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zmfzj

至尊木虫 (著名写手)

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[交流] 玉米褪绿斑驳病毒(Maize chlorotic mottle virus，MCMV)

The genome organization of the single viral RNA of MCMV is most similar to panicum mosaic virus (PMV). Key taxonomic features of the genus Machlomovirus are a unique open reading frame(ORF) at the 5′end of the genome that largely overlaps the pre-readthrough portion of the viral replicase gene, and a readthrough ORF preceding and overlapping the CP gene near the 3′end of the genome.
The virion consists of a 4437 nt single-stranded RNA surrounded by 25.1 kDa CP subunits that lack the protruding domain found on CPs of viruses from many genera in the family Tombusviridae. Sequence similarity to the CPs of PMV, tobacco necrosis virus genus Necrovirus, family Tombusviridae, and southern bean mosaic virus genus Sobemovirus suggest that MCMV is a T = 3 icosahedral virion with 180 copies of its CP in the viral shell.
The plus-sense RNA is 4437 nt long and contains seven overlapping ORFs that encode proteins of 7 kDa or larger. The RNA was reported to have no poly(A) tail and an m7 G cap at the 5′end. However, in common with other viruses in the family, the encoded MCMV replicase does
not have any motifs characteristic of the methyltransferase domain found in viral replicases of capped RNA viruses. None of the other MCMV-encoded proteins contain a methyltransferase domain, so it is likely that the RNA in MCMV, like in other members of the family, is in fact uncapped. The 5′UTR is 117 nt long, and ORF1 encodes a 32 kDa highly acidic protein. ORF2 begins 19 nt downstream and encodes a 50 kDa highly basic protein so the migration of these two proteins in sodium dodecyl sulfate (SDS) polyacrylamide gels is likely to be anomalous. Suppression of the UAG stop codon of ORF2 would produce a 111 kDa protein. A cluster of four ORFs is encoded in the 30 third of the viral RNA downstream of the transcription start site for
the 1467-nt-long subgenomic RNA1 (sgRNA1). ORF4 encodes a 7.5 kDa protein (p7a), and suppression of its UGA stop codon would produce a 31 kDa protein. The second AUG of sgRNA1 begins ORF7 which encodes the viral CP. ORF6 was identified by similarity of its gene product p7b to small peptides encoded in similar locations on carmoviruses, necroviruses, and PMV, and it begins with a noncanonical start codon. In vitro translation of MCMV virion RNA in rabbit reticulocyte lysate produces p32, p50, p111, and p25. The two 7 kDa peptides and p31 were not detected in rabbit reticulocyte lysate translations. The 3′UTR is 343 nt long and encodes a 337-nt-long sgRNA2.
The replication strategy of MCMV has not been completely determined, but inoculation of maize protoplasts with transcripts from wild type and mutant versions of an infectious cDNA has provided some information. Transcripts with mutations in the 30third of the genome that stop expression of one or more of the proteins encoded on sgRNA1 are capable of replication. Additionally, mutations just upstream of the sgRNA1 transcription start site that stop expression of sgRNA1 but do not alter the sequence of p111 are capable of replication, indicating that none of the proteins encoded on sgRNA1 are necessary for replication. Based on the replication mechanisms of other tombusvirus family members it is likely that after virion disassembly, MCMV viral RNA is translated to produce the viral replicase which then synthesizes the negative strand of genomic RNA after recognizing sequences and structures located at the viral 3′terminus that have sequence and structural similarities to the promoters of carmoviruses. The com-
plementary strand is then used as template for synthesis of progeny viral RNA strands. sgRNA synthesis mechanisms differ between genera in the family Tombusviridae, and it is not known which mechanism is used by MCMV. sgRNA1 synthesis may initiate by replicase binding internally to the sgRNA promoter on the genomic complementary strand. Alternatively, occasional premature termination of viral complementary strand synthesis at a specific location may produce separate complementary strand copies of sgRNA1 that are used as templates to synthesize many copies of sgRNA1. Although sgRNA2 accumulates in infected maize plants and inoculated protoplasts, its function and method of transcription are not known.

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1楼 2010-09-26 17:00:36

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wxy20052002

木虫 (著名写手)

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2楼2010-09-29 09:24:43

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wxy20052002

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zmfzj(金币+85, 翻译EPI+1):谢谢 2010-09-30 08:21:43

