Expression of Potato Virus Y Coat Protein Gene in Transgenic Potato

Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium-mediated gene transfer. A large number of regenerated putative transgenic plants were obtained based on kanamycin resistance. Using total DNA purified from transgenic plants as templates and two oligonucleotides synthesized from 5' and 3' of the PVY coat protein gene as primers, the authors carried out polymerase chain reaction (PCR) to check the presence of this gene and obtained a 0. 8 kb specific DNA fragment after 35 cycles of amplification. Southern blot indicated that the PCR product was indeed PVY CP gene which had been integrated into the potato genome. Enzyme-linked immunosorbent assay (ELISA) of our transgenic plants showed that CP gene was expressed in at least some transgenic potato plants.     

转WMV-2外壳蛋白基因西瓜植株的病毒抗性

西瓜是夏季的重要水果,病毒病是影响其品质和产量的重要原因之一。植物基因工程的发展为抗病育种提供了新途径。利用外壳蛋白(coat protein)基因转化高等植物,赋予转基因植物以相应抗病性的成功例子已很多。本文报道WMV-2CP基因在自交子一代的分离符合孟德尔3：1的分离比。经过连续4代的选择鉴定,已从T7、T11和T323个独立转化子的后代中筛选获得8个转基因纯合株系,性状表现整齐一致。Western blot结果表明,R4T7-1、T4T11-3以及R4T32-73个不同来源的株系均能表达产生外壳蛋白。转基因纯合株系WMV-2感染后的病毒抗性实验表明,与未转基因对照相比,转基因株系可以推迟发病时间,减轻发病程度。实验筛选获得的转基因株系R4T32-7表现出对WMV-2的高度抗性,为利用植物转基因技术选育抗病新品种奠定了基础。     

马铃薯S病毒外壳蛋白基因的克隆与原核表达

依据马铃薯S病毒 (PotatovirusS ,PVS)外壳蛋白 (CP)基因序列 (885bp)设计合成了两对引物 ,通过RT PCR扩增得到长 0 .8kb的目的片段 ,将目的片段转入大肠杆菌 ,酶切鉴定证明得到了含有目的片段的重组子 ,测定序列结果与其他PVS分离物CP基因的序列比较 ,发现其核苷酸同源性达 95 %左右 ;构建了含PVSCP基因的融合蛋白原核表达载体 ,并在大肠杆菌中得到表达 ,SDS PAGE测定融合蛋白的分子量为 5 8kD。     

应用核酶体外切割马铃薯卷叶病毒外壳蛋白基因的研究

针对马铃薯卷叶病毒外壳蛋白基因第356～358位点“GUC”．设计、合成了一种“锤头状”核酶。将核酶基因克隆在体外转录载体PSPT19的SP6启动子下游;同时将PLRVCPcDNA亚克隆在体外转录载体pSPT18的SP6启动子下游。利用SP6RNA聚合酶分别体外转录,获得核酶分子和靶RNA序列。在41℃保温进行核酶切割反应,检测到预期大小且被切开的两个RNA短片段。     

马铃薯X病毒外壳蛋白的表达水平与变偶密码子使用频率的相关性

把经密码子修饰的马铃薯X病毒（Potato Virus X, PVX）外壳蛋白（Coat Protein, CP）基因和未修饰的野生型外壳蛋白基因与CaMV 35S启动子融合后,构建成相应的植物表达载体,利用农杆菌介导转化烟草。分别对修饰CP和野生CP的转基因烟草进行Western blot和ELISA分析,结果表明经密码子修饰的PVX外壳蛋白的表达量是野生型蛋白表达量的1/3～1/5。Northern blot结果表明修饰和未修饰的外壳蛋白在转录水平上是一致的。以上结果暗示外源基因中稀有密码子的数量可能是限制外源基因表达的一个因素。改变基因中稀有密码子的数量有可能成为控制基因表达的一种有效途径。     

