转基因玉米株系纯合系选育及其农艺性状表现

目的：以携带水稻矮缩病毒（RDV）运动蛋白缺陷型（MP-）基因的转基因玉米种子T0为试验材料,通过分子分析、抗病鉴定及农艺性状筛选得到转基因纯合株系。方法：首先对转化种子进行潮霉素抗性筛选,其后对各代转基因材料进行PCR检测、农艺性状调查和抗病鉴定。结果：从645粒T0转化种子得到抗潮霉素植株246株,即T1转基因植株。T1、T2、T3、T4代材料的PCR检测阳性率分别为56.9%、83.9%、94.6%和99.8%,证明RDVMP-基因已被导入玉米自交系中,且目的基因可以稳定遗传到转基因植株及其后代。田间人工接种抗病鉴定结果表明,经过连续筛选,转基因后代植株（系）的抗病性不断提高,T1、T2、T3和T4代中的高抗病材料分别占总材料数的8.9%、31.5%、70.7%和100%;所选纯合系连续2年的发病率均为0,抗病性比相应对照株系提高4级。农艺性状调查结果表明,转基因株系的株高比对照株系高15.0～33.4cm,穗位高提高13.4～20.2cm,;穗长增加2.0～3.8cm,穗粒数增加10.2～22.8粒。结论：根据分子检测、田间抗病鉴定及农艺性状鉴定结果,选育到96C0502、96C0507和96C0513等抗矮花叶病转基因玉米纯合株系。     

玉米粗缩病的分子研究新进展

粗缩病是一种世界性玉米(Zea mays)病害,造成玉米产量降低和品质下降。已有的研究表明,导致玉米粗缩病的病毒有4种,均属于植物呼肠孤病毒科、斐济病毒属(Fijivirus)第2组的成员,它们的全基因组均由10条双链RNA片段组成,编码13个蛋白分子;迄今未发现对粗缩病完全免疫的研究材料,但已筛选出少量在不同环境下均表现高抗的种质。玉米抗粗缩病为多基因控制的数量性状,每条染色体上均有可能存在与粗缩病抗性有关的基因座(QTLs)。粗缩病毒侵染玉米后,引起细胞防御系统中相关基因表达、蛋白质合成和激素含量等生物途径发生变化。该文对玉米粗缩病病原分子特征和遗传变异、抗性种质遗传基础及致(抗)病机理等方面的研究成果进行了阐述,旨在为玉米抗粗缩病分子育种提供理论依据。     

外源RNA干涉基因在烟草中的转化及表达

依据RNA干涉机制,以TMV复制酶基因为靶标基因,针对TMV 5个株系复制酶基因间高度同源序列设计引物,经RT-PCR反应获得靶序列,构建靶序列反向重复结构的RNA干涉双元载体.用根癌农杆菌介导将外源基因转化至烟草品种K326基因组中,培育RNA干涉转基因烟草.人工接种病毒验证转基因烟草中外源基因在植物抗病毒能力方面的表达效果,实时荧光定量PCR分析转基因烟草抗病毒能力.结果表明,实验培育的RNA干涉转基因烟草67%对TMV呈现高度抗性;荧光定量PCR分析显示,对TMV具高度抗性的转基因烟草中病毒复制酶基因转录产物mRNA存在很大程度的降解,证实了RNA干涉技术在培育抗病毒烟草品种中的效果.     

转水稻NibT基因玉米植株的获得及抗病性研究

以玉米(Zea mays L.)自交系'金黄96B'为受体材料,供体为质粒pWM101并携带有水稻矮缩病病毒复制酶基因Nib的提前终止突变体基因NibT,采用超声波处理花粉介导植物基因转化方法将NibT基因导入受体,经PCR检测和Southern杂交分析证实获得转基因植株,进而对T1～T3代转基因植株(株系)进行分子分析、田间抗病鉴定和农艺性状调查.逐代分子检测分析结果证明,目的基因可稳定遗传.抗病鉴定结果证明转基因植株(株系)各代抗病水平基本一致,抗病性比对照提高3级.农艺性状调查分析表明,与对照比较,转基因植株株高增加7～18 cm、穗位高增高0～13 cm、穗长增加0.7～2.1 cm、穗粒数多8～35粒、百粒重增加1.1～2.6 g,转基因株系与阴性对照间、各代转基因株系相互间都差异显著(P＜0.05);调查还发现转基因植株的株高和穗位高随着世代的增加,与对照间的差异逐代减少.研究也说明,超声波处理花粉介导植物基因转化方法是一种简捷、快速和有效的植物转化工具.     

