别出来。结论:SCAR标记可作为准确稳定的DNA水平的烟草品种鉴定方法,可对种植、复烤和配方品种的烟叶或叶片进行鉴别。     

苹果炭疽菌的分子鉴定与检测

测定苹果炭疽菌rDNA全序列,比对苹果炭疽菌和其它炭疽菌ITS序列以及构建系统关系树,发现苹果炭疽菌与胶孢炭疽菌的ITS序列相似性高达99．8％,并与胶孢炭疽菌聚在一起,可以明确苹果炭疽菌应属于胶孢炭疽菌。进一步的序列比对发现,苹果炭疽菌的18S rDNA3’端比其它胶孢炭疽菌多出一段379bp的序列,根据这一特有片段设计引物CgF1与通用引物ITS4配对,结果仅能从苹果炭疽菌中扩增出1232bp的特异性条带。用苹果炭疽菌接种离体苹果,以接种发病的病组织总DNA为模板,利用引物CgF1／ITS4进行PCR扩增,同样可以扩增出1232bp的特异性条带,而健康苹果组织DNA中未能扩增出任何条带,表明该方法可用于苹果炭疽菌的鉴定和快速检测。     

入侵中国大陆的红火蚁的鉴定及发生为害调查

根据形态特征鉴定结果首次证实红火蚁SolenopsisinvictaBuren入侵中国大陆 ,并调查了广东省吴川市红火蚁发生为害情况。调查结果表明 ,发生区内部分地点红火蚁发生密度较高 ,主要发生于较稳定的生态环境中 ,如荒坡、草地、长满杂草的田埂等。红火蚁已对当地农业生产、人们的身体健康、日常生活等造成了不利影响。此外 ,Cytb基因序列分析结果表明 ,采自美国佛罗里达和广东吴川的红火蚁与采自广东 4个地方的热带火蚁S .geminata (Fabr.)两物种间有 61个变异位点 ,种内变异为 0。限制性酶切多态实验 (RFLP)的结果显示 ,BamHI酶在红火蚁的特异扩增片段中有一个识别位点 ,而在热带火蚁中无酶切位点 ;MspI酶在热带火蚁的扩增片段中也有一个识别位点 ,而在红火蚁中无识别位点。有酶切位点的扩增产物在酶切后分别获得近 2 0 0bp与 2 5 0bp的片段各一带。因此 ,用PCR RFLP的方法可以简单快速地鉴别红火蚁和热带火蚁。     

Genetic diversity of <Emphasis Type="Italic">Salanx ariakensis</Emphasis> (Salangidae) from Korea and Japan inferred from AFLP

The genetic diversity and population structures within and between Korean and Japanese populations of Salanx ariakensis were investigated using AFLP (amplified fragment length polymorphism). Using seven primer pair sets, 411 fragments were amplified from 31 specimens of S. ariakensis, 393 fragments (96%) being polymorphic. The expected heterozygosities were 0.211 and 0.245 for the Japanese and Korean populations, respectively, levels similar to those of other freshwater and diadromous fishes, but lower than those of marine fishes. The difference in genetic diversity between the two populations may result from their different habitat sizes. Principal coordinate analysis using 393 polymorphic fragments resulted in specimens from each population being plotted separated, suggesting an absence (or little if present) gene flow between them since Tsushima Strait has been open. Therefore, each population of S. ariakensis is relevant from the conservation perspective and should at least be treated as a “management unit” for conservation purposes.     

