沙特阿拉伯Al-Makhwah省大豆黄花叶病毒衣壳蛋白基因的核苷酸序列

Azza G. Farag, E. Khattab, Ibtesam M. Al-sham
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摘要

本研究首次在沙特阿拉伯Al-Makhwah省的豆科植物(Phaseolus vulgaris)中鉴定到大豆黄花叶病毒(Bean Yellow Mosaic Virus, BYMV),并测定了BYMV衣壳蛋白基因的核苷酸序列。采用BYMV机械接种了12科30种植物和栽培品种。其中17例出现全身花叶、黄化、静脉清净、发育不良、斑纹花叶、畸形及严重花叶症状。接种4 ~ 6天后,发现苋菜藜和藜麦藜是局部病害寄主。两种蚜虫,桃蚜。以蚕豆蚜虫为研究对象,研究BYMV的传播。蚕豆蚜是最有效的媒介,占BYMV传播的60%。采用酶联免疫吸附试验(ELISA)、组织印迹免疫结合试验(TBIA)和斑点印迹免疫结合试验(DBIA)等免疫技术对BYMV进行研究。得到阳性反应,证实该病毒在花和种子部位流行。采用BYMV1和BYMV2特异引物(基于BYMV NIb-CP保守序列设计)从大豆组织总RNA中扩增RT-PCR产物。扩增出全长700 bp的核包涵体和外壳蛋白基因区引物(5′- nib - cp3′)cDNA片段。采用Southern blot和Dot blot杂交技术,利用地高辛标记的BYMV cDNA探针检测BYMV侵染大豆植株。大豆和蚕豆感染BYMV后均有较强的阳性反应。对BYMV Potyvirus Al-Makhwah KSA分离物(700 nt) nib区3′端和CP区5′端进行了部分测序和分析(DNA序列提交GenBank accc .no.)。LC025531)。Al-Makhwah KSA分离物BYMV Potyvirus (NIb-CP)与日本分离物的同源率为99%,与美国剑兰分离物和另一日本分离物的同源率分别为95%和93%,证实了BYMV病毒群主要在基因组的某些特定部位多样化,特别是在CP区,而NIb基因非常保守。
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Nucleotide Sequence of Capsid Protein Gene of Bean yellow mosaic potyvirus in Bean Plants from Al-Makhwah Governorate, Saudi Arabia
The current study represents the first identification of Bean Yellow Mosaic Virus (BYMV) in bean plants (Phaseolus vulgaris) from Al-Makhwah Governorate, Saudi Arabia and nucleotide sequencing of capsid protein gene of BYMV. Thirty plant species and cultivars to twelve different families were mechanically inoculated by BYMV. Seventeen of them showed systemic symptoms mosaic, yellowing, vein clearing, stunting streaking mosaic, malformation and severe mosaic as a result of BYMV infection. Chenopodium amaranticolor and Chinopodium quinoa L. were found to be local lesion host after 4-6 days of inoculation. Two aphids, Myzus persicae Sluz. and Aphis faba were used to study the transmission of BYMV. Aphis faba was found to be the most effective vector with 60% of BYMV transmission. Immunological techniques namely Enzyme Linked Immuno Sorbent Assay (ELISA), Tissue Blot Immuno Binding Assay (TBIA) and Dot Blot Immune Binding Assay (DBIA) were amplified to study BYMV. Positive reaction was obtained and the prevalence of the virus in the flowers and seed parts was confirmed. The RT-PCR products were amplified from total RNA of bean plant tissues using specific primer BYMV1 and BYMV2 (designed on conserved sequences of BYMV NIb-CP). A cDNA fragment of 700 bp nuclear inclusion body and coat protein gene region primer (5'-NIb-CP 3') was amplified. Digoxigenin-labelled BYMV cDNA probe through Southern blot and Dot blot hybridization techniques were employed for the detection of BYMV infected bean plants. A strong positive reaction was observed with bean and faba bean infected with BYMV. A part of the 3' end of Nib-region and the 5' end of the CP region of BYMV Potyvirus Al-Makhwah KSA isolate (700 nt) was sequenced and analyzed (The DNA sequence submitted in GenBank acc.no. LC025531). Identity percentage of Al-Makhwah KSA isolate BYMV Potyvirus (NIb-CP) with a Japanese isolate was 99% and with USA gladiolus isolate and another Japanese isolate were 95% and 93%, respectively confirming that BYMV viral group is diversified mainly in some specific parts of genome, especially in the CP region, whereas NIb gene is very conservative.
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