辽宁省玉米矮花叶病毒原鉴定及cDNA克隆与序列分析

Identification cDNA Cloning and Sequence Analysis on Maize Dwarf Mosaic Virus(MDMV)In Liaoning Province

田间采集玉米矮花叶病标样 ,常规摩擦接种检测 ,几个分离物寄主范围局限于禾本科植物 ,可汁液摩擦、蚜虫 (棉蚜和桃蚜 )传毒、种子带毒 (带毒率 3% )。病叶汁液体外稳定性测定稀释限点 (DEP)为 10 -3 ～ 10 -4 、失毒温度 (TIP)为 5 5～ 6 0℃、体外存活期 (LIV)为 1～ 2d。病毒粒子线状约 430～ 75 0× 13～ 15nm ,病叶超薄切片内可见风轮状、环状内含体。病毒提纯制剂紫外最大吸收为 2 6 2nm ,最小吸收为 2 45nm ,A2 6 0 /A2 80 =1 2 ,病毒提纯制剂制备抗血清与MDMV_B(对照为北京分离物 )和所采集的其它分离物均呈阳性反应。以朝阳、大连两分离物病毒RNA为模板 ,按文献报道的MDMV_B外壳蛋白 (CP)基因序列合成引物反转录PCR ,cDNA序列分析表明 ,和已报道的MDMV_B外壳蛋白基因序列同源率达 98 7% ,推导的氨基酸序列存在 2个氨基酸差异 ,其同源率为 99 4%。通过病原鉴定及病毒外壳蛋白基因克隆与cDNA序列分析 ,结果认为引起辽宁地区玉米矮花叶病的病原病毒是MDMV 。

This is the report which treats of the identification,genetic cloning of the virus coat protein and sequence analysis of cDNA in Liaoning Province.Two specimens of Maize dwarf mosaic Diseased leaves has been collected,isolated and purified for the studies.The host ranges of these 2 isolates were all limited to live in the plant of Gramineae,transmited by sap;aphids(cotton aphid and peach aphid)and through seed transmission(only about 3%).The basic virus characterization and storage tests shown that:Dilution end-point(DEP)10 -3 ～10 -4 ;Thermal inactivation point(TIP)55～60℃and longevity in vitro(LIV)1～2days.The virus particles are filamentous form;it ’s size is about 430～750nm×13～15nm.There are conical pinwheels,ringlike inclusion bodies in the plant cytoplasm cells.After the centrifugation;the purified virus sap has tested,the violate absorbaece(A):Max.262nm and Min.245nm.A260/A280=1.2.The antiserum which made fromm these isolates,when tested with Beijing isolate(i.e.CK.virus,MDMV_B),and some other isolates,all shown postive reactions.Using virus PNA as template,through CP genetic sequence of MDMV_B,to synthesize the primer;by reverse transcription to synthesize cDNA.Again by cDNA for template to make PCR,then amplified about 1kb CP genetic fragment;using this fragment;using this fragment cloning to the carrier pUC19,after transformation in E.coli DH5a strain,obtained CP genetic clone.Through sequence analysis of cDNA shown that the homologous percentage was 98.7% to the CP genetic sequence of MDMV_B.Thus inferred by us,that the differences of amino acid ;it’s homologous percentage was 99.4%.According to the biological assay,serological test and cDNA sequence analysis of the above mentioned virus isolate,we suggested that the virus induced maize dwarf mosaic virus disease in Liaoning Province is Maize Dwarf Mosaic Virus(MDMV);and B Strain is also the prevalent strain in this area.