烟草花叶病毒装配起始位点反义RNA表达型中间载体的构建

利用基因工程方法培育抗病毒植物新品种的途径之一,是在植株中建立一个产生病毒基因组功能片段的反义RNA的系统。本工作设计并合成了一段烟草花叶病毒(TMV)装配起始位点反义RNA的基因,再以pBR 325为基本质粒,构建了包含带有花椰菜花叶病毒(CaMV)35S启动子和PolyA信号的反义RNA表达单元,以及为筛选转基因植株所必须的NPT-Ⅱ表达单元的中间载体,为以后经土壤农杆菌而获得转基因烟草植株打下了基础。     

放线菌素D对烟草花叶病毒RNA在离体烟叶中复制的影响

本文报道放线菌素D(AMD)在离体烟叶中对烟草花叶病毒(TMV)的增殖及其核酸(TMV-RNA)复制的影响。按每克叶片用40μgAMD处理,能抑制70％以上叶组织总RNA的合成,在此剂量下AMD对病毒增殖及其核酸复制的影响与用药的时间有密切关系。在接种病毒前5小时或于接种同时给药对病毒增殖和病毒RNA复制都有强烈的抑制作用,可抑制90％以上;而接种后8小时用药就不再表现抑制作用了;接种后24小时用药不但不表现抑制作用,相反对病毒增殖和TMV—RNA的复制都有一定的刺激作用。AMD对TMV增殖和对TMV-RNA复制的影响完全一致。     

流行性出血热病毒R22株cDNA克隆及其特异性鉴定

用家鼠型流行性出血热病毒R22株RNA,经polyA接尾,以Oligo-dT做引物,合成cDNA。用pUC18为载体转染E.coli Mc1061,建立cDNA克隆。再经菌落杂交,选择病毒特异性的5个阳性克隆制成缺口翻译探针,与病毒RNA3个片段进行反杂交,确定RNA片段的特异性。结果表明,3个克隆为中(M)片段的cDNA,另两个分别为大(L)和小(S)片段cDNA。核苷酸序列分析证明,克隆的DNA中含病毒特异的核苷酸序列。     

烟草花叶病毒RNA双链复制型的分离和鉴定

用”P标记感染了TMV‘的普通烟草,从叶组织中提取总RNA,井用纤维素柱层析分离,得到了TMV-RNA的复制型。它在高离子强度下抗Rnase,在低离子强度下对Rnase敏感,并具有能自我退火和能与TMV-RNA杂交等性质。用聚丙烯酰胺凝胶电泳法测得其分子量为3．8×106。根据以上性质可以确认它是对TMV-RNA有专一性的双链RNA。     

定量测定黑鲷生长激素受体mRNA的液相杂交/RNase保护法

目的　建立液相杂交 核糖核酸酶保护测定 (RibonucleaseProtectionAssay,RPA)技术定量检测黑鲷(Sparusmacrocephalus)生长激素受体mRNA水平。方法　将黑鲷生长激素受体cDNA片断亚克隆至pGEM T载体 ,制备特异性的放射性反义RNA探针及正义RNA ,将反义RNA探针与正义RNA及样品总RNA进行液相杂交 ,用RNaseA和RNaseT1 降解杂交产物的单链RNA ,双链杂交体得到保护 ,然后检测杂交体的分子大小及放射强度。结果　检测出了黑鲷生长激素受体反义RNA探针与正义RNA及样品总RNA的特异性杂交片断 ,由此建立了定量测定黑鲷生长激素受体mRNA的液相杂交 RNase保护法 ,并采用该方法在黑鲷的多种组织中检测出了生长激素受体基因的表达 ,表达水平在肝脏中最高。该结果与我们曾采用生长激素放射受体分析法对黑鲷生长激素受体所研究的结果相吻合。结论　为进一步深入研究鱼类生长激素受体分子内分泌的调控理论提供了有力手段。     

登革热病毒基因组末端cDNA的克隆及序列分析

采用磁性分离技术从登革热病毒(D2-04株)感染的C6/36细胞中分离了D2-04病毒RNA.以该RNA为模板进行RT-PCR,分别扩增了D2-04 RNA 5′和3′端cDNA片段,该cDNA片段分别克隆到pGEM-3Z质粒多聚接头的HincⅡ位点得到含有5′端284 bp及3′端525 bp cDNA的重组质粒.通过荧光标记引物及双脱氧核苷酸PCR方法测定了上述cDNA插入片段的序列.同源性比较结果证明D2-04株与其他不同株间的同源性较高,可达93%～98%;不同型间的同源性较差, 仅80%左右; 属间的同源性更低.     

