玉米ZmNPR1基因的克隆及表达分析

该研究从玉米高抗自交系D863F中克隆了ZmNPR1基因(GenBank登录号为MH619241)的gDNA和cDNA序列,开放阅读框长1 866 bp,编码621个氨基酸,相对分子质量67.61 kD,等电点为5.46。系统进化树比对表明,玉米ZmNPR1蛋白和高粱SbNPR1蛋白的亲缘关系较近,相似性高达97%。实时荧光定量PCR结果表明,ZmNPR1在叶片中能够被水稻黑条矮缩病毒诱导并显著上调表达。同时ZmNPR1在玉米叶片、茎、根、雄穗、雌穗以及花丝中均有表达,在雌穗和叶片中的表达量较高。研究表明,ZmNPR1可能在玉米粗缩病抗病过程中起着重要作用。     

Abscisic acid negatively modulates plant defence against rice black‐streaked dwarf virus infection by suppressing the jasmonate pathway and regulating reactive oxygen species levels in rice

Abscisic acid (ABA) plays a multifaceted role in plant immunity and can either increase resistance or increase susceptibility to some bacterial and fungal pathogens depending on the pathosystem. ABA is also known to mediate plant defence to some viruses. In this study, the relationship between the ABA pathway and rice black‐streaked dwarf virus (RBSDV) was investigated in rice. The expression of ABA pathway genes was significantly reduced upon RBSDV infection. Application of exogenous hormones and various ABA pathway mutants revealed that the ABA pathway plays a negative role in rice defence against RBSDV. Exogenous hormone treatment and virus inoculation showed that ABA inhibits the jasmonate‐mediated resistance to RBSDV. ABA treatment also suppressed accumulation of reactive oxygen species by inducing the expression of superoxidase dismutases and catalases. Thus, ABA modulates the rice–RBSDV interaction by suppressing the jasmonate pathway and regulating reactive oxygen species levels. This is the first example of ABA increasing susceptibility to a plant virus.     

水稻黑条矮缩病毒基因组片段S9的cDNA克隆和全序列分析

应用RT PCR技术克隆了 3个中国水稻黑条矮缩病毒 (riceblack streakeddwarfvirus,RBSDV)分离株基因组片段S9,并测定了它们的全序列。结果表明 :RBSDV浙江分离株 (RBSDV Zj)基因组片段S9全长 190 0nt(EMBL登录号为AJ2 97430 ) ,RBSDV河北分离株 (RBSDV Heb)基因组片段S9全长 1898nt(EMBL登录号为AJ2 9742 9) ,湖北分离株S9全长 190 0nt(EMBL登录号为AJ2 9170 6 )。 3个分离株S9均含有两个开放阅读框(ORF) ,分别编码约 40kD和 2 4kD的多肽。3个中国分离株之间的核苷酸同源性高达 98.5 %～ 98.8% ,与日本分离株S9的核苷酸同源性均为 89.9%～ 90 .2 % ,而与意大利MRDVS8同源性仅为 85 .3%～ 86 .4%。我们发现ORF2十分保守 ,4个RBSDV分离株S9的ORF2同源性高达为 97.6 %～ 10 0 % ,与意大利MRDVS8的ORF2同源性也高达 94.3%。     

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.     

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。     

Transmission by Laodelphax striatellus Fallen of Rice black‐streaked dwarf virus from Frozen Infected Rice Leaves to Healthy Plants of Rice and Maize

Rice black‐streaked dwarf virus (RBSDV) is transmitted naturally to important crops such as rice, maize, barley and wheat in a persistent manner by the planthoppers, Laodelphax striatellus, Unkanodes sapporona and Unkanodes albifascia. Insect vector transmission tests are the basis for identifying viral incidence, evaluating the resistance of varieties and selecting resistance sources for rice and maize breeding. A simple, rapid and reliable method is described by which virus‐free small brown planthoppers (L. striatellus) acquired RBSDV from frozen infected rice leaves and transmitted it to healthy rice and maize plants. After feeding on frozen infected rice leaves, the planthoppers were tested by RT‐PCR for the presence of virus after 10, 15, and 22 days, respectively. The percentages of RBSDV‐containing insects were 0, 25 and 71.43% of L. striatellus fed on frozen infected rice leaves compared to 0, 28.25 and 71.43% of L. striatellus fed on fresh infected rice leaves, respectively. In transmission tests, three of eight rice seedlings (37.5%) and four of eight maize seedlings (50%) were inoculated by the planthoppers that had fed previously on frozen leaves and had allowed a 22 days latent period and showed typical disease symptoms. As a positive control, four of eight rice seedlings (50%) and four of six maize seedlings (66.67%) became infected. All rice and maize plants expressing disease symptoms were identified as virus‐positive by RT‐PCR. These results indicated that the planthoppers acquired RBSDV from frozen infected leaves and transmitted the virus to healthy plants.