甜菜坏死黄脉病毒外壳蛋白基因在甜菜转基因植株中的表达

甜菜坏死黄脉病毒(Beet Necrotic Yellow Vein Virus,BNYVV)是一种由甜菜多粘菌(Polymyxo be tae)传播的多分体植物病毒．基因组由4～5条单链正意RNA构成^[1]。60年代末,由Tamada首次报道^[2],这种病毒可对甜菜造成严重危害,侵染甜菜后产生丛根症状(Rhizomania),并导致甜菜产量和含糖 量的大幅度下降。除欧洲、北美及日本的严重发生以外,我国自70年代以来在东北、内蒙古及西北许多省区也有大量甜菜丛根病的发生报道^[3]。由于尚无有效药剂及措施用于甜菜丛根病或病毒传播介体的防治．在我国也无法采用大面积轮作作为防治手段,所以目前在世界各地及我国上述地区甜菜丛根病的发病面积逐年扩展,对甜菜生产和制糖业造成直接威胁。针对这一情况．本文报道了含有甜菜坏死黄脉病毒外壳蛋白基因的甜菜植株的转化再生工作,以期在甜菜亲本育种中获得新的抗性材料．为抗病毒品种的培育打下基础。     

宁夏甜菜丛根病的研究

发生在宁夏甜菜上的一种病毒病的病株叶丛主要表现为黄化、焦桔和叶脉黄化坏死。从其分离的病毒粒子呈杆状,宽约20nm,长度为65—110nm、270—300nm和390—420nm,能侵染甜菜、菠菜、昆诺阿藜、苋色藜、番杏,与甜菜坏死黄脉病毒(BNYVV)抗血清呈阳性反应。综上所述,认为该病害是由BNYVV引起的。     

甜菜几丁质酶和β-1,3-葡聚糖酶活性与其对丛根病抗性的关系

应用比较生理学方法,以抗丛根病(rhizomania)性不同的4个甜菜品种为材料,研究了丛根病地和无丛根病地上抗、感病品种的几丁质酶(chitinase)和β-1,3-葡聚糖酶(β-1,3-glucanase)活性与甜菜抗丛根病的关系.抗病品种在病地和无病地上皆比感病品种具有较高的几丁质酶和β-1,3-葡聚糖酶活性,表明这两种酶与甜菜抗丛根病性有关;另外,病地上两种酶的活性均不同程度地高于无病地上,是病原物侵染诱导抗性的表现.     

抗甜菜坏死黄脉病毒单抗轻链可变区基因的克隆及全序列测定

甜菜是温带地区主要糖料作物,甜菜丛根病是其一种毁灭性病害,在世界范围内广泛蔓延,发病田块可以造成２０％—５０％以上的减产,甚至绝收,糖度可以降低４—８度。甜菜坏死黄脉病毒（ＢＮＹＶＶ）是丛根病的主要病源,目前没有好的防治方法。为了探索用基因工程抗体技术与植物转化技术防治丛根病的可行性,首先要把抗ＢＮＹＶＶ的单克隆抗体的可变区基因扩增和克隆出来,本文报道轻链可变区基因的扩增、克隆和全序列分析的结果。材料和方法抗甜菜坏死黄脉病毒的单抗杂交瘤（３Ｃ４）由北京农业大学植物病毒研究室制备［１］。细胞培养于…     

甜菜坏死黄脉病毒外壳蛋白基因的克隆和序列分析

以甜菜坏死黄脉病毒(BNYVV)内蒙分离物的RNA为模板,通过PcR扩增获得外壳蛋白(CP)基因的目的片段。将其重组到pGEM一7zf(+)并转化JMl01得到了含有完整CP基因的重组子。采用双脱氧终止法进行序列分析,结果表明CP基因为567nt,与文献(1]报道相比,氨基酸和核苷酸的同源性分别为98．4％和96．7％。     

生物分子相互作用分析法测定甜菜组织中的钙调素

以甜菜为实验材料,经甜菜坏死黄脉病毒（beet necrotic yellow vein virus, BNYVV）侵染后,用生物分子相互作用分析（biomolecular interaction analysis,BIA）技术测定植物组织中钙调素（calmodutin,CaM）含量,分析在BNYVV侵染下植物组织中CaM的变化。该方法可以实时检测生物分子之间的相互作用,受杂质影响小,可简化样品的前处理,能够快速高通量分析大量的蛋白质样品,灵敏度高,准确性好。     

砂培体系中甜菜多粘菌生物学特性的研究

在改进的砂培体系中，甜菜多粘菌完成生活循环只需７天。利用砂培体系研究了多粘菌在不同ＰＨ值、光照，接种材料和接处量条件下，对寄主的侵染以及在其中繁殖的情况。 多粘菌完成侵染所需时间，侵染的游支孢子最初释放时间，游协孢子体外存活期和休眠孢子对温度的敏感性等生物学特性。     