植物抗病毒分子机制

在与植物病毒的长期斗争中,植物进化出多种抗病毒机制,其中RNA沉默和R基因介导的病毒抗性是最受人们关注的两种机制.一方面,RNA沉默是植物抵抗病毒侵染的重要手段.植物在病毒侵染过程中可形成病毒来源的双链RNA,经过DCL蛋白的切割、加工形成sRNA,与AGO蛋白结合形成RISC指导病毒RNA的沉默,用于清除病毒.相应地,病毒在与植物的竞争中进化出RNA沉默抑制子,抑制宿主RNA沉默系统以逃避宿主RNA沉默抗病毒反应,增强致病能力.另一方面,植物也进化出R基因介导植物对包括病毒在内的多类病原的抗性.R蛋白直接或间接识别病毒因子,通过一系列的信号转导途径激活植物防御反应,限制病毒的进一步侵染.对植物抗病毒的研究有助于人们对植物抗病分子基础的理解,有重要的科学意义和潜在应用价值.本文综述了植物抗病毒分子机制的重要进展.     

植物转基因沉默研究进展

随着转基因技术在植物中的广泛应用,转基因沉默受到越来越多的重视。转基因沉默可发生在转录和转录后两种水平,其基本特征就是依赖于同源的重复序列。转基因的重复拷贝间,转基因与同源的同源基因间及RNA病毒与同源转基因间都会发生基因沉默。可能有不同的机制导致转基因沉默。本文综述了转基因沉默的机理研究及转基因沉默在植物抗病基因工程和植物功能基因组方面的应用。     

浙江和河北发生的一种水稻、小麦、玉米矮缩病是水稻黑条矮缩病毒引起的

以浙江省的水稻黑条矮缩病和河北省的玉米粗缩病的病毒分离物为材料,对我国南北方两种病毒分离物进行了比较研究。两种病毒分离物的粒子大小形态相似,且在血清学上密切相关;二者的介体、寄主相同,并引起相同的症状,提纯两种病毒分离物的基因组片段凝胶电泳显示它们相应的基因组片段之间大小极相似。根据水稻黑条矮缩病毒（Rice black strecaked dwarf virus),RBSDV的S7和S8设计引物,利用PR－PCR技术,在两种病毒分离物中均可特异扩增到预期大小的片段,序列测定比较分析表明：它们的同源性达97．0％－98．9％,与日本RBSDV的同源性（92．2％－95．5％）高于与意大利MRDV的同源性（76．6％－88．4％）。从而认为我国南方的水稻黑条矮缩病和北方和玉米粗缩病都是同一种病毒－RBSDV引起的,也就是说我国北方的玉米粗缩病病原实际上是水稻黑条矮缩病毒,而不玉米粗缩病毒。     

植物MicroRNA介导的基因调控在作物改良中的应用潜能

植物Micro RNAs(mi RNAs)是长度约22个核苷酸的內源、单链、非编码、小分子RNA,通过降解或抑制靶m RNA介导转录后沉默。随着高通量测序技术及各种研究技术的进步,越来越多的micro RNA被测序和验证,为利用基因工程手段进行作物改良提供了丰富的候选基因资源。综述了mi RNAs在生物胁迫、非生物胁迫和植物生长发育等方面的最新研究进展,同时分析了mi RNAs介导的基因调控在作物改良中的应用潜能,并介绍了几种基于mi RNA作用机制的植物基因工程改良作物的转基因新技术,如靶基因RNAi,人工mi RNAs(ami RNAs),Target Mimics(TM),超表达mi RNA-resistant tagerts;也讨论了基于mi RNA作用机制的转基因技术所面临的风险和挑战。     