烟草环斑病毒外壳蛋白基因的原核表达及抗血清的制备

烟草环斑病毒(Tobaccoringspotvirus,TRSV)是我国二类进境检疫危险性有害生物,对农业生产危害较大。本研究依据TRSV外壳蛋白基因cp序列设计合成了2条引物,通过RT-PCR扩增得到长约1500bp的目的片段。将目的片段与质粒pET-22b( )连接,构建了含TRSVcp基因的融合蛋白原核表达载体pETRSV-CP。序列分析表明,TRSV-SD1的cp基因全长1548bp,编码515个氨基酸与GenBank中其它TRSV分离物cp基因相比,核苷酸及推导的氨基酸序列同源性为90.7%~94.6%。将pETRSV-CP转入大肠杆菌,诱导表达。SDS-PAGE结果显示,表达的TRSVCP融合蛋白的相对分子质量约为58kDa。以此融合蛋白制备的抗血清的效价为1/1024,抗血清与TRSV具有良好的特异性反应。     

马铃薯卷叶病毒外壳蛋白基因的合成,分子克隆和全序列分析

以分离自马铃薯主栽品种“紫花白”的马铃薯卷叶病毒(PLRV)分离物的RNA为模板,用人工合成的引物,用反转录和随后PCR扩增的方法合成了PLRV外壳蛋白(CP)基因的cDNA,并克隆于pUC19中。进一步用限制酶切和核苷酸序列分析表明,合成的cDNA由627个核苷酸组成(包括起始和终止密码),序列中有Hinc Ⅱ和BamH Ⅰ两个酶切位点,与国外报道一致。和国外的4个PLRV分离物CP基因序列对比结果,具有高度同源性,其同源率达99.0—99.7％。     

翻译和非翻译马铃薯Y病毒外壳蛋白基因介导的抗病性比较

利用RT－PCR方法克隆获得马铃薯Y病毒烟草叶脉坏死株系（PVY^N）的可翻译和不可翻译外壳蛋白（CP）基因,并分别插入pROKⅡ质粒中获得重组双元表达载体。通过根癌农杆菌（Agrobacterium tumefaciens）介导的基因转化方法,将可翻译和不可翻译的PVY^N-CP基因分别导入烟草栽培品种NC89叶片组织中,得到抗卡那霉素的再生植株,对所得到的抗卡那霉素的再生苗进行PCR检测表明,被导入可翻译和不可翻译CP基因的植株分别占卡那霉素抗性植株的95％和98％。攻毒实验表明,两种类型的转基因烟草对PVY^N的抗病性具有相似性,其表现型为：免疫、抗病和感病。免疫型转基因植株的抗病性不受接种物类型及其剂量的影响。Southern印迹杂交结果显示,目的基因已经整合到烟草基因组中。Northern印迹杂交证明,两种类型的CP基因都已在RNA水平上得到了表达,但细胞内RNA的积累量与转基因植株的抗病强度成负相关。Western印迹杂交表明,在表达不可翻译PVY^N－CP基因的转基因植株内 以CP蛋白,而在导入可翻译的PVY^N-CP基因的植株内检测到了CP蛋白,且CP蛋白含量与抗病性不存在正相关。本研究结果证明,表达可翻译与不可翻译PVY^N-CP基因的转基因烟草对PVY^N的抗病性均为RNA介导的抗病性。     

黄瓜花叶病毒衣壳蛋白基因转化辣椒研究

The plant expression vector of the coat protein(CP) gene of cucumber mosaic virus (CMV) BS strain was used to transform three kinds of pepper (Capsicum annuum) tissues (cotyledon, stem and root) by agrobacterium-mediated co-cultivation. 53%-68.4% of the total tissues (639) can be induced to be calli, but only cotyledon calli can be further regenerated to form shoots (regenerated efficiency 39.7%). 70%(42/60) of the putative transformed plants were confirmed to have CP gene in their genomes by Southern blot. The mRNAs and the CP were respectively found in 80% of transgenic plants by Northern blot and DAS-ELISA. 24 of the transgenic plants expressing CP gene of BS strain showed three kinds of resistant level (severe symptom, delay of symptomatic development, no symptom) to infection of CMV-BS and of CMV-P. However, there was distinctly higher resistance to inoculation of CMV-BS than that with CMV-P in these transgenic plants.     