砂培体系中甜菜多粘菌生物学特性的研究

在改进的砂培体系中,甜菜多粘菌（Polymyxabetae）完成生活循环只需7天。利用砂培体系研究了多粘菌在不同pH值、光照、接种材料和接种量条件下,对寄主的侵染以及在其中繁殖的情况,研究了多粘菌完成侵染所需时间,侵染的游动孢子最初释放时间,游动孢子体外存活期和休眠孢子对温度的敏感性等生物学特性。     

病毒粒子转移到植物原生质体和小孢子中

丹麦Danisco公司科学家M.Joersbo和J.Brunstedt最近描述了利用轻微的声处理转化植物原生质体的报道.他们声称,已采用“新型而有效”的声处理技术把甜菜坏死黄脉病毒(BNYVV)粒子导入甜菜原生质体中,包裹的病毒粒子能在原生质体内复制. 尽管在超声波处理之前通过BNYVV粒子与原生质体的混合可获得最佳的有效的病毒转移,但当原生质体声处理一小时后通过把病毒粒子加入原生质体也可获得显著水平的感染.     

甜菜坏死黄脉病毒75kDa通读蛋白基因构建与表达

利用DNA重组技术,将甜菜坏死黄脉病毒(BNYVV)内蒙分离物的CP基因和54kDa通读区片段拼接。构建了BNYVV 75kDa通读蛋白基因。序列分析表明,构建的75kDa通读蛋白基因与野生型相比．只有4个核苷酸发生了改变(包括将CP基因的终止密码子TAG改造为ATG),相应地2个氨基酸也发生了改变。将75kDa通读蛋白基因及其54kDa片段分别克隆到pJw2上,构建了这两十基因的原核表达载体。SDS—PAGE和western blotting检测结果表明,75kDa通读蛋白基因在E coli BL21(DE3)中经温度(42℃)诱导后除可特异地表达75kDa蛋白外。还产生两种小蛋白。75kDa通读蛋白基因的54kDa片段只表达出37kDa的蛋白。     

Studies on the Biology of Polymyxa betae, the Vector of Beet Necrotic Yellow Vein Virus

The development of Polymyxa betae within the roots of a sugar beet variety susceptible to Rhizomania was observed in hydroponic culture over a period of 10 days. Light microscope studies showed that at an average temperature of 20 °C the life cycle of the fungus, containing beet necrotic yellow vein virus (BNYW), was completed within 10 days. A change from the multiplication phase to the survival phase of P. betae became evident. At the beginning of the life cycle the fungus produced mainly zoospores whereas at a later stage plasmodia developed into resting spores. Zoospore density in the nutrient solution reached a maximum between the 5th and 7th day after inoculation and then declined to the initial concentration. The number of zoospores attached to the root surface increased progressively at 48 h intervals, correlated with a parallel increase in BNYW-content of the roots. Light- and fluorescence microscopy revealed that zoospores of P. betae often attach near the point of release and do not move very long distances. In addition it became evident that zoospores may attach to the thallus wall inside the zoosporangium that they have developed in.     

In vitro dual culture of Polymyxa betae in Agrobacterium rhizogenes transformed sugar beet hairy roots in liquid media

Polymyxa betae is a soil-borne protist and an obligate parasite of sugar beet that transmits the beet necrotic yellow vein virus. Sugar beet hairy roots, transformed by Agrobacterium rhizogenes, were inoculated with surface-sterilized root fragments infected by P. betae. After 10 wk in a liquid medium, typical structures of P. betae were observed in this in vitro system. This first in vitro culture of P. betae in liquid medium will contribute to a better understanding of this protist's biology through providing a way to conserve and produce purified isolates of the protist.     

Arabidopsis thaliana, a new tool to investigate Polymyxa betae-host interactions

Little is known about the genome of Polymyxa betae and its interactions with sugar beet, due partly to the obligate nature of the protist and the patents on Beta vulgaris sequences. The identification of an ecotype of Arabidopsis thaliana compatible with the protist would help to improve this knowledge. The infection and development of P. betae in 14 worldwide ecotypes of A. thaliana were studied. The detection of plasmodia and resting spores and the production of zoospores in the roots of A. thaliana were obtained in three bioassays, using automatic immersion systems and individual glass tubes. Detection was done using molecular detection and microscopy. Compatible interactions were established between 13 A. thaliana ecotypes of the 14 that were tested and the monosporosoric Belgian strain of P. betae, A26-41. The ecotype Cvi-0 (N1096), from the Cape Verde Islands, was the most compatible with the protist. This ecotype is also susceptible to Plasmodiophora brassicae, another plasmodiophorid. Polymyxa betae infection in A. thaliana was relatively very low compared with B. vulgaris, but every stage of the life cycle of the protist was present. The spore-forming phase was promoted at the expense of the sporangial phase, probably caused by the stress of this new environment. In addition, the protist revealed a new phenotype. This new model study will allow molecular tools available for A. thaliana to be used in order to gain a better understanding of the P. betae-plant interaction during the spore-forming phase.     