基于CRISPR/Cas9系统的拟南芥双链结合蛋白AtHyl1基因编辑

RNA沉默是植物重要的抗病毒防御机制,双链RNA结合蛋白(dsRNA-binding proteins, DRB)是RNA沉默信号途径中的关键蛋白。DRB1/HYL1是拟南芥基因组编码的7个DRBs之一,本研究将人工合成含2个AtHyl1靶位点序列的串联t RNA-gRNA片段导入CRISPR/Cas9表达载体中,构建双靶点的CRISPR/Cas9表达载体,通过转化拟南芥dcl2drb4双突变体获得36株转基因阳性植株。对经测序分析可能已发生基因编辑的3株进行单克隆测序分析,测序结果表明均已发生编辑,获得了AtHyl1基因被编辑的拟南芥dcl2drb4突变体T_1代转基因植物。该结果为研究AtHyl1是否参与DCL4介导的抗病毒RNA沉默通路提供了帮助。     

转WMV-2外壳蛋白基因西瓜植株的病毒抗性

西瓜是夏季的重要水果,病毒病是影响其品质和产量的重要原因之一。植物基因工程的发展为抗病育种提供了新途径。利用外壳蛋白(coat protein)基因转化高等植物,赋予转基因植物以相应抗病性的成功例子已很多。本文报道WMV-2CP基因在自交子一代的分离符合孟德尔3：1的分离比。经过连续4代的选择鉴定,已从T7、T11和T323个独立转化子的后代中筛选获得8个转基因纯合株系,性状表现整齐一致。Western blot结果表明,R4T7-1、T4T11-3以及R4T32-73个不同来源的株系均能表达产生外壳蛋白。转基因纯合株系WMV-2感染后的病毒抗性实验表明,与未转基因对照相比,转基因株系可以推迟发病时间,减轻发病程度。实验筛选获得的转基因株系R4T32-7表现出对WMV-2的高度抗性,为利用植物转基因技术选育抗病新品种奠定了基础。     

Enhanced Virus Resistance in Transgenic Maize Expressing a dsRNA-Specific Endoribonuclease Gene from E. coli

Maize rough dwarf disease (MRDD), caused by several Fijiviruses in the family Reoviridae, is a global disease that is responsible for substantial yield losses in maize. Although some maize germplasm have low levels of polygenic resistance to MRDD, highly resistant cultivated varieties are not available for agronomic field production in China. In this work, we have generated transgenic maize lines that constitutively express rnc70, a mutant E. coli dsRNA-specific endoribonuclease gene. Transgenic lines were propagated and screened under field conditions for 12 generations. During three years of evaluations, two transgenic lines and their progeny were challenged with Rice black-streaked dwarf virus (RBSDV), the causal agent of MRDD in China, and these plants exhibited reduced levels of disease severity. In two normal years of MRDD abundance, both lines were more resistant than non-transgenic plants. Even in the most serious MRDD year, six out of seven progeny from one line were resistant, whereas non-transgenic plants were highly susceptible. Molecular approaches in the T12 generation revealed that the rnc70 transgene was integrated and expressed stably in transgenic lines. Under artificial conditions permitting heavy virus inoculation, the T12 progeny of two highly resistant lines had a reduced incidence of MRDD and accumulation of RBSDV in infected plants. In addition, we confirmed that the RNC70 protein could bind directly to RBSDV dsRNA in vitro. Overall, our data show that RNC70-mediated resistance in transgenic maize can provide efficient protection against dsRNA virus infection.     

Efficient Inoculation of Rice black‐streaked dwarf virus to Maize Using Laodelphax striatellus Fallen

Maize rough dwarf disease caused by Rice black‐streaked dwarf virus (RBSDV) is the most important disease of maize in China. Although deploying disease resistant hybrids would be the most effective way to control the disease, development of resistant hybrids has been limited by virus transmission rates that are too low for effective screening. An efficient inoculation technique for RBSDV was developed using Laodelphax striatellus Fallen, in which a virus‐free planthopper colony was developed and viruliferous planthoppers were obtained by allowing a 3‐ to 4‐day acquisition access period on RBSDV‐infected wheat plants. Planthoppers were then allowed a 25‐ to 28‐day latent period on wheat seedlings followed by a 3‐day inoculation access period on two‐to‐three‐leaf stage maize seedlings. By 35 days postinoculation, susceptible hybrid ‘Zhengdan 958’, inbred lines of ‘Ye 107’ and ‘Ye 478’ plants showed 100% RBSDV infection with symptoms of stunting plants, darkening leaves and white waxy swellings on underside of leaves. At tasseling stage, average disease indices were from 96.4 to 100.0%. Enzyme‐linked immunosorbent assays were correlated with the presence of symptoms. The high efficiency of RBSDV transmission obtained using this technique provides a reliable procedure to screen for RBSDV resistance in maize.     