登革病毒2型全长cDNA感染性转录体的构建和鉴定

为构建登革病毒感染性克隆, 针对登革病毒2型基因组全长cDNA的体外转录方法及感染性转录体进行研究。采用长链RT-PCR技术, 扩增DEN2 NGC株全长基因组cDNA, 以之为模板, 用SP6 RNA聚合酶系统制备体外转录RNA转录体, 分别经乳鼠脑内接种及电穿孔转染BHK-21细胞, 观察其感染效应。并从受染鼠脑和病变细胞中提取总RNA, 进行RT-PCR扩增、克隆测序以及电镜观察。结果发现, 从感染鼠脑和细胞中经RT-PCR均可扩增出病毒特异的基因片段, 大小与预期一致; 并从乳鼠脑组织和BHK-21细胞中观察到恢复病毒颗粒。上述结果表明本文成功构建的DEN2 NGC株病毒全长cDNA的体外转录体具有感染性, 乳鼠脑内接种途径与电穿孔转染细胞一样可成为体外转录体感染宿主细胞、获得恢复病毒的方法。     

猪瘟病毒反义基因在培养细胞上的效应研究——反义基因对猪瘟病毒的抑制作用

应用免疫荧光抗体技术检测了整合有猪瘟病毒反义基因的PK-15细胞克隆对猪瘟病毒的抑制效应。结果表明,五个不同的反义基因片段对猪瘟病毒的抑制效率存在很大差异,其中A片段的抑制效率最高(94％～98％),B片段次之(58％～76％),C片段再次(～64％),D片段和E片段未见明显的抑制效应。抑制效率的差异可能与反义基因片段的位置、长短以及反义RNA表达质粒载体的差异有关。     

利用优化的5′-RACE实验定位副溶血弧菌基因的转录起始位点

目的:优化5′-cDNA末端快速扩增(5′-RACE)实验平台,用于定位副溶血弧菌(VP)基因的转录起始位点。方法:提取VP的总RNA,用rDNaseⅠ消化去除可能污染的基因组DNA;利用T4 RNA连接酶将已知序列的寡核苷酸片段连接至RNA的5′端,进而将其逆转录成cDNA;以cDNA为模板,采用巢式PCR技术扩增目的基因DNA片段,并将其直接克隆入T载体;最后通过测序比对的方法确定靶基因的转录起始位点。利用引物延伸实验进一步研究VPA1027的转录起始位点,以检验5′-RACE实验结果的可靠性。结果:5′-RACE实验结果表明,VPA1027、scrG、scrA、cpsA及VPA0198的转录起始位点分别为G(-103)、G(-70)、T(-205)、C(-129)和G(-238)(翻译起始位点为+1);引物延伸结果显示,VPA1027的转录起始位点也为G(-103)。结论:优化后的5′-RACE实验可以精确定位VP基因的转录起始位点。     

烟草花叶病毒的核糖核酸5′-端帽子结构在病毒颗粒中的位置与病毒的感染力

兔抗m~7GMP血清与烟草花叶病毒(TMV)制剂反应能产生免疫沉淀、并抑制TMV的感染力达90%以上。~(32)pCp在RNA连接酶作用下与TMV制剂反应,分离~(32)p标记的TMV再经过核糖核酸酶(RNaseT_2)水解,电泳分离得到~(32)pm~7G~(5′)ppp~(5′)Gp,这些结果说明TMV病毒颗粒中的RNA的5′-端帽子结构可能暴露在病毒颗粒的外部,因而容易与抗m~7GMP血清及~(32)pCp反应,同时也说明TMV的RNA5′-端帽子结构与TMV的感染力有密切关系。     