玉米褪绿斑驳病毒
玉米褪绿斑驳病毒单一RNA的基因组组成方式与黍花叶病毒属（PMV）非常相似。玉米褪绿斑驳病毒属的关键分类学特征是在基因组5’端具有一个独一无二、与病毒复制酶基因复制前通读部分有较大重叠的开放阅读框；此外，在基因组3’端附近，具有一个通读的开放阅读框，该开放阅读框位于病毒外壳蛋白CP基因上游，并于CP基因重叠。
玉米褪绿斑驳病毒颗粒由一个4437nt的单链RNA组成，周围有分子量为25.1KDa的CP蛋白亚基环绕。在番茄丛矮病毒家族的许多属中，其病毒颗粒的CP蛋白都具有一个凸出的结构域，但玉米褪绿斑驳病毒颗粒的CP蛋白不具有这样的凸出结构。分析玉米褪绿斑驳病毒与黍花叶病毒属、烟草坏死病毒属、番茄丛矮病毒科以及南方菜豆花叶病毒属的序列相似性，研究表明，玉米褪绿斑驳病毒衣壳亚基的组装方式遵循二十面体立体对称T=3的规则，病毒颗粒表面由180个拷贝的CP蛋白组成。正链RNA长4437nt，包含7个重叠的开放阅读框，这些开放阅读框编码的蛋白质分子量为7kDa或者更大。具帽状结构RNA病毒的重组酶序列中，常具有甲基转移酶结构域，但玉米褪绿斑驳病毒重组酶与家族中的其它病毒一样，不具有该结构域的任何特征基序。玉米褪绿斑驳病毒编码的其它蛋白也都不具有甲基转移酶的结构域，因此，与家族中其它成员一样，玉米褪绿斑驳病毒的RNA的帽状结构很可能已经被去除。5’端非翻译区长117nt，开放阅读框1编码一个分子量为32kDa的蛋白，该蛋白具有高度的酸性。开放阅读框2的第一个密码子位于下游19nt处，编码一个分子量为50kDa的蛋白，该蛋白具有高度的碱性。因此这两个蛋白质分子在SDS-PAGE胶中的迁移率很可能是不一致的。若抑制开放阅读框中的UAG终止密码子，使其作用不能发挥，则可以产生一个111 kDa的蛋白质分子。玉米褪绿斑驳病毒的1／33序列处编码一个由4个开放阅读框组成的基因簇，位于转录起始点下游，该基因簇编码一个长1467nt的亚基因组RNA1(sgRNA1)。开放阅读框4编码一个7.5 kDa的蛋白质分子（p7a），若抑制其开放阅读框中的UGA终止密码子，使其作用不能发挥，则可以产生一个31kDa的蛋白质分子。开放阅读框7由亚基因组sgRNA1的第二个AUG处开始，编码病毒外壳蛋白。开放阅读框6具有一个非典型的起始密码子，编码产物是p7b，p7b是一个小肽。在香石竹斑驳病毒属、坏死病毒属和黍花叶病毒属病毒基因组的相同位点处，都编码与p7b相似的小肽。体外翻译玉米褪绿斑驳病毒颗粒的RNA，在兔网织红细胞裂解物中获得四个产物p32，p50，p111和p25。未发现两个7kDa的肽和p31。3′非翻译区长343nt，编码一个337 nt的亚基因组sgRNA2。
玉米褪绿斑驳病毒颗粒的复制机制尚未彻底阐明，然而，通过给玉米原生质体接种一个致病性玉米褪绿斑驳病毒cDNA的野生型和突变型转录产物，人们已经获得了一些数据。终止病毒cDNA亚基因组sgRNA1序列中的一个或多个蛋白质表达，同时在基因组1／33序列处引入突变，病毒基因组转录产物仍然能够复制。另外，在亚基因组sgRNA1转录起始点上游引入突变，同时终止亚基因组sgRNA1的表达，但未改变序列p111，病毒仍然能够复制；表明亚基因组sgRNA1不编码病毒复制的必需蛋白。综合考虑番茄丛矮病毒家族中其它成员的复制机制，很可能在病毒粒子解体后，玉米褪绿斑驳病毒RNA被翻译产生了病毒复制酶，复制酶识别3’末端与香石竹斑驳病毒启动子类似的序列和结构，进而合成了基因组RNA的负链。其互补链再被用作模板以合成子代RNA链。番茄丛矮病毒家族中，不同属有不同的病毒亚基因组RNA合成机制，玉米褪绿斑驳病毒亚采用何种亚基因组RNA合成机制尚不明确。重组酶与亚基因组互补链上的启动子内部结合后，起始亚基因组sgRNA1的合成。另外，病毒互补链的合成过程偶尔会在一个特定位点处发生成熟前终止现象，由此导致产生不同的亚基因组sgRNA1的互补链，这些亚基因组sgRNA1的互补链可以用作合成多种亚基因组sgRNA1的模板。尽管亚基因组sgRNA2可以在感病玉米植株及接种了病毒的玉米原生质体中累积，但其转录机制和功能仍然未知。

图2 玉米褪绿斑驳病毒基因组组成及蛋白质产物。用方框标出了基因组RNA和亚基因组RNA（sgRNA1）上每个阅读框中的7个开放阅读框。同时标出了两个被抑制掉的终止密码子（UAG和UGA），开放阅读框6的非典型的起始密码子用短划线表示。蛋白质用深黑色条形表示，位于其所在的mRNA下方。深蓝色方框代表编码复制酶的区域，该复制酶的蛋白质组成与番茄丛矮病毒家族中具有单分体基因组的其它成员具有高度相似性；淡蓝色方框区域也代表编码复制酶的区域，该复制酶的序列组成仅与黍花叶病毒复制酶相似。菱形表示编码“GDD”基序的区域。开放阅读框4中的绿色方框表示编码保守小肽的序列，黍花叶病毒、香石竹斑驳病毒、坏死病毒中都具有该小肽。弯曲的红色箭头表示sgRNA转录起始点。sgRNA2未包含任何重要的开放阅读框。

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3楼2010-09-29 12:12:59

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