p2 of rice stripe virus (RSV) interacts with OsSGS3 and is a silencing suppressor

A rice cDNA library was screened by a galactosidase 4 (Gal4)-based yeast two-hybrid system with Rice stripe virus (RSV) p2 as bait. The results revealed that RSV p2 interacted with a rice protein exhibiting a high degree of identity with Arabidopsis thaliana suppressor of gene silencing 3 (AtSGS3). The interaction was confirmed by bimolecular fluorescence complementation assay. SGS3 has been shown to be involved in sense transgene-induced RNA silencing and in the biogenesis of trans-acting small interfering RNAs (ta-siRNAs), possibly functioning as a cofactor of RNA-dependent RNA polymerase 6 (RDR6). Given the intimate relationships between virus and RNA silencing, further experiments were conducted to show that p2 was a silencing suppressor. In addition, p2 enhanced the accumulation and pathogenicity of Potato virus X in Nicotiana benthamiana. Five genes that have been demonstrated to be targets of TAS3-derived ta-siRNAs were up-regulated in RSV-infected rice. The implications of these findings are discussed.     

广西林朵林场栽针（杉）保阔混交造林的效果研究

广西林朵林场栽针（杉）保阀混交造林试验表明：１９龄和３０龄混交林林分总蓄积量分别为１８４．７８和５１５．５８ｍ３ｈｍ（－２）,比同龄杉木纯林的依次高２３．０％和２１．８％．混交林中主要树种杉木的树高、胸径和蓄积量随混交树种株数比例的增加而降低．混交林林地枯枝落叶层现存量平均为１５．１８ｔｈｍ（－２）,Ｎ、Ｐ、Ｋ、Ｃａ和Ｍｇ５种元素的积累量平均为４２２．２２ｋｇｈｍ（－２）,分别比杉木纯林的高２５．３％和３２．３３％．混交林林地土壤有机质、全氮、水解氮、有效磷和速效钾的平均含量以及孔隙度和持水量都高于杉木纯林．     

水稻条纹病毒蛋白与核酸的特性

提纯的水稻条纹病毒(云南宜良分离物)在电镜下的形态为多型性,但主要是宽8-10 nm,长80-250的分枝丝状体,有些为直径3 nm或8 nm的开环环状体,有些为13 nm宽,130-190 nm长的丝状体,但其基本结构应是直径3 nm、长度不等的丝状体.经聚丙烯酰胺凝胶电泳分析,vRNA4编码的病害特异蛋白(SP)分子量为19.9 kDa,而vcRNA3编码的外壳蛋白(CP)约为33.6 kDa.在非变性条件下,RSV的4条ssRNAs大小分别为3.0×106(ssRNA1)、1.2×106(ssRNA2)、0.9×106(ssRNA3)和0.8×106 Da(ssRNA4),有时出现一条大小为0.58×106 Da的单链RNA(ssRNA5);而4条dsRNAs的分子量分别为4.9×106(dsRNA1)、2.8×106(dsRNA2)、2.0×106(dsRNA3)和1.7×106 Da(dsRNA4).利用制备电泳分离提纯的外壳蛋白免疫家兔,得到了高特异性的抗血清.A蛋白夹心ELISA检测结果表明,RSV-CP与水稻草状矮化病毒(RGSV)CP抗血清有微弱的反应,但与RSV、RGSV的SP抗血清没有反应,而RSV-CP抗血清与RSV-SP及RGSV的SP、CP都无血清学关系,这个结果表明RGSV与RSV之间在进化上具有一定的亲缘关系.     

Molecular characterization of the microRNA-138-Fos-like antigen 1 (FOSL1) regulatory module in squamous cell carcinoma

MicroRNA-138 is one of the most frequently down-regulated microRNAs in cancer. We recently identified 51 candidate targets of microRNA-138 (Jiang, L., Dai, Y., Liu, X., Wang, C., Wang, A., Chen, Z., Heidbreder, C. E., Kolokythas, A., and Zhou, X. (2011) Hum. Genet. 129, 189-197). Among these candidates, Fos-like antigen 1 (FOSL1) is a member of Fos gene family and is a known proto-oncogene. In this study, we first confirmed the microRNA-138-mediated down-regulation of FOSL1 in squamous cell carcinoma cell lines. We then demonstrated the effect of this microRNA-138-FOSL1 regulatory module on downstream genes (homolog of Snail 2 (Snai2) expression and the Snai2-mediated repression of E-cadherin expression), as well as its contributions to tumorigenesis. The microRNA-138-directed recruitment of FOSL1 mRNA to the RNAi-induced silencing complex was confirmed by a ribonucleoprotein-immunoprecipitation assay. Three canonical and three high affinity non-canonical microRNA-138 (miR-138) targeting sites were identified on the FOSL1 mRNA: one in the 5'-UTR, three overlapping sites in the coding sequences, and two overlapping sites in the 3'-UTR. The direct targeting of miR-138 to these sites was confirmed using luciferase reporter gene assays. In summary, we describe an important microRNA regulatory module, which may play an important role in cancer initiation and progression. Our results also provide evidence that microRNAs target both canonical and non-canonical targeting sites located in all areas of the mRNA molecule (e.g. 5'-UTR, coding sequences, and 3'-UTR).     

