利用酵母双杂交膜系统筛选与小麦蓝矮植原体免疫膜蛋白互作的异沙叶蝉蛋白

正小麦蓝矮病(Wheat blue dwarf,WBD)是我国西北麦区一种重要植原体病害,在我国西部地区危害严重。该病害由异沙叶蝉(Psammotettix alienus L.)专化性传播,介体传毒成为病害流行的重要中心环节[1]。本实验室前期通过免疫荧光标记研究发现WBD植原体免疫膜蛋白(immunodominant membrane protein, IMP)与介体异沙叶蝉肌动蛋白互作,说明IMP在植原体传播和致病过程中起关键作用。     

小麦蓝矮植原体SWP1效应蛋白的抗血清制备及检测应用

 小麦蓝矮病是西北冬麦区一种重要的植原体病害,造成严重的产量损失。为进一步探究小麦蓝矮植原体的致病机理,通过原核表达获得其致病因子SWP1的重组蛋白并制备抗血清。将PCR扩增到的SWP1基因片段插入到原核表达载体pET-30a(+)上,导入E.coli BL21 (DE3)中,表达出约12 kDa含His标签的融合蛋白。用纯化的融合蛋白注射大白兔皮层,获得了特异性较强的SWP1抗血清,其效价为1∶4 000,并成功应用于SWP1在本氏烟中的检测。制备的抗血清在SWP1蛋白结构、功能及其与寄主的互作研究中具有重要意义。     

基于酵母双杂交系统筛选灰飞虱体内与大麦黄条点花叶病毒核衣壳蛋白互作的蛋白质

 为了获取影响大麦黄条点花叶病毒(BYSMV)在灰飞虱体内增殖、积累和传播的相关介体因子,本研究利用分离泛素酵母双杂交膜系统,以BYSMV核衣壳蛋白(N)为诱饵对灰飞虱cDNA文库进行了筛选。 将BYSMV N基因构建到诱饵载体pDHB1上进行表达检测和功能验证,结果表明重组载体pDHB1-N能在酵母内正常表达并行使功能。利用诱饵载体筛选pPR3-N空文库对文库筛选条件进行优化,确定3-氨基-1,2,4-三唑(3-AT)浓度为12 mmol·L^-1的QDO平板为筛选文库培养基条件,去除可能存在的轻微筛库背景。在此筛选条件下以诱饵载体从灰飞虱cDNA文库中筛选得到57个阳性克隆,序列比对结果表明这些阳性克隆编码17种候选蛋白,包括表皮蛋白、泛素B、核糖体膜相关蛋白、细胞色素b5以及海藻糖转运蛋白等。 经酵母双杂交共转验证和β-半乳糖苷酶检测进一步确认了这17个候选蛋白与BYSMV N发生互作。本研究成功从灰飞虱分离泛素酵母双杂交膜系统cDNA文库筛选到与BYSMV N互作的蛋白质,为进一步探索弹状病毒与介体昆虫的分子互作机制奠定了基础。     

大豆花叶病毒诱导的应用于膜蛋白酵母双杂交的大豆cDNA文库构建

Membrane-baesd yeast two-hybrid system is an effective method for research on interaction between Soybean mosaic virus-encoded membrane-associated proteins and host factors, while the Gateway technology without the use of restriction enzyme cloning techniques is easier for construction of virus-induced host cDNA library. In this study, both membrane-based yeast two-hybrid system and Gateway^TM systems were used. With TRIZOL regent, total RNA was extracted from soybean leaves infected with soybean mosaic virus. SMV-induced soybean primary cDNA library constructed by Gateway technology was recombined into a reconstructed prey vector for membrane-based yeast two-hybrid system. The capacity and quality tests showed that the library titer was 1.7 ? 10⁶cfu/mL and the length of inserted cDNA fragments ranged from 0.5 to 2 kb. It is available for research on interaction between the virus-encoded membrane protein and host.     

Expression of phytoplasma‐induced witches’ broom disease symptoms in acid lime (Citrus aurantifolia) trees is affected by climatic conditions

Witches’ broom disease (WBD), caused by ‘Candidatus Phytoplasma aurantifolia’, is a serious disease of acid lime (Citrus aurantifolia) in Oman and the UAE. However, little is known about the distribution of phytoplasma and the expression of WBD symptoms in different geographical locations. A survey was carried out in 18 districts in Oman and the UAE covering 143 orchards and 5823 acid lime trees. ‘Candidatus Phytoplasma aurantifolia’ was detected in acid lime in all the 18 surveyed districts. However, the development of typical symptoms of WBD was only observed in 12 districts. Districts in which the phytoplasma was present but symptoms were not expressed were located either in desert areas or in areas characterized by semitropical conditions. Phylogenetic analysis of 16 phytoplasma isolates from trees developing WBD symptoms and six phytoplasma isolates from trees with no WBD symptoms showed that all isolates share an identical 16S rRNA sequence, belonging to subgroup II‐B. Quantitative PCR analysis showed that the concentration of phytoplasma is significantly higher (8800–801 000 copies) in leaves developing WBD symptoms compared to 2–268 copies in symptomless leaves from the same trees and 8–874 copies in acid lime trees from areas where disease symptoms were not expressed. The lack of expression of WBD symptoms under certain environmental conditions may suggest that symptom development and phytoplasma are affected by certain unfavourable environmental conditions. These findings could provide a basis for managing WBD through encouraging lime cultivation under climatic conditions less conducive to WBD symptom expression.     