The interaction between tylophorine B and TMV RNA

Tylophorine B exhibits 60% inhibition against tobacco mosaic virus (TMV) at a concentration of 1.0 x 10(-6) g/ml. In our study, high affinity for TMV RNA and assembly origin of TMV RNA (oriRNA) was revealed, accompanied by the conformational change of RNA. Considering that TMV assembly begins with the specific recognition by the coat protein aggregate of oriRNA, and that tylophorine B has favorable interaction with oriRNA, we speculate that tylophorine B likely exerts its virus inhibition by binding to oriRNA and interfering with virus assembly initiation. This work may shed light on the possible molecular inhibition mechanism against TMV by tylophorine B, and provide clues in rational design of sequence-specific RNA binding antivirus drugs.     

Cooperative Thermal Denaturation of the Assembly Origin Region of TMV RNA

Abstract

The assembly origin (AO) region of the tobacco mosaic virus RNA melts in an unusually narrow(2.5°C) temperature range. In an 0.01 M phosphate buffer the melting temperature of AO was found to be 41.5°C. This value corresponds to the regions with the most stable secondary/tertiary structure of the whole TMV RNA molecule. It is assumed that the AO region has a specific tertiary structure, which is maintained by the long-range interactions as well as by interactions of the pseudoknot type.     

烟草—TMV相互作用中质膜NADPH氧化酶的组装

比较 Ｎ Ｎ 烟草（与烟草花叶病毒（ Ｔ Ｍ Ｖ）发生非亲和相互作用）和普通烟草 ３００２ 品种（与 Ｔ Ｍ Ｖ 发生亲和相互作用）在烟草— Ｔ Ｍ Ｖ 的相互作用中质膜 Ｎ Ａ Ｄ Ｐ Ｈ 氧化酶的组装激活、产生活性氧的差异．用两相法制备密闭的正向型质膜（ Ｐ Ｍ）囊泡,以 Ｓ Ｏ Ｄ 敏感的 Ｎ Ａ Ｄ Ｐ Ｈ 依赖的 Ｃｙｔ ｃ 的还原表示 Ｎ Ａ Ｄ Ｐ Ｈ 氧化酶的活性,用人类噬中性白细胞 Ｎ Ａ Ｄ Ｐ Ｈ 氧化酶亚基 ｐ４７ ｐｈｏｘ 的抗体对烟草叶片蛋白进行免疫学检测．结果显示在两种烟草叶片胞质中均存在与 ｐ４７ ｐｈｏｘ 亚基的抗体发生免疫交叉反应的相同分子量的蛋白,该蛋白在 Ｔ Ｍ Ｖ 侵染 Ｎ Ｎ 基因烟草后可向质膜发生转移,且伴随有氧化酶活性的升高．而对于普通烟草则无氧化酶膜组分和酶活性的明显变化．以上结果表明,烟草叶片质膜上存在与哺乳动物 Ｎ Ａ Ｄ Ｐ Ｈ 氧化酶相类似的氧化酶,它的组装和激活可能是烟草— Ｔ Ｍ Ｖ 非亲和相互作用早期活性氧的主要来源．     

Efficiency of Nitrous Acid as An Inactivating and Mutagenic Agent of Intact Tobacco Mosaic Virus and Its Isolated Nucleic Acid

When tobacco mosaic virus (TMV) and its isolated nucleic acid (TMV-RNA) were treated with nitrous acid, the nucleic acid was inactivated six times faster than the intact virus. Inactivation of both the infectious entities was exponential with treatment time to 0.1% level of survival. Eight different mutant phenotypes were scored after inactivation of TMV and TMV-RNA to 50, 10, 1.0, and 0.1% survival levels. Significantly more mutants in relation to unaltered isolates were induced at all levels of survival upon nitrous acid treatment of TMV than of TMV-RNA. Furthermore, the proportion of two specific mutant phenotypes was significantly greater in treated TMV than in treated TMV-RNA. No qualitative differences, however, were observed between the mutational spectra of nitrous acid-treated TMV and TMV-RNA. These results indicate that, in the intact virus, the viral capsid protects some of the sites involved in lethality; thus, proportionately more mutants are induced on nitrous acid treatment of TMV versus TMV-RNA.     

Specific encapsidation of fragments of TMV RNA.

