dsRNA介导的PRSV-CP基因3''-端同源序列瞬时表达对番木瓜环斑病毒侵染的影响

体外合成我国番木瓜主产区环斑病毒外壳蛋白(PRsv-CP)基因3'-端278 bp同源区段,构建包含278 bp正义链、内含子(PDK内含子)及278 bp反义链的RNA介导的植物表达载体p2301-CPU,直接转化根癌农杆菌EHA105.通过根癌农杆菌介导的瞬时表达法,研究同源dsRNA对番木瓜环斑病毒(PRSv)侵染的干扰作用.结果表明,瞬时表达的dsRNA能够特异地干扰PRSV侵染.     

MicroRNA用于创制抗PRSV番木瓜新种质的展望

微小RNA(microRNA, miRNA)是真核生物体内调控基因表达的一类非常重要的小分子RNA,在植物逆境适应过程中发挥着重要的作用。总结番木瓜环斑病毒(papaya ring spot virus, PRSV)致病机理研究的最新进展,探讨miRNA在植物抗病毒中的研究进展和应用前景,提出研究番木瓜miRNA参与抗PRSV机制的途径,为开展番木瓜miRNA研究和创制抗PRSV番木瓜新种质提供理论参考。     

dsRNA介导的PRSV-CP基因3＇-端同源序列瞬时表达对番木瓜环斑病毒侵染的影响

体外合成我国番木瓜主产区环斑病毒外壳蛋白（PRSV-CP）基因3＇-端278bp同源区段,构建包含278bp正义链、内含子（PDK内含子）及278bp反义链的RNA介导的植物表达载体p2301-CPU,直接转化根癌农杆菌EHA105。通过根癌农杆菌介导的瞬时表达法,研究同源dsRNA对番木瓜环斑病毒（PRSV）侵染的干扰作用。结果表明,瞬时表达的dsRNA能够特异地干扰PRSV侵染。     

利用dsRNA介导的抗病性获得抗马铃薯Y病毒（PVY）的转基因烟草

为了获得抗马铃薯Y病毒(PVY)转基因烟草,分别扩增PVY HC-Pro基因3’端正、反向片段,构建反向重复植物表达载体.利用农杆菌介导法转化普通烟草(Nicotiana tabacum)云烟85,经抗性筛选及抗病性鉴定,获得T0代转基因抗病烟草株系.繁殖T0代抗病株系,抗病性鉴定发现,部分抗病株系的T1代烟株发生了一定比例的抗感分离.Real-time PCR检测发生抗感分离的T1代烟株,发现抗病烟株中PVY HC-Pro基因RNA积累水平显著低于感病烟株,说明抗病烟株中HC-Pro基因发生了RNA沉默.利用dsRNA技术沉默HC-Pro基因,获得了烟草品种云烟85的T1代转基因抗PVY株系.     

苎麻纤维发育相关的3个miRNA鉴定与表达分析

研究以苎麻品种中苎1号为材料,开展其茎皮小RNA(miRNA)测序.结果显示,在苎麻茎皮中,共检测到378个miRNA,分析了4个管家基因在苎麻6个部位中的表达水平,发现18s在各组织中稳定表达.以18s基因作为内参基因,分析了3个候选miRNA(miR156、miR166和miR828)在苎麻6个部位中的表达谱,发现...     

植物中miRNA及其靶基因的检测与鉴定方法

miRNAs(microRNA,微RNA)作为植物体内一类重要的内源性非编码小分子RNA,能够显著调控细胞发育的多个生物学过程,对植物生长起重要调控作用。诸多研究表明:在植物生长发育过程中miRNA的表达具有组织特异性和时空特异性。因此,检测和分析不同组织或样品中miRNA及其靶基因的种类与表达特征,可为研究miRNA的生物学功能提供重要信息和依据。本文综述了近年来植物中miRNA及其靶基因的检测与鉴定方法及其存在的优缺点,为进一步开展miRNA及其靶基因的功能研究提供借鉴和参考。     

