水稻黑条矮缩病毒第七号片段的cDNA克隆及其在大肠杆菌中的表达

运用RT-PCR技术,根据玉米粗缩病毒S6的末端序列设计引物,从玉米粗缩病感病材料中克隆得到一条2.2kb长的cDNA片段,序列分析表明,该片段全长2193bp,含两个开放阅读框,分别编码41.0kD和36.3kD的多肽。序列分析结果表明,该cDNA片段及其编码产物与水稻黑条矮缩病毒S7的cDNA序列及编码产物存在最高的相似性,推测该cDNA片段为水稻黑条矮缩病毒的S7片段,而不是玉米粗缩病毒的S6。分别将该片段的两个阅读框克隆到原核表达载体pET21d及pGEXKG中,经IPTG诱导后,两种蛋白均得到了高效的表达。表达产物回收后制备并得到了高效价的抗血清。     

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。     

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.     

Understanding the epidemiological factors that intensify the incidence of maize rough dwarf disease in Spain

Currently the dominant limiting factor to maize production in Spain is caused by Maize rough dwarf virus (MRDV). This study aimed to evaluate the epidemiology factors involved in the increased incidence of MRD disease in Spain. We examined the maize planthopper dynamics and MRDV incidence throughout two maize growing seasons in six locations using a set of eight maize varieties: four Bt‐varieties (BT‐var) and their isogenic counterparts (NBT‐var). Our results indicate that MRDV incidence is greatly influenced by the first colonisation of maize by Laodelphax striatellus but not by Metadelphax propinqua and by the susceptibility of the maize varieties. No significant differences were observed between the BT‐var and NBT‐var, although BT‐var exhibited 1% less MRDV infection than NBT‐var. Cultivated wheat and Lolium perenne were found for the first time to be natural hosts of MRDV. However, wheat does not seem to be a preferred host for the development of L. striatellus. Partial sequencing of genome segments S1–S9 and full sequencing of segment S10 revealed that the Spanish MRDV isolate shares nucleotide identities ranging from 93% to 97% with the available sequences of segments S7–S10 of the Italian MRDV isolate. The highest nucleotide identities with other fijiviruses were observed with Rice black‐streaked dwarf virus. Molecular variability analysis of MRDV isolates collected over a ten years period showed high nucleotide (>97%) and amino sequence identities (>99%) on segment S10, suggesting a low temporal variability.     

玉米粗缩病毒基因组第七组份的cDNA克隆及序列分析

从采自中国河北滦城感病的玉米材料中提取玉米粗缩病毒(MRDV)的双链RNA。根据已知MRDV的部分序列设计引物,反转录、PCR扩增,克隆并测序分析了MRDV的第七片段(S7)cDNA序列。结果表明,S7 cDNA序列全长为1936bp,与国外所测的MRDV S7的序列长度相等,而且S7包含的两个阅读框(ORF1和ORF2)位置无变化。它们的核苷酸和最大开放阅读框(ORF1)同源性分别为877%和91.6%,然而,MRDV S7的片段与水稻黑条矮缩病毒(RBSDV)S8片段的核苷酸和最大开放阅读框(ORF1)有更高的同源性,分别为95.5%和93.5%。     

Genome-Wide Association Implicates Candidate Genes Conferring Resistance to Maize Rough Dwarf Disease in Maize

Maize rough dwarf disease (MRDD) is a destructive viral disease in China, which results in 20–30% of the maize yield losses in affected areas and even as high as 100% in severely infected fields. Understanding the genetic basis of resistance will provide important insights for maize breeding program. In this study, a diverse maize population comprising of 527 inbred lines was evaluated in four environments and a genome-wide association study (GWAS) was undertaken with over 556000 SNP markers. Fifteen candidate genes associated with MRDD resistance were identified, including ten genes with annotated protein encoding functions. The homologous of nine candidate genes were predicted to relate to plant defense in different species based on published results. Significant correlation (R² = 0.79) between the MRDD severity and the number of resistance alleles was observed. Consequently, we have broadened the resistant germplasm to MRDD and identified a number of resistance alleles by GWAS. The results in present study also imply the candidate genes in defense pathway play an important role in resistance to MRDD in maize.     