Movement protein Pns6 of rice dwarf phytoreovirus has both ATPase and RNA binding activities

Cell-to-cell movement is essential for plant viruses to systemically infect host plants. Plant viruses encode movement proteins (MP) to facilitate such movement. Unlike the well-characterized MPs of DNA viruses and single-stranded RNA (ssRNA) viruses, knowledge of the functional mechanisms of MPs encoded by double-stranded RNA (dsRNA) viruses is very limited. In particular, many studied MPs of DNA and ssRNA viruses bind non-specifically ssRNAs, leading to models in which ribonucleoprotein complexes (RNPs) move from cell to cell. Thus, it will be of special interest to determine whether MPs of dsRNA viruses interact with genomic dsRNAs or their derivative sRNAs. To this end, we studied the biochemical functions of MP Pns6 of Rice dwarf phytoreovirus (RDV), a member of Phytoreovirus that contains a 12-segmented dsRNA genome. We report here that Pns6 binds both dsRNAs and ssRNAs. Intriguingly, Pns6 exhibits non-sequence specificity for dsRNA but shows preference for ssRNA sequences derived from the conserved genomic 5'- and 3'-terminal consensus sequences of RDV. Furthermore, Pns6 exhibits magnesium-dependent ATPase activities. Mutagenesis identified the RNA binding and ATPase activity sites of Pns6 at the N- and C-termini, respectively. Our results uncovered the novel property of a viral MP in differentially recognizing dsRNA and ssRNA and establish a biochemical basis to enable further studies on the mechanisms of dsRNA viral MP functions.     

水稻条纹病毒病害特异蛋白的提纯及血清学特性

应用差别ｐ Ｈ 值沉淀蛋白质的原理,建立了水稻条纹病毒病特异蛋白（ Ｓ Ｐ） 的两种提纯方法。这两种方法都可以从病叶中提纯到大量的 Ｓ Ｐ,其粗提纯量分别为０ ．８ 和２ ．０ ｍｇ／ｇ 病叶。通过 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 分离后得到了精提纯的蛋白,其分子量为２０ ．１ ｋ Ｄａ 。将粗提纯和精提纯的 Ｓ Ｐ 分别免疫兔子,制备出效价为５１ ２００ 和６ ４００ 的抗血清。将效价为６ ４００ 的高度特异性的抗血清用于研究 Ｒ Ｓ Ｖ Ｓ Ｐ 与 Ｒ Ｓ Ｖ Ｃ Ｐ 及同属的水稻草状矮化病毒（ Ｒ Ｇ Ｓ Ｖ） Ｓ Ｐ、 Ｃ Ｐ 之间的血清学关系,结果表明, Ｒ Ｓ Ｖ Ｓ Ｐ 的抗血清与 Ｒ Ｇ Ｓ Ｖ Ｃ Ｐ、 Ｒ Ｓ Ｖ Ｃ Ｐ 之间无反应,但可与 Ｒ Ｇ Ｓ Ｖ Ｓ Ｐ 微弱反应;而 Ｒ Ｇ Ｓ Ｖ Ｓ Ｐ、 Ｃ Ｐ 及 Ｒ Ｓ Ｖ Ｃ Ｐ 的抗血清与 Ｒ Ｓ Ｖ Ｓ Ｐ 之间都无血清学反应。结果证实了 Ｒ Ｓ Ｖ 和 Ｒ Ｇ Ｓ Ｖ 之间存在着进化上的亲缘关系     

Development of an insect vector cell culture and RNA interference system to investigate the functional role of fijivirus replication protein

An in vitro culture system of primary cells from white-backed planthopper, an insect vector of Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus, was established to study replication of the virus. Viroplasms, putative sites of viral replication, contained the nonstructural viral protein P9-1, viral RNA, outer-capsid proteins, and viral particles in virus-infected cultured insect vector cells, as revealed by transmission electron and confocal microscopy. Formation of viroplasm-like structures in non-host insect cells upon expression of P9-1 suggested that the matrix of viroplasms observed in virus-infected cells was composed basically of P9-1. In cultured insect vector cells, knockdown of P9-1 expression due to RNA interference (RNAi) induced by synthesized double-stranded RNA (dsRNA) from the P9-1 gene strongly inhibited viroplasm formation and viral infection. RNAi induced by ingestion of dsRNA strongly abolished viroplasm formation, preventing efficient viral spread in the body of intact vector insects. All these results demonstrated that P9-1 was essential for viroplasm formation and viral replication. This system, combining insect vector cell culture and RNA interference, can further advance our understanding of the biological activities of fijivirus replication proteins.     

猪肺炎支原体p97 C末端基因重组腺病毒的构建及其免疫效果

摘要：【目的】构建携猪肺炎支原体（Mycoplasma hyopneumoniae,Mhp）黏附因子p97 C末端基因的重组腺病毒并研究其诱导小鼠产生的免疫反应,为研制新型Mhp疫苗奠定基础。【方法】从Mhp基因组中扩增p97 C末端基因,并将其克隆到穿梭载体pShuttle-CMV中,该重组穿梭质粒经Pme I线性化后电转化到BJ5183-AD-1细胞中进行同源重组获得重组腺病毒DNA,纯化后的重组腺病毒质粒经Pac I酶切线性化后转染AD293细胞以获得重组腺病毒。对该重组腺病毒进行RT-PCR、间接     

植物抗病基因的研究进展

近年来,已有１０多个植物抗病基因被克隆并定序。植物抗病基因编码的蛋白,大多含有富氨酸重复单位（ＬＲＲ）和核苷酸结合位点（ＮＢＳ）等结构。在植物与病原物的互作中,这些蛋白可作为受体识别由病原物无毒基因编码的激发子,从而激发一系列防卫反应,使植物表现出抗病性。克隆的植物抗病基因可用于培育基因工程植株而大大加快育种速度。本文对目前植物抗病基因研究中存在的问题及发展前景也进行了探讨。