刺葡萄DFR基因植物表达载体构建及转化茉莉花愈伤组织的研究

本研究以从刺葡萄克隆获得的DFR基因为目的基因，同时从质粒pCAMBIA1302中扩增获得CaMV35S启动子和NOS终止子，并利用双酶切法构建由CaMV35S启动子驱动DFR基因且带有NOS终止子的表达载体，验证测序结果显示成功构建了pCAMBIA1302-35S-DFR-NOS植物表达载体，并将该载体转化到农杆菌EHA105中。采用农杆菌注射渗透法转化烟草叶片进行瞬时表达，经鉴定，转化后的烟草叶片中能检测到GFP基因的表达。采用农杆菌介导法将该植物表达载体转化受体茉莉花愈伤组织，经多轮抗性筛选获得了转基因抗性愈伤组织，经PCR鉴定，茉莉花转基因抗性愈伤组织中检测到GFP、Hyg和DFR基因等的存在，初步证明目的基因DFR已成功整合到宿主的基因组中。茉莉花愈伤组织遗传转化体系的建立，验证了花色苷合成相关基因转化茉莉花愈伤组织的可行性，为今后通过植物转基因技术丰富茉莉花的花色奠定基础。     

过量表达大豆MIR319基因提高烟草的低磷耐受性

MicroRNA是一类非编码小RNA分子,在植物磷信号通路中发挥重要的调控作用,前期研究中发现microRNA319(miR319)在低磷胁迫的大豆根中上调表达。为了进一步验证miR319的功能,本文从大豆中克隆了编码大豆miR319b的基因Gm MIR319并将其转化到烟草中,获得阳性转基因植株后鉴定其对低磷的耐受性。过量表达Gm MIR319的转基因烟草在低磷胁迫时根长明显较对照长,地上部和地下部鲜重也显著高于对照,表明GmMIR319能够提高烟草对低磷的耐受性。通过荧光定量PCR分析烟草中miR319推定的靶基因的表达,其中一个靶基因TC18729的表达量随着miR319表达的上升而下降,表明miR319可能通过调控TC18729而调节烟草对低磷胁迫的反应。本文的研究结果将有助于应用大豆miR319调控植物对低磷的胁迫反应,为通过基因工程手段培育磷高效植物提供基因源。     

番木瓜环斑病毒CP基因同源区段的克隆及植物表达载体的构建

采用RT-PCR技术对番木瓜环斑病毒CP基因的同源区段进行了克隆和鉴定分析。序列分析结果表明,获得PRSV-CP基因3′端的278bp DNA片段同源率最高。构建了目的基因包含278bp正义链、内含子(PDK内含子)及278bp反义链的RNA介导的植物表达载体p2301-CPU,并通过电激法导入农杆菌中。经PCR及酶切鉴定,证实质粒已被导入获得了农杆菌工程菌株。利用该植物表达载体对番木瓜的遗传转化工作目前正在进行中。     

利用RNA干扰技术提高大豆脂肪含量

利用冻融法将大豆11S球蛋白GY1基因RNA干扰表达载体转入农杆菌EH105中,以大豆"吉农28"为受体,通过农杆菌介导的大豆子叶节转化法导入大豆,获得T1代转基因苗12株,并对得到的转基因植株进行分子生物学鉴定。PCR和Southern杂交检测表明,RNAi植物表达载体p3301-Gy1已成功插入到转基因大豆植株的基因组DNA中;RT-PCR和SDS-PAGE检测结果表明RNA干扰在转录后水平发挥了作用,11S球蛋白表达含量降低;利用BUCHI N-500型近红外谷物分析仪对转化的大豆蛋白质和脂肪含量进行检测,结果转化植株的籽粒蛋白质含量(36.07%)平均降低1.43个百分点,脂肪含量(21.28%)平均提高0.76个百分点。因此,利用RNA干扰技术提高大豆脂肪含量是可行的。     

Genetic Identification of a New Small Grain Dwarf Gene in Rice

The plant material used in the study was rice line 162d, a new small grain dwarf mutant. Polymorphic analysis of 221 SSR loci demonstrated that 162d derived from a semidwarf variety Shuhui 162 through mutation, and 162d and Shuhui 162 were just a pair of near isogenic lines. Genetic analysis of Fl and F2 populations suggested that dwarfism in 162d was controlled by a single recessive gene. Phenotypic characteristics of the mutant gene were that plant height was about a quarter of normal height, grain size about a quarter of normal size, leaf was short and broad, and seed setting rate was very low,compared with the near isogenic line Shuhui 162. The mutant gene was sensitive to gibberellin (GA3) treatment and did not located on the region near the centromere of rice chromosome 5, where dl gene located. Therefore, it was concluded that the mutant gene of 162d was a new small grain dwarf gene in rice.     