玉米粗缩病改良新抗源T877的抗性评价

玉米粗缩病是我国玉米产区的一种重要病害。本研究利用自然接虫法初步鉴定了41份玉米自交系对粗缩病的抗性,并对其中有代表性的10份材料进行了3个播期的试验。筛选出3份高抗、4份抗、3份中抗材料,大部分材料(占75.6%)表现为感和高感,抗性较好的材料属于PB亚群。高抗粗缩病自交系T877在不同年份、不同播期间抗性稳定,以此为亲本育成苏玉19等新品种。     

水稻黑条矮缩病毒基因组片段S7的cDNA克隆及全序列分析

应用RTPCR技术克隆了2个水稻黑条矮缩病毒 (rice blackstreaked dwarf virus,RBSDV)中国分离物,即浙江分离物和河北分离物的基因组片段S7,并测定了他们的全序列。结果表明：RBSDV浙江分离物(RBSDVZj)基因组片段S7全长2193nts(EMBL登录号为AJ297427),RBSDV河北分离物基因组片段S7全长2190nts(EMBL登录号为AJ297428),二者均含有两个开放阅读框(open reading frame,ORF),分别编码约41kD和36kD多肽,2个中国分离物核苷酸同源性高达99%,相应的ORF编码的多肽同源性分别为100%和94.4%,与日本RBSDV基因组片段S7核苷酸同源性为93.4%和93.8%,相应ORF编码的多肽同源性分别为98.1%(ORF1)、96.5%和97.8%(ORF2),与意大利MRDV S6核苷酸同源性为85.1%和85.3%,相应多肽同源性分别为92.3%(ORF1)、85.5%和86.8%(ORF2)。     

Proteome profile of maize (Zea Mays L.) leaf tissue at the flowering stage after long-term adjustment to rice black-streaked dwarf virus infection

Maize rough dwarf disease (MRDD) is a viral disease and causes great yield loss. To better understand the effects of MRDD on plant growth and metabolism, comparative proteomic analysis of leaves from virus-infected and normal plants was performed. In order to eliminate the interference of Ribulose-1, 5-bisphosphate carboxylase with low-abundance proteins, total proteins were pre-fractionated by 15% PEG and the proteins from supernatant and precipitated fractions were analyzed by 2-DE, subsequently. Out of approximately 1200 protein spots detected, less than 2% of the spots on the gels were overlapping between the fractions of precipitation and supernatant. We identified 91 differentially accumulated proteins that belong to multiple metabolic/biochemical pathways in plants. Further analysis of these identified proteins indicated that MRDD resulted in dramatic changes in the fundamental metabolism, including glycolysis and starch metabolism, and eventually the significant differences in morphology and development between virus-infected and normal plants. Moreover, MRDD occurrence increased the demands for G-proteins, antioxidant enzymes, lipoxygenases and UDP-glucosyltransferase BX9, which may play important roles in response of plant against virus infection. The results also suggested that MRDD is a complicated disease controlled by multigene participating in different pathways.     

江苏水稻黑条矮缩病毒S10的cDNA克隆序列分析

从来自江苏连云港并在本实验室保存的水稻黑条矮缩病毒接种的病株玉米中提取dsRNA ,采用改进的单引物扩增技术获得了病毒基因组片段S10的cDNA克隆并测定了其全序列。结果表明S10全长 180 1bp ,含有一个ORF ,组织结构与日本报道的RBSDV基本一致 ,核苷酸和推导的氨基酸序列与MRDV的相似性分别为 87.5 %和92 .6 % ,与RBSDV的相似性分别为 93.3%和 96 .4%。该研究也为病毒dsRNA克隆和序列分析奠定了基础     

粗缩病对玉米产量和籽粒品质的影晌

以6个对玉米粗缩病（MRDV）表现不同抗性的玉米品种为材料,研究了粗缩病对玉米产量性状和籽粒品质的影响。结果表明,在供试品种中,‘青农105’和‘青农8’为抗病品种,‘登海3622’和‘农大108’为中抗品种,‘先玉335’和‘郑单958’为感病品种。感病后,玉米果穗穗长、行粒数、穗粒重和产量显著降低,且损失程度表现为抗病品种〈中抗品种〈感病品种：籽粒中粗淀粉含量显著降低,粗蛋白含量升高,粗脂肪含量变化不明显。回归分析表明,通过旃情指数可以准确预测玉米粗缩病导致的产量损失。     

Identification of a major quantitative trait locus for resistance to maize rough dwarf virus in a Chinese maize inbred line X178 using a linkage map based on 514 gene-derived single nucleotide polymorphisms

