水稻雄性不育诱导和突变体基因等位测验

用 30 0Gy60 Coγ射线辐照诱变水稻恢复系测 48、测 49、测 64和明恢 77的干种子 ,从M2代中选得 7株雄性不育突变株。遗传观察表明 ,他们分别为单基因隐性遗传 ,双列杂交测验结果分属于 4个不等位的雄性不育基因突变体 :测 64ms、明恢 77ms 1、明恢 77ms 2和测 48ms 1。     

Fast and efficient discrimination of the Isotoma viridis group (Insecta: Collembola) by PCR-RFLP

Identification of collembolan species is generally based on specific morphological characters, such as chaetotaxy and pigmentation pattern. However, some specimens do not match to described characters because these refer to adult specimens, often of one specific sex, or the characters are highly variable in adults (e.g. pigmentation, setae or furcal teeth). Isozymes have frequently assisted species discrimination, and also these may vary with developmental stage or environmental conditions. For identification of single species of the Isotoma viridis group, we present both direct sequencing of the cytochrome oxidase subunit II (COII) gene and a simple DNA-based molecular method.

Five PCR primers amplifying the COII region (717 bp) of the mitochondrial DNA were used. The sequences clearly separated the species I. viridis, I. riparia and I. anglicana, irrespective of colour varieties within the first species. DNA amplification products of different species can also be distinguished by digestion with restriction endonucleases, followed by gel electrophoresis for separation of fragments. This restriction fragment length polymorphism (RFLP), obtained after digestion with the endonucleases TaqI, VspI, MvaI and Bsp143I, revealed specific fragments that separated the three species from each other. Since restriction enzymes are sensitive to single base mutations, we suggest to use a combination of enzymes with at least two species-specific restriction sites when using the RFLP technique. For the I. viridis complex, VspI and Bsp143I appear to be an appropriate combination.     

Analysis of 18S rRNA gene of Octostigma sinensis (Projapygoidea: Octostigmatidae) supports the monophyly of Diplura

The phylogenetic position of Diplura within Hexapoda has been controversial. There are three major lineages in Diplura: Campodeoidea, Projapygoidea, and Japygoidea. However, most of the previous studies were restricted to Campodeoidea and Japygoidea. Until now, only preliminary morphological study on Projapygoidea was reported, and no sequence data from Projapygoidea was available. The main aim of the present study was to investigate the phylogenetic position of Octostigmatidae, one of the three families of Projapygoidea, in Diplura and to test if Diplura are monophyletic. The complete 18S rRNA gene sequences of Octostigma sinensis (Projapygoidea: Octostigmatidae) from subtropical China, together with representative species of Campodeoidea and Japygoidea, and several species of Protura and Collembola were analyzed. The phylogenetic trees were obtained by different methods (neighbor-joining, maximum parsimony, and maximum likelihood) with a chelicerate species as outgroup. Our results suggested that Octostigma was closer to the genus Parajapyx (Japygoidea: Parajapygidae) than to the representative genus of Campodeidae (Campodeoidea). All phylogenetic trees supported the monophyly of Diplura.     