Genetic Identification of a New Small Grain Dwarf Gene in Rice

The plant material used in the study was rice line 162d, a new small grain dwarf mutant. Polymorphic analysis of 221 SSR loci demonstrated that 162d derived from a semidwarf variety Shuhui 162 through mutation, and 162d and Shuhui 162 were just a pair of near isogenic lines. Genetic analysis of F1 and F2 populations suggested that dwarfism in 162d was controlled by a single recessive gene. Phenotypic characteristics of the mutant gene were that plant height was about a quarter of normal height, grain size about a quarter of normal size, leaf was short and broad, and seed setting rate was very low,compared with the near isogenic line Shuhui 162. The mutant gene was sensitive to gibberellin (GA3) treatment and did not located on the region near the centromere of rice chromosome 5, where dl gene located. Therefore, it was concluded that the mutant gene of 162d was a new small grain dwarf gene in rice.     

利用SSR标记分析玉米P-162近缘系的遗传多样性

利用SSR标记技术分析47个玉米P-162近缘系的亲缘关系及遗传多样性,研究P-162自交系的亲缘关系。结果表明,22对多态性较好的随机引物中共检测到154个等基因变异,每对引物检测到等位基因3~12个,平均7.48个。利用UPGMA聚类分析法将供试自交系划分为7类,47个玉米P-162近缘系材料之间具有一定的遗传差异,总体供试材料遗传基础相对较狭窄。     

Occurrence of Telosma mosaic virus causing passion fruit severe mosaic disease in Thailand and immunostrip test for rapid virus detection

The cause of leaf and fruit severe mosaic disease in purple passion fruit (Passiflora edulis) in Chiang Mai province, Thailand, was identified and virus diagnostic kit was developed. Symptomatic plants collected from diseased mother plants in the nursery at the Royal Pangda research station (RPRS) was used for virus isolation through single lesion transfer in Chenopodium amaranticolor, followed by virus propagation for purification in Nicotiana benthamiana, and back inoculation to purple passion fruit. Electron microscopy of leaves with typical symptoms revealed potyvirus-like flexuous rod particles, ca. 750 nm long and pinwheel inclusion bodies in the cytoplasm of infected cells. The deduced amino acid sequence of the complete coat protein revealed that the isolated virus was a strain of Telosma mosaic virus with which it shared 84% identity. A rabbit polyclonal antiserum was produced against purified virions, and used to develop a gold-labeled immunostrip for rapid virus diagnosis. The test strip could detect the virus in diseased passion fruit sap up to a dilution of 1: 640, and positive test line could be read within 3–5 min. Application of strip test for virus assay in RPRS nursery's mother plants help screen clean stocks. This strip test kit supports RPRS program of producing virus-free planting materials for farmers.     

一个新的水稻小粒矮秆突变基因的遗传鉴定

水稻品系162d是一个新发现的水稻小粒矮秆突变体。通过对221个SSR标记位点的多态性分析证明162d是由蜀恢162突变产生的,162d和蜀恢162是一对近等基因系。对F1和F2代的遗传分析表明162d的矮生性由一对隐性基因控制。该基因的表型特点是株高为正常高度的1/4左右,籽粒为正常大小的1/4左右,结实很差,叶短而宽。该基因对赤霉素GA[sub]3[/sub]敏感,不位于d1基因所在的水稻第5染色体着丝点附近区域。因此,认为162d突变基因是一个新的水稻小粒矮秆基因。     

利用农杆菌介导合子胚转化法将Bar基因转入玉米自交系的研究

基于农杆菌介导的合子胚转化方法是一种具有自主知识产权的转基因新方法。利用该方法转化玉米合子胚,可直接获得转化种子。以东北春玉米区玉米自交系8902、Pa91、丹340作为转化受体,通过合子转化法进行抗草丁膦除草剂基因Bar的转基因研究。在获得的3 560份材料中,通过对T_1代种子苗叶面喷施草丁膦除草剂Basta,共获得153株抗性植株,抗性频率为4.3%。PCR鉴定阳性植株为102株,转化频率为2.87%。对PCR检测出的阳性植株进行自交获得T_2代,对材料继续进行抗性筛选,对抗除草剂表型的分离比率进行抗性遗传分析。     