Maize rough dwarf disease (MRDD) is a worldwide viral disease and causes significant yield losses in maize (Zea mays L.) production. In this study, we mapped and characterized quantitative trait loci (QTL) conferring resistance to MRDD using 89 F8 recombinant inbred lines derived from a cross between X178 (resistant parent) and B73 (susceptible). The population was evaluated for MRDD resistance in Baoding, Hebei Province, China (a hot spot of MRDD incidence) under natural infection in 2008 and 2009 and artificial inoculation in 2010. Genotypic variances for disease severity index (DSI) were highly significant in the population. Heritability estimates for DSI evaluation were 0.472 and 0.467 in 2008 and 2009, respectively. The linkage map was constructed using 514 gene-derived single nucleotide polymorphisms (SNPs) and 72 simple sequence repeat markers, spanning a genetic distance of 1,059.72?cM with an average interval of 1.8?cM between adjacent markers. Multiple-QTL model mapping detected a major QTL for MRDD resistance on chromosome 8, explaining 24.6?C37.3% of the phenotypic variation across three environments. In 2010, an additional QTL was detected on chromosome 10, explaining 15.8% of the phenotypic variation. The major QTL on chromosome 8 and the SNP markers (SNP31, SNP548, and SNP284) co-located with the QTL peak have potential for further functional genomic analysis and use in molecular marker-assisted selection for MRDD resistance in maize.     

The use of insecticides to control maize rough dwarf virus (MRDV) in maize*

Insecticides were screened in the laboratory for fast action against the delphacid planthopper, Laodelphax striatellus, the vector of maize rough dwarf virus (MRDV) in Israel. In these tests the synthetic pyrethroids were more efficient than oxydemeton-methyl or endosulfan giving 50% mortality after 5 h compared to 10% in the non-pyrethroid insecticides. Selected insecticides were then tested in maize fields for their effect on disease spread and crop yield. The synthetic pyrethroids cypermethrin, fluvalinate and biphenthrin reduced MRDV incidence by 22–59% and disease was less severe in the treated plots. Total crop yield (stems, leaves, ears) was 28% higher in the biphenthrin-and fluvalinate-treated plots than in untreated plots.     

Identification of promoter motifs regulating ZmeIF4E expression level involved in maize rough dwarf disease resistance in maize (Zea Mays L.)

Maize rough dwarf disease (MRDD, a viral disease) results in significant grain yield losses, while genetic basis of which is largely unknown. Based on comparative genomics, eukaryotic translation initiation factor 4E (eIF4E) was considered as a candidate gene for MRDD resistance, validation of which will help to understand the possible genetic mechanism of this disease. ZmeIF4E (orthologs of eIF4E gene in maize) encodes a protein of 218 amino acids, harboring five exons and no variation in the cDNA sequence is identified between the resistant inbred line, X178 and susceptible one, Ye478. ZmeIF4E expression was different in the two lines plants treated with three plant hormones, ethylene, salicylic acid, and jasmonates at V3 developmental stage, suggesting that ZmeIF4E is more likely to be involved in the regulation of defense gene expression and induction of local and systemic resistance. Moreover, four cis-acting elements related to plant defense responses, including DOFCOREZM, EECCRCAH1, GT1GAMSCAM4, and GT1CONSENSUS were detected in ZmeIF4E promoter for harboring sequence variation in the two lines. Association analysis with 163 inbred lines revealed that one SNP in EECCRCAH1 is significantly associated with CSI of MRDD in two environments, which explained 3.33 and 9.04 % of phenotypic variation, respectively. Meanwhile, one SNP in GT-1 motif was found to affect MRDD resistance only in one of the two environments, which explained 5.17 % of phenotypic variation. Collectively, regulatory motifs respectively harboring the two significant SNPs in ZmeIF4E promoter could be involved in the defense process of maize after viral infection. These results contribute to understand maize defense mechanisms against maize rough dwarf virus.     

粗缩病对不同抗性玉米品种叶片生理特性的影响

以6个对玉米粗缩病（MRDV）表现不同抗性的玉米品种为材料, 研究了粗缩病对玉米叶片叶绿素含量、超氧化物歧化酶（SOD）活性和可溶性蛋白含量的影响。结果表明, 感病后, 各品种叶片叶绿素含量、SOD活性和可溶性蛋白含量显著降低, 叶绿素含量和SOD活性下降幅度表现为感病品种〉中抗品种〉抗病品种, 可溶性蛋白含量下降幅度表现为抗病品种〉中抗品种〉感病品种（‘青农105’除外）。对病情指数与各生理指标变化幅度的相关分析发现, 叶绿素含量和SOD活性的下降幅度与病情指数均呈显著正相关, 除‘青农105’外的5个品种可溶性蛋白含量的下降幅度与病情指数呈极显著负相关。这说明, 品种对粗缩病的抗病性与感病后各生理指标的变化幅度有关; 品种抗性越强, 感病后叶绿素含量和SOD活性下降幅度越小, 可溶性蛋白含量下降幅度越大。     

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.     

