利用酵母双杂交系统筛选与草莓镶脉病毒移动蛋白P1互作的寄主因子

 构建感染草莓镶脉病毒(SVBV)森林草莓的酵母cDNA文库,利用酵母双杂交系统,筛选出与SVBV P1蛋白互作的15种寄主因子。生物信息学分析发现,这15种寄主因子参与茉莉酸途径、泛素化、光合作用、抗病抗逆、蛋白修饰、蛋白运输和氧化还原等多种生物过程。另外,这些寄主因子还具有其他分子功能,包括氧化还原酶活性、蛋白二硫化物异构酶活性和金属离子结合活性等。本研究初步探讨了P1与寄主因子的互作机理,为揭示SVBV侵染森林草莓以及SVBV在寄主中扩展的分子机制提供理论依据。     

利用酵母双杂交系统筛选与草莓镶脉病毒P6蛋白互作的森林草莓寄主因子

【目的】草莓镶脉病毒(Strawberry vein banding virus,SVBV)是侵染草莓的主要病毒,但其侵染草莓的机制尚不清楚。论文以SVBV的P6蛋白为诱饵筛选森林草莓(Fragaria vesca)c DNA文库的寄主因子,为解析SVBV侵染草莓的分子机制提供理论依据。【方法】SVBV接种森林草莓,提取出现明显症状叶片的总RNA,Dnase I处理后,用SMART法反转录合成ds c DNA,均一化处理c DNA并酶切纯化,将400 bp的短片段去除,其余片段连接到p GAD-T7质粒载体上,构建森林草莓初级c DNA文库。同时将SVBV P6构建到酵母双杂交诱饵载体p GBK-T7上,再将p GBK-P6和p GBK-T7分别转化酵母菌株AH109,阳性酵母菌株接种SD/-Trp液体培养基,鉴定诱饵载体对酵母细胞的毒性。将转化p GBK-P6的酵母菌分别涂布SD/-Trp、SD/-Leu-Trp和SD/-His-Trp平板,测定菌落生长情况,分析P6蛋白对酵母报告基因的自激活活性。然后用森林草莓初级c DNA文库质粒转化含有诱饵载体p GBK-P6的AH109酵母菌株,共转化子依次涂布SD/-Leu-Trp、SD/-Leu-Trp-His和SD/-Trp-Leu-His-Ade/X-α-Gal平板,最终筛选蓝色且长势较好的阳性菌落,提取酵母质粒并测序,Gen Bank中初步比对候选基因,利用Uniprot在线网站的gene ontology(GO)通路注释互作蛋白因子,分析互作蛋白的生物学功能。【结果】3种c DNA文库平均库容超过2.0×106 cfu,平均文库重组率为97%,文库插入片段平均扩增长度1 kb,表明森林草莓c DNA文库符合试验标准。最终利用SD/-Trp-Leu-His-Ade/X-α-Gal培养基筛选得到230个酵母阳性克隆,经过序列相似性比对,除去重复序列、载体序列和移码序列,共筛得15个与SVBV P6互作的寄主因子。GO通路注释结果表明这些寄主因子参与了13种生物过程,包括泛素化、转录因子调节、防御反应、代谢过程、氧化还原和胞内氨基酸代谢等过程;这15个寄主因子的分子功能多样,包括乙酰转移酶活性、萜烯合酶活性、脱氢酶活性、金属离子结合活性、蛋白激酶活性和水解酶活性等。【结论】成功构建了森林草莓酵母c DNA文库,筛选出15个与SVBV P6互作的森林草莓寄主因子,为进一步探明SVBV与森林草莓互作的分子机理提供了理论依据。     

草莓镶脉病毒ORF I基因的克隆、原核表达及抗血清制备

The total DNA was extracted from strawberry leaves infected with Strawberry vein banding virus (SVBV) by CTAB method. Specific primer pairs were designed to amplify the gene ORF I of SVBV-Shenyang isolate by PCR, gene ORF I was cloned into modified prokaryotic expression vector pET-32a (+)-GST, the recombinant plasmid pET-ORF I was transformed into E. coli DH5α, then the positive clones were screened and sequenced. The recombinant plasmid was extracted and transformed into Escherichia coli BL2l (DE3), the fusion protein with an approximate molecular weight of 56 kDa was obtained with IPTG induction and Ni²⁺-NTA affinity column purification. The purified fusion protein was used to immunize the rabbits to prepare the specific antiserum. The result of ELISA showed that the titer of the prepared antiserum is up to 1:520 000, Western blot analysis indicated that the prepared antiserum could reacted specifically with purified recombinant fusion protein. Not only the expression of P1 protein in SVBV infected-strawberry leaves, but also the expression of P1 protein in P1-infiltrated Nicotiana benthamiana leaves could be detected, using 2 000 times diluted antiserum.     