Polycistronic artificial miRNA‐mediated resistance to Wheat dwarf virus in barley is highly efficient at low temperature

Infection of Wheat dwarf virus (WDV) strains on barley results in dwarf disease, imposing severe economic losses on crop production. As the natural resistance resources against this virus are limited, it is imperative to elaborate a biotechnological approach that will provide effective and safe immunity to a wide range of WDV strains. Because vector insect‐mediated WDV infection occurs during cool periods in nature, it is important to identify a technology which is effective at lower temperature. In this study, we designed artificial microRNAs (amiRNAs) using a barley miRNA precursor backbone, which target different conservative sequence elements of the WDV strains. Potential amiRNA sequences were selected to minimize the off‐target effects and were tested in a transient sensor system in order to select the most effective constructs at low temperature. On the basis of the data obtained, a polycistronic amiRNA precursor construct (VirusBuster171) was built expressing three amiRNAs simultaneously. The construct was transformed into barley under the control of a constitutive promoter. The transgenic lines were kept at 12–15 °C to mimic autumn and spring conditions in which major WDV infection and accumulation take place. We were able to establish a stable barley transgenic line displaying resistance to insect‐mediated WDV infection. Our study demonstrates that amiRNA technology can be an efficient tool for the introduction of highly efficient resistance in barley against a DNA virus belonging to the Geminiviridae family, and this resistance is effective at low temperature where the natural insect vector mediates the infection process.     

Improving panicle exsertion of rice cytoplasmic male sterile line by combination of artificial microRNA and artificial target mimic

The adoption of hybrid rice caused the second leap in rice yield after the ‘green revolution’ and contributes substantially to food security of China and the world. However, almost all cytoplasmic male sterile lines (A lines) as females of hybrid rice have a natural deficiency of ‘panicle enclosure’, which blocks pollination between the A line and the fertility restorer line as the male (R line) of hybrid rice and decreases seed yield. In hybrid rice seed production, exogenous ‘920’ (the active ingredient is gibberellin A₃) must be applied to eliminate or alleviate panicle enclosure of the A line; however, this not only increases production cost and pollutes the environment, it also decreases seed quality. In this study, we designed a transgenic approach to improve plant height and panicle exsertion of the A line to facilitate hybrid rice production and maintain the semi‐dwarf plant type of the hybrid. This approach comprising two components—artificial microRNA (amiRNA) and artificial target mimicry—can manipulate the differential expression of the endogenous Eui1 gene that is associated with rice internode elongation in the A line and the hybrid. amiRNA is a recently developed gene silencing method with high specificity, while target mimicry is a natural mechanism inhibiting the miRNA function that was also recently characterized. This approach provides a paradigm to tune the expression of endogenous genes to achieve the desired phenotype by combining amiRNA and artificial target mimicry technologies.     

Resistance to Wheat streak mosaic virus generated by expression of an artificial polycistronic microRNA in wheat

Wheat streak mosaic virus (WSMV) is a persistent threat to wheat production, necessitating novel approaches for protection. We developed an artificial miRNA strategy against WSMV, incorporating five amiRNAs within one polycistronic amiRNA precursor. Using miRNA sequence and folding rules, we chose five amiRNAs targeting conserved regions of WSMV but avoiding off-targets in wheat. These replaced the natural miRNA in each of five arms of the polycistronic rice miR395, producing amiRNA precursor, FanGuard (FGmiR395), which was transformed into wheat behind a constitutive promoter. Splinted ligation detected all five amiRNAs being processed in transgenic leaves. Resistance was assessed over two generations. Three types of response were observed in T(1) plants of different transgenic families: completely immune; initially resistant with resistance breaking down over time; and initially susceptible followed by plant recovery. Deep sequencing of small RNAs from inoculated leaves allowed the virus sequence to be assembled from an immune transgenic, susceptible transgenic, and susceptible non-transgenic plant; the amiRNA targets were fully conserved in all three isolates, indicating virus replication on some transgenics was not a result of mutational escape by the virus. For resistant families, the resistance segregated with the transgene. Analysis in the T(2) generation confirmed the inheritance of immunity and gave further insights into the other phenotypes. Stable resistant lines developed no symptoms and no virus by ELISA; this resistance was classified as immunity when extracts failed to transmit from inoculated leaves to test plants. This study demonstrates the utility of a polycistronic amiRNA strategy in wheat against WSMV.     