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.     

Impact of 9 years of Bt-maize cultivation on the distribution of maize viruses

This study assesses the effect of Bt-maize on the distribution of maize viruses. Random surveys were conducted in Spain between 2001 and 2006 to evaluate the occurrence of maize viruses in Bt-maize cultivation areas and in areas where this crop had not been introduced. Maize dwarf mosaic virus (MDMV) was the predominant virus in Bt-areas, and Maize rough dwarf virus (MRDV) was the most predominant one in non-Bt-areas, with MRDV an emergent virus in both types of areas. A decline in the occurrence of MDMV and an increase in that of Sugarcane mosaic virus was observed in Bt-areas. Additionally, data obtained over 6 years in experimental fields showed non-significant differences between the infection rates exhibited by two generations of Bt varieties and the non-transformed isogenics varieties for any of the viruses. Our data suggest that differences in virus distribution are linked to the genetic background of the maize varieties and the distribution of virus reservoirs rather than to Bt-maize cultivation.     

转人工miRNA抗玉米粗缩病毒载体玉米的培育及其抗病能力

玉米是重要的粮食作物,水稻黑条矮缩病毒(RBSDV)是玉米粗缩病的病原,由其引起的玉米粗缩病给玉米生产造成重大损失。利用人工mi RNA构建抗病毒植物的技术已经在多种植物中被证明有效,但是在玉米中的尝试未见报道。实验根据玉米zea-mi R159a的前体序列和RBSDV基因组中编码功能蛋白的基因和基因沉默抑制子的序列信息设计引物,构建了用于沉默RBSDV编码基因和基因沉默抑制子的ami RNA(Artificial mi RNA)基因。构建p CAMBIA3301-121-ami RNA植物表达载体,利用农杆菌介导法转化玉米自交系综31(Z31)。对转基因玉米进行分子检测,选择mi RNA表达量高的纯合体株系进行自然发病实验,按0-4的分级标准调查玉米粗缩病的严重度。结果表明,转抗粗缩病毒人工mi RNA载体玉米纯合体株系的抗病表现好于野生型玉米,其中针对基因组6的S6-mi R159转基因玉米抗病情况较好。研究表明利用人工mi RNA技术构建抗病毒病玉米新品种是可行的。     

Genomic features of intertypic recombinant sabin poliovirus strains excreted by primary vaccinees

The trivalent oral poliomyelitis vaccine (OPV) contains three different poliovirus serotypes. It use therefore creates particularly favorable conditions for mixed infection of gut cells, and indeed intertypic vaccine-derived recombinants (VdRec) have been frequently found in patients with vaccine-associated paralytic poliomyelitis. Nevertheless, there have not been extensive searches for VdRec in healthy vaccinees following immunization with OPV. To determine the incidence of VdRec and their excretion kinetics in primary vaccinees, and to establish the general genomic features of the corresponding recombinant genomes, we characterized poliovirus isolates excreted by vaccinees following primary immunization with OPV. Isolates were collected from 67 children 2 to 60 days following vaccination. Recombinant strains were identified by multiple restriction fragment length polymorphism assays. The localization of junction sites in recombinant genomes was also determined. VdRec excreted by vaccinees were first detected 2 to 4 days after vaccination. The highest rate of recombinants was on day 14. The frequency of VdRec depends strongly on the serotype of the analyzed isolates (2, 53, and 79% of recombinant strains in the last-excreted type 1, 2, and 3 isolates, respectively). Particular associations of genomic segments were preferred in the recombinant genomes, and recombination junctions were found in the genomic region encoding the nonstructural proteins. Recombination junctions generally clustered in particular subgenomic regions that were dependent on the serotype of the isolate and/or on the associations of genomic segments in recombinants. Thus, VdRec are frequently excreted by vaccinees, and the poliovirus replication machinery requirements or selection factors appear to act in vivo to shape the features of the recombinant genomes.