改造的马铃薯Y病毒复制酶基因介导高度抗病性

提取马铃薯Y病毒中国分离株(PVY—c)的mRNA作为模板,随机六聚脱氧核苷酸和寡聚dT为引物合成了单链cDNA。通过聚合酶链式反应(PcR)获得了PVY—C的核内含体b(Nib)全长cDNA克隆。在对其进行全序列分析的基础上,构建了PVY—CNIb基因全长．5’端缺失381个碱基和Nib反义RNA三种不同形式高等植物表达载体。在土壤农杆菌LBA4404的介导下,转化烟草生产品种NC89,获得了所有三种表达载体的转基因植株。通过分子生物学检测和抗性分析发现不同形式的Nib基因序列的转基因植株对马铃薯Y病毒表现不同程度的抗性。其中,以5’端缺失的Nlb的基因转化植株表现最好,从总共20个这类转化株系中筛选到4个株系至少在100μg／m1 PVY—C接种浓度下,表现完全的抗病效果。从总共39个全长Nib基因转化株系中,仅有一个株系,在100μg／ml PVY—c的攻毒接种下具有完全的抗病性。所有33个Nib基因反义RNA的转化植株中,无一株系表现完全的抗病效果,但是有部分株系能不同程度地延缓或减轻发病程度,并有部分植株在发病后50d左右有恢复健康的趋势。虽然能够在上述3种形式的Nib基因序列的转基因植物中检测到相应的RNA的转录产物,但是均未能检测到其相应的蛋白表达产物。     

转望江南核糖体失活蛋白基因cassin烟草及其对烟草花叶病毒的抗性

通过构建植物表达载体,由农杆菌介导,将望江南核糖体失活蛋白基因cassin转入烟草。PCR和Southern blot杂交结果证明：外源基因已经以单拷贝整合到烟草基因组内,并且在后代发生遗传分离。RT—PCR和Northern blot杂交结果显示：外源基因可以正常转录。用不同浓度的TMV机械摩擦接种转基因T1、T2代各3个自交株系,以非转基因烟草为阴性对照,实验结果表明转基因烟草对TMV表现出不同程度的抗性。     

番木瓜环斑病毒YsCP基因的序列分析和植物表达载体的构建

用双脱氧链终止法分析了克隆的番木瓜环斑病毒（PRV）Ys株系外壳蛋白（CP）基因的序列,结果表明YsCP基因全长858nt。对PRV国内外16个株系或分离物CP基因的比较发现,YsCP基因与国内株系或分离物CP基因的同源性较高（94．44％～97．68％）,而与国外株系或分离物CP基因的同源性较低（88．88％～92．70％）。CP基因之间的差异主要靠近基因5’端,特别是在YSCP基因第63nt后连续缺失6nt,SmGTHAI和SRI的CP基因也在此处缺失3nt。将YsCP基因插入中间质粒pRokⅡ的CaMV35S启动于和nos终止序列之间形成CP基因的植物表达载体pRPCY,通过三亲交配使pRPCY进入农杆菌LBA4404,与其中的pAL4404构成双元载体系统。     

黄瓜花叶病毒衣壳蛋白存在于被其侵染的烟草叶绿体中

用原生质体法制备出高纯度的完整叶绿体经SDS-PAGE电泳,银染后,发现黄瓜花叶病毒(CMV)侵染的烟草病叶叶绿体蛋白质图谱和健叶叶绿体相比,多出一条染色较弱的迁移率与CMV衣壳蛋白质相同的带,经Western转移,用CMV游离衣壳蛋白亚基抗血清进行斑点酶联(Immunoblot)检测,证明这条带就是CMV衣壳蛋白质。健康叶绿体中加入去掉叶绿体的病叶汁液而制备出的叶绿体中无CMV衣壳蛋白质,说明这不是在叶绿体提纯过程中得到的假象,即衣壳蛋白质存在于被CMV侵染的完整叶片叶绿体中。这个结果否认了以往报道的CMV衣壳蛋白质不存在于烟草叶绿体中的结论。另外还发现,叶绿体中的衣壳蛋白质浓度与叶片症状严重程度呈正相关。     