苏云金芽胞杆菌WB5中营养期杀虫蛋白3Aa基因(vip3Aa)的克隆、表达与纯化

营养期杀虫蛋白(vegetative insectcidal protein,Vip)是由苏云金芽胞杆菌(Bacillus thuringiensis,Bt)在营养生长指数中期至稳定期期间分泌产生的一类新型杀虫因子,分为Vip1、Vip2和Vip3三种,以Vip3的研究最为深入。Vip3A对鳞翅目(Lepidoptera)害虫具有广谱杀虫活性,具有重要研究意义。本研究以Bt WB5菌株总DNA为模板,采用PCR方法扩增vip3Aa(Gen Bank登录号:AAM22456)全长,将该片段纯化回收后与p MD18-T连接并转入大肠杆菌(Escherichia coli)DH5α,进行酶切验证和序列测定。将vip3Aa基因片段与经同样酶切的表达载体p Czn1连接,构建重组表达载体p Czn1-vip3Aa,转入大肠杆菌Arctic ExpressTM(DE3),异丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)进行诱导表达。SDS-PAGE结果表明,在沉淀和上清中均有约88 k D VIP3Aa蛋白质表达产物;采用Ni亲和树脂对上清中的VIP3Aa融合表达蛋白进行了纯化,获得了纯化蛋白。本研究成功亚克隆了vip3Aa基因,并表达纯化了VIP3Aa蛋白,为后续研究VIP3Aa蛋白在昆虫中肠的作用受体提供了基础资料。     

小麦光周期基因TaCO9-1A的克隆、功能标记开发及其与冬春性关系的研究

光周期是植物从营养生长转为生殖生长的重要影响因子,CO(constans)基因在模式植物拟南芥(Arabidopsis thaliana)的光周期途径中起重要作用。本研究利用同源克隆技术从普通小麦(Triticum aestivum L.)宁春4号中克隆了CO同源基因Ta CO9-1A(Gen Bank登录号:KM236233)的g DNA(genomic DNA)及c DNA(complementary DNA)。Ta CO9-1A的全长编码区(coding sequences,CDS)为876 bp,编码291个氨基酸,含有CO-like蛋白家族典型的CCT结构域,但不含B-box结构域;系统进化分析表明,Ta CO9-1A蛋白与水稻(Oryza sativa L.)Ghd7及大麦(Hordeum vulgare L.)Hv CO9位于同一分支;蛋白质空间结构分析表明,其CCT结构域的NF-YA2区域较为保守;Ta CO9-1A原核表达蛋白分子量为31 k D,与预测结果一致;实时荧光定量PCR结果显示,Ta CO9-1A在普通小麦抽穗期的根、茎、叶及幼穗均有表达,但根部表达量较低,相对表达量依次为叶茎幼穗根。比较该基因在冬春性不同品种中的核酸序列发现,冬性品种第二外显子存在6个碱基的缺失,针对该差异开发了分子标记,在25份冬春性不同的小麦品种中扩增出511和517 bp两种带型,分别与这些品种的冬春性及在杨凌地区两年的抽穗及开花时间早晚显著相关。本研究结果表明,Ta CO9-1A在小麦春化作用和光周期途径中扮演着重要角色,为研究小麦光周期途径和春化作用途径提供了基础资料,有助于揭示Ta CO9-1A调控小麦冬春性及成熟期的分子机理。     

陆地棉小GTP结合蛋白基因GhROP6的克隆及表达初步分析

为探究ROP基因在棉花抵御逆境胁迫中的生物学功能,利用同源克隆的方法获得一个陆地棉GhROP6基因,通过生物信息学方法分析其理化性质、结构及进化关系,利用实时荧光定量PCR(qRTPCR)技术探究GhROP6基因的组织表达特异性及不同逆境胁迫和外源激素处理下的表达模式,构建GhROP6基因的VIGS载体并转化棉花,利用qRT-PCR技术检测其沉默效率。结果显示,GhROP6基因开放阅读框（ORF）为597 bp,编码一个含198个氨基酸的I类ROP蛋白;多重序列比对结果显示,GhROP6符合ROP蛋白结构特征,且与其他物种ROP蛋白高度同源;进化树分析结果显示GhROP6蛋白与拟南芥AtROP6蛋白同源性最高;GhROP6基因在棉花根、茎、真叶及子叶中均有表达,且在真叶中表达量最高;GhROP6基因对干旱、高盐、低温、高温等胁迫和外源脱落酸（ABA）、生长素（IAA）处理均有不同程度的响应,可能在棉花抗逆反应中扮演着重要角色。GhROP6在棉花的叶片和根部均得到有效沉默,表明已获得GhROP6基因沉默植株。本研究为进一步了解GhROP6基因的分子生物学功能奠定了基础。     

Soil bacterial communities under slash and burn in Mozambique as revealed by a metataxonomic approach

The slash-and-burn system is a subsistence agronomical practice widespread in tropical areas worldwide. This system has been extensively studied,especially for its impacts on agronomical aspects and soil physicochemical properties; however, knowledge of soil microbial diversity under slash and bum is scarce. In this study, for the first time, soil bacterial diversity of three locations from Central Mozambique, where slash and burn has been practiced for different durations of the forest fallow p...     