太空诱变百子莲SP1代植株的性状鉴定及观赏性评价

太空搭载诱变是选育新品种的有效方法。本研究以太空搭载种子诱变的百子莲SP₁代实生苗为材料，以国际百子莲新品种测试标准为指导，对29株8~10年生苗的形态指标进行测量。采用主成分分析法从15个形态指标中提取出6个主成分，利用模糊综合评价法对受试植株进行整体观赏性评价；以多样性较高的观赏部位——花（花序）为对象，采用层次－关联分析法对百子莲花部的观赏性进行评价。结果表明，受试植株在15个形态指标上的差异显著，各指标之间存在极显著或显著的相关性，相关性系数集中在0.2~0.6之间。综合主成分分析和主因子分析表明，在百子莲诱变群体中，与株高、花序型相关的因子对观赏性影响较大，这些也是易产生变异的性状。建立综合评价函数对29个受试植株的观赏性进行评价，筛选出总体观赏性较高的植株，如植株506、445、319的花量、花序高度等指标均较高，适合进一步无性繁殖保持其优良性状。对花部的15个指标进行加权灰度关联系数计算，排序前6的指标为花量C₁、花期C₁₃、花色C₇、花序直径C₂、初花度C₁₄和小花苞色C₈，每个指标权重均大于6%，权重总和大于70%。结合连续2 a的调查发现，花色、花梗色、花量、初花度等指标相对稳定，不易受环境影响，由此筛选出花色具有渐变特征的植株445，适合作为杂交育种的材料，通过杂交选育获得更具观赏性的后代。通过单一指标和整体评价相结合的方式进行单一指标的变异株选择，利用热图法评价太空诱变的百子莲成年植株在生长过程中的性状变化。本研究初步建立了百子莲植株和花部的观赏性评价体系，对百子莲新品种的选育具有较好的借鉴意义。     

拟南芥胱硫醚-γ-合成酶( D-AtCGS)抗血清制备及对D-AtCGS转基因大豆的检测

构建了N端缺失拟南芥CGS基因(D-AtCGS)的原核表达载体,对其进行了原核表达、蛋白纯化和抗血清制备,并利用获得的抗血清对国家大豆改良中心北京分心中心获得的组成型表达、种子特异性表达的D-AtCGS转基因大豆及转全长AtCGS( F-AtCGS)和D-A TCGS的豆科模式植物百脉根进行了Western blot检...     

Phenotypic variation in root development of 162 soybean accessions under hypoxia condition at the seedling stage

Soybean is often damaged by hypoxia caused by waterlogging at the seedling stage. Hypoxia severely inhibits root development and retards plant growth. We aimed to clarify phenotypic variation in root development under hypoxia condition at the seedling stage using diverse soybean accessions. Root development in 162 accessions was evaluated in hydroponic culture. Substantial changes under hypoxia were investigated by means of WinRHIZO analysis before and after the treatment. We found significant phenotypic variation in hypoxia tolerance in root among the 162 accessions. A principal components analysis indicated an association between hypoxia tolerance and the country of origin. We found three new accessions which have a high ability to develop roots under hypoxia (Kokubu 7, Maetsue zairai 90B, and Yahagi). Root development in selected accessions was also evaluated in soil culture. Root development levels in hydroponic and soil culture were significantly correlated. These results will provide important information on waterlogging damage in regions where waterlogging occurs. The three accessions with hypoxia-tolerant roots might be useful for genetic improvement of waterlogging tolerance of modern soybean varieties.     

IFS转基因大豆的鉴定及高异黄酮植株的筛选

大豆异黄酮是一种广泛应用的保健性活性物质,近年来已成为衡量大豆品质的重要指标之一。异黄酮合酶是大豆异黄酮合成途径中的关键酶基因之一,其在植物中的表达效率直接影响异黄酮含量。为进一步验证该基因的功能并获得高异黄酮稳定遗传转基因植株,本试验基于已有的IFS转基因材料开展研究。将其扩繁至T2代,考种分析植株农艺性状,发现转基因植株的性状指标未发生明显变化,PCR鉴定IFS转基因后代的结果显示:在125株转基因植株中,60株为阳性,占比48%,说明IFS在后代中可稳定遗传。选取IFS转基因吉林35、Willimas 82品种T2代的41株,利用改良后的三波长法测定籽粒中大豆异黄酮的含量,结果显示:IFS转基因植株的平均异黄酮含量为1.2 mg·g~(-1);其中15株的异黄酮含量高于非转基因植株,占比达到36.6%,说明从转入IFS基因转基因大豆能够筛选出高异黄酮植株。本研究获得了稳定遗传的高异黄酮植株,为大豆遗传育种提供优异的种质资源;改良后的三波长法较原有方法更为精准、快速。