Genome structure and variability of Fijiviruses

The Fijivirus [Fiji disease virus, rice black-streaked dwarf virus (RBSDV), maize rough dwarf virus (MRDV), and pangola stunt virus (PaSV), the mal de Rio Cuarto virus (MRCV) strain of MRDV, and oat sterile dwarf virus] are distributed worldwide except for north America, and some of them cause serious disease. However, their genomes have not been extensively studied, and limited molecular data are available only for RBSDV, MRDV, PaSV and MRCV. ALl Fijiviruses have 10 segments, with an aggregate genome size larger than in other plant reovirus genera. All viruses analysed possess the same terminal conserved sequences, which differ from those of the phytoreoviruses and oryzaviruses. There are also sequence-specific inverted repeats adjacent to the terminal sequence. With MRDV and RBSDV, at least two of the segments are bicistronic. Homology studies suggest that MRDV and RBSDV, although known as separate viruses, should be considered as geographical races of the same virus. In contrast, limited data suggest that PaSV and MRCV are less close to each other and to MRDV/RBSDV. Electropherotyping has revealed variation among field isolates, with RBSDV, MRDV and MRCV.     

Mapping quantitative trait loci conferring resistance to rice black-streaked virus in maize (Zea mays L.)

Maize rough dwarf disease (MRDD) is one of the most serious virus diseases of maize worldwide, and it causes great reduction of maize production. In China, the pathogen was shown to be rice black-streaked virus (RBSDV). Currently, MRDD has spread broadly and leads to significant loss in China. However, there has been little research devoted to this disease. Our aims were to identify the markers and loci underlying resistance to this virus disease. In this study, segregation populations were constructed from two maize elite lines '90110', which is highly resistant to MRDD and 'Ye478', which is highly susceptible to MRDD. The F(2) and BC(1) populations were used for bulk sergeant analysis (BSA) to identify resistance-related markers. One hundred and twenty F(7:9) RILs were used for quantitative trait loci (QTL) mapping through the experiment of multiple environments over 3 years. Natural occurrence and artificial inoculation were both used and combined to determine the phenotype of plants. Five QTL, qMRD2, qMRD6, qMRD7, qMRD8 and qMRD10 were measured in the experiments. The qMRD8 on chromosome 8 was proved to be one major QTL conferring resistance to RBSDV disease in almost all traits and environments, which explained 12.0-28.9 % of the phenotypic variance for disease severity in this present study.     

基于烟草瞬时表达体系对amiRNA沉默效果快速有效的预验证

amiRNA(artificial microRNA)作为一种诱导基因发生特异性沉默的技术已在多种植物中应用,但设计出的不同amiRNAs在所转化株系中的沉默效率难以预测,因此对amiRNA载体的沉默效率进行预验证是非常必要的。本实验以丹参(Salvia miltiorrhiza)的1个MYB类转录因子基因SmPAP1的mRNA序列为amiRNA作用对象,并挑选2个经在线软件WMD3(Web MicroRNA Designer)设计的amiRNAs,分别命名为amiRNA1-SmPAP1和amiRNA2-SmPAP1,然后通过农杆菌介导将构建的2个amiRNA载体和SmPAP1过表达植物载体在烟草叶片细胞中进行瞬时共表达。结果显示,amiRNA2的表达丰度约是amiRNA1的2倍;amiRNA2对靶标SmPAP1的沉默效率约是amiRNA1的2.5倍;SmPAP1在mRNA和蛋白水平上均与相应amiRNA的表达水平呈显著负相关。因此,amiRNA在烟草细胞中的瞬时表达可快速、有效地对不同amiRNA沉默效果进行预验证,从而为后续的植物遗传转化研究提供重要参考。