山羊IGFBP-3基因多态性与体重、屠体性状的关系

以波尔山羊及其杂交后代为实验材料,通过PCR-RFLP技术检测到每个群体均有X spⅠ多态,测序后发现A→G转换是导致出现多态性的原因。对该位点与体尺性状进行最小二乘分析时发现:该位点与断奶重和眼肌面积有关(P<0.05):AG型显著大于GG型(P<0.05),且有AG>AA>GG规律。     

猪体细胞核移植重构胚发育异常及印迹基因表达特征研究的进展

由于猪的器官极可能成为人类未来器官的供应源,猪体细胞核移殖及转基因等方面的研究已成为全球的热点。近几年来,猪体细胞核移植研究取得了一定的进展,但总体来看核移植效率还很低(1%~2%),还需要不断完善核移植程序及对一些基础问题的深入研究。本文主要对猪体细胞核移植重构胚发育异常及“印迹”基因表达特征作一综述与分析,旨在为研究者提供一些有益的启示。     

多位点基因打靶技术的鳜鱼体内实验研究

在转基因动物的研究中,一般采用随机插入的方法。基因在受体动物的基因组中随机整合,严重制约了转基因动物外源基因的稳定遗传,是转基因动物研究亟待解决的问题。转基因定点整合技术,即基因打靶技术是解决上述问题的重要途径,但是基因打靶存在打靶效率太低的问题。本研究小组已建立了体外动物细胞的多位点基因打靶技术,并构建了用于鳜鱼的体内多位点基因打靶的打靶载体,本文开展多位点基因打靶技术的鳜鱼体内实验研究。以鳜鱼rDNA基因(编码rRNA的基因)及其间隔序列作为同源重组引导序列,构建含干扰素基因的打靶载体,以其间隔序列为靶位点,针对鱼类胚胎发育特点,采用精子介导技术进行多位点基因打靶,最终获得定点整合干扰素基因的鳜鱼,并建立一套适用于动物的基因定点敲入的体内基因打靶技术。主要研究内容如下:(1)采用组织培养方法,获得鳜鱼头肾淋巴细胞和脾淋巴细胞,采用脂质体介导法使多位点基因打靶载体在鳜鱼的细胞上实行基因转移。采用正负筛选方法,利用筛选药物G418和GCV(更昔洛韦)筛选转染后的细胞两周后,对存活细胞进行DNA水平和RNA水平鉴定。结果证明了干扰素基因在鳜鱼细胞上实现了定点整合和稳定表达。(2)利用精子介导法将经线性化处理后的多位点基因打靶载体在鳜鱼的胚胎上实行基因转移。(3)根据靶位点定点整合的正负筛选原理,利用筛选药物G418和GCV筛选与富集定点整合后的阳性鱼胚或鱼苗,结果成功孵化出106尾转基因鳜鱼鱼苗。(4)培育6个月后,取20尾转基因鳜鱼进行鉴定。采用PCR、RT-PCR、测序等方法进行定点整合的鉴定。结果表明:6尾检测到已转入的基因——干扰素基因,其中有3尾实现了定点整合并表达。本研究率先将多位点基因打靶技术应用到转基因鱼的研究中,建立一套适用于鱼类的高效、安全的多位点基因打靶技术,并获得外源基因定点整合并表达的鳜鱼。