Expression of Rice Stripe Virus Coat Protein Gene in Transgenic Rice Plants

Rice stripe virus (RSV) is a pathogen of rice stripe disease causing great damage to rice. The disease is transmitted by Laodelphax striatellus and three other planthoppers. RSV infects as much as 37 cereals including rice, wheat, maize and results in a significant reduction in yield in epidemic year. In order to develop efficient means of controlling the disease, authors have studied the amino acid composition of RSV coat protein (CP), synthesized and cloned the cDNA to CP, sequenced the full-length CP gene. Having inserted the RSV CP gene into plant expression vector pROK Ⅱ, authors transformed rice suspension culture via microprojectile bombardment and obtained transgenic plants expressing the CP gene. The suspension culture was initiated by inoculating yellowish, compact and embryogenic calli derived from seeds into suspension medium containing proline and maltose. After being cultured at 26℃ in the dark for about half a year, finely-dispersed and embryogenic suspension culture was estabolished. Before bombardment the suspension culture was evently applied onto three-layered filter-paper discs in a petri dish. CaCl2 and spermidine was employed to coat tungsten particle with plasmid DNA. 2.5 μl of coated particle was loaded onto bullet and each dish was bombarded three times. Immediately after being bombarded, the suspensions were cultured in modified N6 medium. 2 days later the suspensions were transferred to the same medium but containing G418, which were subcultured weekly. Being subject to G418 selection for two months, white and fast-growing clones were emerged from the brownish cultures. Green plants regenerated when the resistant calli were transferred to differentiation medium. The regenerated plants were firm enough to grow well in the greenhouse. 10 plants regenerated from G418 resistant calli were tested for their transformed nature by Southern blot using 32P-labelled CP gene as a probe. Among the plants tested, 2 plants showed clearly hy bridizing bands with a molecular weight corresponding to RSV CP gene. Western blot further demonstrated that RSV CP gene was expressed in transgenic rice plants. At present tests on the antiviral effects of transgenic plants by feeding plantphoppers infccted with RSV are being underway.     

悬浮细胞再生表达外壳蛋白的转基因Indica水稻对水稻条纹病毒的抗病性(英文)

合成、克隆了水稻条纹病毒中国株的外壳蛋白基因并进行了序列分析,由Indica水稻成熟胚的愈伤组织形成胚性运浮细胞。用含有CP基因的pROK2表达载体的DNA包被1.09μm直径钨粉颗粒轰击培养细胞。被轰击的培养物在含有G418（40mg／mL,）的培养基中进行选择培养,由对G418抵抗的愈伤组织中获得10株再生株。用32P－dCTP标记的CP基因作为探针,以Southernblot测定其转化特性。由抗病的和对照的植株抽提基因组DNA用EcoRI和BamHI进行酶切,其中两个植株显示出0.6kh和0.7kb两条条交带．其大小与CP基因相对应。Westernblot和ELISA测定进步证明CP（32kDa）在转基因水稻中表达。16株转基因植株和100株对照植株用带毒的叶蝉接种,接种病毒后24d只有37.5％的CP转基因植株产生病毒症状,而对照植株为96％。进一步证明转基因水稻植株具有对RSV的抗病性。转基因植株T1代CP的表达分离比例为3.6:1。     

大蒜花叶病毒外壳蛋白基因cDNA的克隆和序列分析

我们从自然发病的大蒜中分离得到了大蒜花叶病毒。以其基因组ＲＮＡ为模板合成了３＇末端部分ｃＤＮＡ。从中选出一批插入片段在２．０ｋｂ以上的重组克隆,经Ｎｏｒｔｈｅｒｎ点杂交分析证实了所选克隆与基因组ＲＮＡ同源。通过对若干个克隆的插入片段两端部分序列的测定,选出一个克隆ｐＧＭ４９５,其插入片段的长度约为２．４ｋｂ,３′末端存有一个Ｐｏｌｙ（Ａ）结构,它应包含了编码该病毒外壳蛋白全部序列。序列测定的结果表明,这个ｃＤＮＡ片段全长为２３７９ｂｐ,其中含有与酶切图谱分析结果相符的ＥｅｏＲＩ、ＰｓｔＩ及ＢａｍＨＩ酶切位点。第一个终止密码子ＴＡＡ与３′ｇ末端相距２６４ｂｐ,我们根据碱基序列推定的氨基酸序列与其它已发表的Ｐｏｔｙｖｉｒｕｓ的外壳蛋白氨基酸序列以及外壳蛋白翻译后加工的蛋白酶专一切点相比较后推测,编码该病毒外壳蛋白序列可能起始于３′末端上游的１１７０ｂｐ处,共编码３０２个氨基酸,其分子量为３６ｋＤ,略大于ＳＤＳ－ＰＡＧＥ所测定的３３ｋＤ,非编码区域长２６４ｂｐ,富含ＡＴ,并有多个终止密码子的存在。趾３′末端３２～２７ｂｐ处有一个ＡＡＴＡＡ序列。