Changes in Ribonuclease and Glucose-6-Phosphate Dehydrogenase Activities Induced by Beet Necrotic Yellow Vein Virus in Sugar Beet

Activities of host ribonucleases and glucose-6-phosphate dehydrogenase were studied in three cultivars (Monosvalof, Steffi and Rimini) of sugar beet differing in their resistance to beet necrotic yellow vein virus (BNYVV). No differences were found in the susceptibility of cultivars to BNYVV between mechanically inoculated and Polymyxa betae (a natural fungal vector of the virus) infected plants, but the culmination of reproduction curves of BNYVV in mechanically inoculated plants was observed one week earlier than in plants inoculated by means of P. betae. The activities of ribonucleases corresponded with virus multiplication. In roots, activities of ribonucleases reached a maximum at day 7; in leaves, maximum activity was found at day 21 in cv. Monosvalof, and at day 14 in cv. Steffi. The relatively resistant cultivar Rimini showed much lower activities. The activity of glucose-6-phosphate dehydrogenase was only slightly increased at the time of culmination of the BNYVV reproduction curve in cvs. Monosvalof and Steffi. This revised version was published online in June 2006 with corrections to the Cover Date.     

甜菜多粘菌基因组片段的克隆及PCR检测

根据资料报导设计引物,扩增Ｐｏｌｙｍｙｘａ　ｂｅｔａｅ的基因组片段,将其克隆在ｐＧＥＭ－３Ｚｆ（＋）质粒载体上,并通过双酶切、ＰＣＲ扩增和部分序列测定,证明克隆片段为Ｐ．ｂｅｔａｅ基因组片段。用扩增Ｐ．ｂｅｔａｅ基因组片段的引物对由Ｐ．ｇｒａｍｉｎｉｓ侵染小麦和Ｏ．ｂｒａｓｓｉｃａｅ侵染豇豆根系抽提的总ＤＮＡ进行ＰＣＲ扩增,均未获得任何ＤＮＡ产物,进一步证实了上述结果。对移入病土２、３．５天的甜菜苗单株根系进行ＤＮＡ粗提,移入病土３．５天的甜菜苗ＰＣＲ检测其已被Ｐ．ｂｅｔａｅ侵染,对确定Ｐ．ｂｅｔａｅ的早期侵染有重要意义。     

Evidence that Polymyxa species may infect Arabidopsis thaliana

Polymyxa spp. are obligate biotrophs belonging to the plasmodiophorid group, responsible for transmitting a large number of plant viruses to many crop species. Their obligate nature makes them difficult to study. Controlled environment experiments were used to investigate the potential of infection of Arabidopsis thaliana by Polymyxa spp. to provide a more tractable system. Two ecotypes of Arabidopsis, Columbia and Landsberg erecta, were grown in soils known to be infested with Polymyxa. At the end of a 2-month growth period, both ecotypes were found to harbour Polymyxa-like structures or spores. These findings were confirmed by Polymyxa-specific PCR tests and rDNA sequencing, which positively identified the presence of Polymyxa in the roots of both ecotypes of Arabidopsis. Both Polymyxa graminis and Polymyxa betae were identified. This is the first report of infection of Arabidopsis by Polymyxa spp. and shows the possibility of using this system for studies of infection biology and host-parasite interactions.     

Progress towards the understanding and control of sugar beet rhizomania disease

Rhizomania is a soil-borne disease that occurs throughout the major sugar beet growing regions of the world, causing severe yield losses in the absence of effective control measures. It is caused by Beet necrotic yellow vein virus (BNYVV), which is transmitted by the obligate root-infecting parasite Polymyxa betae . BNYVV has a multipartite RNA genome with all natural isolates containing four RNA species, although some isolates have a fifth RNA. The larger RNA1 and RNA2 contain the housekeeping genes of the virus and are always required for infection, whereas the smaller RNAs are involved in pathogenicity and vector transmission. RNA5-containing isolates are restricted to Asia and some parts of Europe, and these isolates tend to be more aggressive. With no acceptable pesticides available to restrict the vector, the control of rhizomania is now achieved almost exclusively through the use of resistant cultivars. A single dominant resistance gene, Rz1 , has been used to manage the disease worldwide in recent years, although this gene confers only partial resistance. More recently, new variants of BNYVV have evolved (both with and without RNA5) that are able to cause significant yield penalties on resistant cultivars. These isolates are not yet widespread, but their appearance has resulted in accelerated searches for new sources of resistance to both the virus and the vector. Combined virus and vector resistance, achieved either by conventional or transgenic breeding, offers the sugar beet industry a new approach in its continuing struggle against rhizomania.