森林草莓转录因子MYB 12基因克隆及蛋白特性分析

采集森林草莓(Fragaria vesca)植株叶片,TRIzol法提取样本总RNA,设计特异性引物RT-PCR扩增转录因子MYB 12基因,获得1条约1 kb大小的特异性片段,克隆并测序。序列分析表明,该片段序列全长988 nts,含有完整的MYB 12基因开放阅读框(ORF),ORF长度为855 nts,编码285个氨基酸。序列比对发现,森林草莓MYB 12基因与来源于5种蔷薇科植物MYB基因的序列相似性较高,为75%~76%,而与其他7种非蔷薇科植物MYB基因的序列相似性相对较低,为47%~68%。构建MYB基因系统关系树,发现森林草莓与其他5种蔷薇科植物的MYB基因聚成一个分支,说明森林草莓与蔷薇科植物亲缘关系较近。生物信息学分析表明,MYB 12蛋白具有一定的亲水性,其N-端有2个MYB结构域,属于R2R3-MYB亚族。MYB 12蛋白二级结构主要以α-螺旋为主,并含有一些不规则的卷曲(Random coil)结构。这为研究该基因在植物生长发育及生理代谢过程中的调控机制奠定了基础。     

草莓镶脉病毒ORF I基因的克隆、原核表达及抗血清制备

The total DNA was extracted from strawberry leaves infected with Strawberry vein banding virus (SVBV) by CTAB method. Specific primer pairs were designed to amplify the gene ORF I of SVBV-Shenyang isolate by PCR, gene ORF I was cloned into modified prokaryotic expression vector pET-32a (+)-GST, the recombinant plasmid pET-ORF I was transformed into E. coli DH5α, then the positive clones were screened and sequenced. The recombinant plasmid was extracted and transformed into Escherichia coli BL2l (DE3), the fusion protein with an approximate molecular weight of 56 kDa was obtained with IPTG induction and Ni²⁺-NTA affinity column purification. The purified fusion protein was used to immunize the rabbits to prepare the specific antiserum. The result of ELISA showed that the titer of the prepared antiserum is up to 1:520 000, Western blot analysis indicated that the prepared antiserum could reacted specifically with purified recombinant fusion protein. Not only the expression of P1 protein in SVBV infected-strawberry leaves, but also the expression of P1 protein in P1-infiltrated Nicotiana benthamiana leaves could be detected, using 2 000 times diluted antiserum.     

南方水稻黑条矮缩病毒安徽分离物S5片段的克隆及S5-2基因的原核表达

【目的】探讨南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)安徽分离物(SRBSDVAn Hui-HN2)的遗传特性,并获得原核表达的P5-2蛋白。【方法】RT-PCR扩增SRBSDV S5片段,克隆、测序并进行序列分析。将S5-2基因插入原核表达载体,重组载体转化大肠埃希菌并用IPTG诱导,Ni2+-NTA亲和柱纯化融合蛋白,SDS-PAGE分析P5-2蛋白的表达情况。【结果】SRBSDV-An Hui-HN2 S5片段全长3 167 bp,包含S5-2基因全长612 bp,编码204个氨基酸。序列比对结果显示,SRBSDV-An Hui-HN2 S5片段与其他SRBSDV分离物S5片段的序列相似性极高,达99.0%~99.7%,而与斐济病毒属(Fijivirus)其他成员S5片段的序列相似性较低,仅为38.0%~71.3%;构建的S5片段系统发育树表明SRBSDV和RBSDV聚成1个分支,其中6个SRBSDV分离物聚成1个亚分支。原核表达获得相对分子质量约为47 000的重组蛋白,Western blot分析显示,GST单抗能够与重组融合蛋白发生特异性反应。【结论】SRBSDV各分离物之间亲缘关系非常近,而与Fijivirus其他成员亲缘关系较远,原核表达获得的融合蛋白为靶标蛋白。     

南方水稻黑条矮缩病毒安徽分离物S7片段的克隆及其S7-1基因编码蛋白的原核表达

为探讨南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)的种群变异并获得S7-1原核表达蛋白,采用RT-PCR技术扩增SRBSDV安徽分离物S7片段,并进行克隆、测序及序列分析,再利用特异性引物扩增该分离物S7-1基因并克隆到原核表达载体p ET-GST上,重组质粒p ET-S7-1转化大肠杆菌BL21(DE3),IPTG诱导及Ni~(2+)-NTA亲和柱纯化融合蛋白,再进行Western blot检测。结果显示,SRBSDV安徽分离物S7片段与其它SRBSDV各个分离物S7片段的序列相似性极高,达99.3%~99.9%,而与斐济病毒属Fijivirus其它种之间的序列相似性较低,为35.5%~73.3%。SRBSDV安徽分离物与其它SRBSDV各个分离物聚成1个单独的分支,彼此间亲缘关系较近。试验获得了分子质量约为68 k D的S7-1融合蛋白,且GST单抗能够与S7-1融合蛋白发生特异性反应,表明本研究得到的融合蛋白确为靶标蛋白。