The in vitro reconstitution of tobacco mosaic virus (TMV) is initiated by the binding of a disk of TMV protein to the 'disk recognition site', a region of the RNA chain at or near the 5'-terminus for which the disk has special affinity. In order to gain insight into the recognition process, we have studied the ability of disks to encapsidate short RNA fragments produced by partial pancreatic or T1 RNase digestion of TMV RNA. The disk is capable of dicriminating among such fragments, encapsidating only a few of the many present in the digest. The products of encapsidation are short nucleoprotein rods of the same diameter as TMV and of length proportional to that of the encapsidated RNA fragment. The particles differ from TMV, however, in one significant aspect (apart from their length): they possess rings of RNA-free protein at one or both extremities of the rod. In the case of T1 RNase digestion the principal encapsidated fragments were fragments T1 (105 nucleotides) and a family of smaller fragments containing elements of the same sequence. Partial digestion with pancreatic RNase generated only one major fragment (fragment P1; 150 nucleotides) with affinity for the disk. Fragment T1 has been sequenced and shown to represent a portion of the coat protein cistron. Fragment P1 has been partially sequenced but its function is not yet known. Several lines of evidence indicate that fragment T1 is not the disk recognition site. The portion of the TMV RNA chain from which fragment P1 is derived, on the other hand, is encapsidated early in the reconstitution process; thus fragment P1 may contain the disk recognition site. Fragment T1 and fragment P1 both have purine-rich and cytosine-poor sequences near their termini. In addition, fragment T1, and possibly fragment P1, possess a periodicity of order three in purine residues. It seems likely that one or both of the aforesaid properties are largely responsible for the affinity of these fragments for the disk.     

用不同方法把烟草花叶病毒RNA基因导入植物原生质体的研究

应用电激法和聚乙二醇法以及脂质体协调的上述两种方法对烟草和青菜原生质体进行烟草花叶病毒ＴＭＶ－ＲＮＡ的导入试验,并应用酶标免疫技术、电镜观察、半叶接种和十二烷基磺酸钠－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）等方法对在原生质体中增殖的ＴＭＶ进行鉴定。实验证明,虽然电激法和聚乙二醇法均能有效地将外源病毒基因导入植物原生质体,但经阳离子脂质体处理后的ＴＭＶ－ＲＮＡ,其转染效率可提高１０倍以上。ＴＭＶ在原生质体转染４８小时后达到复制高峰。ＳＤＳ－ＰＡＧＥ显示,原生质体转染４８小时后,除出现ＴＭＶ外壳蛋白明显条带外,尚有１条分子量在５０～５５ｋｄ蛋白质条带也明显增强。这些研究结果对植物遗传工程和抗病毒基因有种研究提供重要的数据和基础。     

检测烟草中烟草花叶病毒的RNA斑点杂交法

用普通烟草花叶病毒OM株3′-端约2kb的cDNA为探针,探索了用RNA斑点杂交法对烟草组织中烟草花叶病毒RNA进行检测的条件。这些条件包括用分子杂交法观察云南烟区和上海烟草上分到的烟草花叶病毒与OM株的同源性,从烟草组织中提取烟草花叶病毒的几种方法的比较,使RNA有效地固定在硝酸纤维素滤膜上的方法,烟草组织中是否有干扰RNA固定和杂交的物质,斑点杂交方法检测烟草花叶病毒的特异性、灵敏度等。     

Studies on the biosynthesis of TMV

Summary RNA synthesis was investigated at different stages of systemic infections in which TMV formation approaches synchrony. The phenol-extracted nucleic acids were chromatographed on methylated albumin coated kieselguhr columns. At the onset of the rapid phase of virus particle formation RNA with the same chromatographic properties as TMV-RNA was predominantly synthesized when the leaves were labeled for 1.5 hours with ¹⁴C-uridine. Only a small amount of label was found in the double-stranded replicative form. At late stages of infection proportionally more replicative form was synthesized.When leaves at the start of the rapid phase of virus particle formation were labeled for 3.5 min only, a considerable amount of label was found in the replicative form. The proportion of radioactivity in this structure decreased with increasing labeling time, suggesting an intermediary function of this RNA.