中国小麦微核心种质资源Psy基因的等位变异

根据已发表的麦族植物体Psy基因序列的保守区设计引物PsyO2,克隆小麦Psy基因（片段）。结果表明,PsyO2引物的扩增产物出现2种带型：196bp和233bp,序列分析表明两条特异条带涵盖了小麦Psy基因第2外显子全部序列,相差的37bp为Psy基因第2内含子中的一段插入序列,可反映不同黄色素含量（YPC）,属小麦风,,基因的等位变异。验证试验表明,248份小麦微核心种质中有153份材料（占样品数的65．7％）扩增出196bp条带,群体内YPC均值7．314mg kg^-1,属高YPC范畴;另有95份材料（占样品总数的38．3％）扩增出233bp条带,群体内YPE均值为5．207mg kg^-1,属低YPC范畴,方差分析表明二者YPC差异达1％极显著水平差异,说明上述37bp的插入序列是导致小麦品种间YPC产生差异的原因之一,因此该引物扩增的Psy基因对小麦YPC具有显著影响,引物PsyO2是对小麦YPC进行分子鉴定的重要标记。     

太空诱变哈密瓜两性花性状连锁标记的RAPD分析

以哈密瓜品种'早皇后'、太空诱变后两性花株突变体及其后代分离群体为试材,采用BSA方法对哈密瓜两性花性状进行了RAPD分析.研究结果表明:在所筛选的490条10-mer随机引物中,只有S1254在两性花株基因池中扩增到750 bp的多态性条带,而在其它基因型的群体中未扩增到此条带.通过对分离群体及其姐妹系进行单株验证,均获得相同的扩增结果,说明S1254750与两性花性状连锁,该标记与两性花性状的遗传距离为4.5 cM.     

Species and Strain Identification of the Predatory Mite Euseius Finlandicus by RAPD-PCR and ITS Sequences

The variation within and between Finnish Euseius finlandicus populations was investigated by RAPD-PCR and ITS sequence analyses. Resin DNA extraction was found to be a suitable method for samples of single mites used in PCR. The banding patterns from 24 RAPD primers and 10 primer pairs were very similar and reproducible in all specimens of the predatory mite studied. However, the E. finlandicus K-strain could be distinguished from organophosphate-resistant predatory mites (R-strain), since almost all of them produced a 1,400 bp RAPD-PCR product, which was missing or very rare in other strains studied. Another RAPD band of ca. 680 bp was in turn much more common in other mites of E. finlandicus than in the K-strain mites. Mite specific primers were designed and used to follow the survival of the R-strain released on apple trees. The 680 bp band obtained with specific primers was specific to the species E. finlandicus mites studied, including those that had been negative with RAPD primers. The 1,400 bp specific primers could be used as a marker for following the survival of R-strain mites on apple trees. At the species level it was possible to distinguish adults and eggs of E. finlandicus from Anthoseius rhenanus and Phytonemus pallidus by RAPD-PCR. In addition, a band at 480bp was found to correspond to DNA of the predatory mite Phytoseius macropilis, when both specific primer pairs were used together. It was not possible to amplify the ITS region of E. finlandicus rDNA using several primer pairs that work in other mites and aphids. However, a basidiomycete rDNA sequence was amplified with one of these ITS primer pairs in K-strain mites. Finally, it was found that fungal rDNA-specific primers amplified an ITS region of ca. 650 bp in several strains of E. finlandicus. Internal primers, designed to amplify the central part of the 650 bp product, successfully amplified this product from all the mites.     

SCAR标记在茶树种质资源鉴定中的应用

SCAR标记是一种在RAPD技术的基础上发展起来的新型分子标记技术,提高了分子标记辅助选择育种的效率,在茶树种质资源的合理开发与利用中具有广阔的应用前景.运用优化后的RAPD反应体系对10个茶树品种的基因组DNA进行遗传差异分析,随机引物S89、S4分别在白毫早和福云6号中扩增得到长度为498 bp、1 622 bp的差异片段,命名为BHZ498、FY1622.根据它们的测序结果分别设计了一对特异引物,BHZ498的特异引物为SB1/SB2;FY1622的特异引物为SC1/SC2,用这两对特异引物对10个茶树品种的基因组DNA进行扩增.引物SB1/SB2和SC1/SC2分别在白毫早和福云6号中扩增出唯一的一条扩增带,而这两对引物在其他供试茶树材料中均无相应的扩增带,结果表明已将BHZ498、FY1622标记成功转化成SCAR标记.