小麦蓝矮植原体免疫膜蛋白(Imp)的跨膜区影响其在大肠杆菌中的表达

根据跨膜区预测结果,设计ImpF1/ImpR1和ImpF2/ImpR2两对特异性引物。以ImpF1/ImpR1,ImpF1/ImpR2,Imp-F2/ImpR1和ImpF2/ImpR2共4个组合经PCR扩增得到4个目标片段,即:免疫膜蛋白Imp基因的全长(Imp-B);切除C-端跨膜区的Imp基因的基因序列(Imp-N);切除N-端跨膜区的Imp基因的基因序列(Imp-C);切除N-和C-端跨膜区的Imp基因的基因序列(Imp-S)。经酶切连接到原核表达载体Pmal-c2x,并转入E.coliBL21(DE3)PlysS菌株中。SDS-PAGE电泳验证,只有切除N-、C-端跨膜区的Imp基因的基因序列(Imp-S)得到大量表达,结果表明:小麦蓝矮植原体免疫膜蛋白的跨膜区影响其在大肠杆菌中的表达。根据跨膜区预测结果,设计ImpF1/ImpR1和ImpF2/ImpR2两对特异性引物。以ImpF1/ImpR1,ImpF1/ImpR2,Imp-F2/ImpR1和ImpF2/ImpR2共4个组合经PCR扩增得到4个目标片段,即:免疫膜蛋白Imp基因的全长(Imp-B);切除C-端跨膜区的Imp基因的基因序列(Imp-N);切除N-端跨膜区的Imp基因的基因序列(Imp-C);切除N-和C-端跨膜区的Imp基因的基因序列(Imp-S)。经酶切连接到原核表达载体Pmal-c2x,并转入E.coliBL21(DE3)PlysS菌株中。SDS-PAGE电泳验证,只有切除N-、C-端跨膜区的Imp基因的基因序列(Imp-S)得到大量表达,结果表明:小麦蓝矮植原体免疫膜蛋白的跨膜区影响其在大肠杆菌中的表达。     

小麦蓝矮植原体寄主范围的鉴定及RFLP分析

 小麦蓝矮是我国首次报道的小麦植原体病害。采用介体接种植物,症状观察和应用植原体16S rDNA基因通用引物对R16mF2/R16mR1进行PCR扩增,在接种小麦和传毒介体中均扩增出1.4kb的特异片段,鉴定出小麦蓝矮植原体新寄主7种。用巢式PCR方法对小麦蓝矮病田自然发病杂草进行分子检测,从表现症状的10种杂草中均扩增出1.2kb的特异片段。利用6种植原体特异性限制性内切酶对10种杂草的扩增片段进行RFLP(restriction fragment length polymor-phism)分析表明:扩增片段的RFLP图谱与目前已知的16Sr I组翠菊黄化植原体的RFLP图谱相近。鉴定出小麦蓝矮植原体田间自然新寄主10种。     

Identification of wheat blue dwarf phytoplasma effectors targeting plant proliferation and defence responses

The identification of effectors from pathogenic microbes is one of the most important subjects for elucidating infection mechanisms. Wheat blue dwarf (WBD) phytoplasma causes dwarfism, witches' broom, and yellow leaf tips in wheat plants, resulting in severe yield loss in northwestern China. In this study, 37 candidate effector proteins were transiently expressed in Nicotiana benthamiana. Plants expressing the SAP11‐like protein SWP1 exhibited typical witches' broom. Interestingly, another protein, SWP11, induced both cell death and defence responses, including H₂O₂ accumulation and callose deposition. Analysis by qRT‐PCR was used to show that a marker gene of the hypersensitive response, HIN1, and three pathogenesis‐related genes, PR1, PR2 and PR3, were significantly up‐regulated in leaves of N. benthamiana expressing SWP11. In addition, SWP12 and SWP21 (TENGU‐like) were shown to suppress SWP11‐, BAX‐, and/or INF1‐induced cell death. These results indicated that SWP21 has a distinct role in virulence compared with TENGU and that WBD phytoplasma possesses effectors that target plant proliferation and defence responses. The ability of these effectors to trigger or suppress plant immunity provides new insights into the phytoplasma–plant interaction.     

白粉菌侵染后的拟南芥酵母双杂交cDNA文库构建及其利用

 拟南芥广谱抗病基因RPW8对拟南芥白粉病菌、霜霉病菌和烟草花叶病毒等均具有抗性。为了深入研究其广谱抗病机制,筛选鉴定与RPW8具有直接相互作用的蛋白,我们以含有RPW8的拟南芥纯合转基因系S5为材料,接种拟南芥白粉菌系UCSC1,36 h后取样,构建了白粉菌侵染初期的拟南芥cDNA文库。为了提高文库对长片段基因5′端的覆盖率,分别使用含有oligo(dT)和oligo(dN)的接头引物反转录cDNA第一链,PCR扩增双链cDNA。将纯化后的双链cDNA与线性化载体pGADT7-Rec混合,利用同源重组技术在酵母菌株Y187中构建cDNA文库。经检测,文库转化效率为5.0×10⁶/3 μg pGADT7-Rec,滴度为2.5×10⁸ CFU·mL^-1,插入片段长度在350～2 000 bp之间,平均插入片段大小为750 bp。用RPW8.1和RPW8.2构建诱饵载体,分别获得11和12个候选互作蛋白。结果表明此cDNA文库质量较好,适用于互作蛋白的筛选。