小麦矮缩病毒(wheat dwarf virus,WDV)引起的小麦矮缩病是一种危害严重的小麦病害。为研究小麦与WDV的互作机制,本研究首先以小麦为供试材料,提取小麦幼苗叶片和根组织的总RNA,采用SMART(switching mechanism at 5′end of RNA transcri...小麦矮缩病毒(wheat dwarf virus,WDV)引起的小麦矮缩病是一种危害严重的小麦病害。为研究小麦与WDV的互作机制,本研究首先以小麦为供试材料,提取小麦幼苗叶片和根组织的总RNA,采用SMART(switching mechanism at 5′end of RNA transcript)技术构建了小麦全长cDNA文库。该文库滴度大于4×10^(9)cfu/mL、初级文库库容量超过3.6×10^(6)cfu,插入片段长度在500~2000 bp之间,平均长度大于1000 bp,重组率约100%,成功获得了高质量的小麦cDNA文库。为筛选与WDV复制蛋白(replication protein,Rep)互作的寄主因子,构建了酵母双杂交诱饵载体pGBKT7-Rep,验证其无毒性和自激活现象后,利用构建的文库初步筛选出32个与WDV-Rep蛋白发生相互作用的候选靶标蛋白。在此基础上,采用酵母双杂交系统对筛选获得的10个候选蛋白进行一对一互作验证,最终确定其中8个蛋白与WDV-Rep存在相互作用。生物信息学分析表明,这些阳性互作蛋白包括锌指转录因子TFⅢA、去SUMO化酶DeSI、E3泛素连接酶BRE1等,其功能涉及基因转录调控、蛋白质泛素化修饰、植物抗逆防御应答、光合作用能量转换和RNA加工等多个关键生物学过程。该研究为深入解析WDV致病机理和寄主抗病机制提供了材料和理论基础。展开更多
An accurate assessment of host and pathogen gene expression during infection is critical for understanding the molecular aspects of host-pathogen interactions.Often,pathogen-derived transcripts are difficult to ascert...An accurate assessment of host and pathogen gene expression during infection is critical for understanding the molecular aspects of host-pathogen interactions.Often,pathogen-derived transcripts are difficult to ascertain at early infection stages owing to the unfavourable transcript representation compared to the host genes.In this study,we compare two sequencing techniques,RNAseq and enrichment sequencing(RenSeq and PenSeq)of cDNA,to investigate gene expression patterns in the doubled monoploid potato(DM)infected with the late blight pathogen Phytophthora infestans.Our results reveal distinct advantages of cDNA RenSeq and PenSeq over traditional RNAseq in terms of target gene representation and transcriptional quantification at early infection stages.Throughout the infection time course,cDNA enrichment sequencing enables transcriptomic analyses for more targeted host and pathogen genes.For highly expressed genes that were sampled in parallel by both cDNA enrichment and RNAseq,a high level of concordance in expression profiles is observed,indicative of at least semi-quantitative gene expression representation following enrichment.展开更多
目的:构建分化潜能更高的神经母细胞瘤肾上腺素能(ADRN)型细胞cDNA表达文库。方法:提取人神经母细胞瘤细胞N型细胞株SK-N-BE(2)、SK-N-SH、SH-sy5y和IMR-32在对数生长期阶段的mRNA,随后应用SMART(Switching Mechanism At5′end of the R...目的:构建分化潜能更高的神经母细胞瘤肾上腺素能(ADRN)型细胞cDNA表达文库。方法:提取人神经母细胞瘤细胞N型细胞株SK-N-BE(2)、SK-N-SH、SH-sy5y和IMR-32在对数生长期阶段的mRNA,随后应用SMART(Switching Mechanism At5′end of the RNA Transcript)技术制备双链cDNA,同时在其两端连接5′Adapter。进一步采用同源重组方法构建cDNA文库,并对电转化后的文库进行扩增,测定文库的滴度和库容量,同时利用PCR方法鉴定插入片段大小,最后再对cDNA文库进行NGS测序(Next Generation Sequencing),检测文库中基因含量。结果:建立的pcDNA3.1(+)cDNA表达文库总库容量为1×10~9 CFU,文库滴度为2.52×10~8 CFU/mL,平均插入片段为1.2 kb,阳性率为100%,NGS测序分析结果显示文库包含9 247个基因。结论:本研究成功构建pcDNA3.1(+)表达文库,具有较好的质量和良好的多态性,为后续特异性基因的筛选研究提供了有力工具。展开更多
酵母双杂交技术是研究蛋白质之间相互作用的常用手段之一。构建高温处理条件下多年生黑麦草叶片酵母双杂交cDNA文库,能为后续利用酵母双杂交技术探究多年生黑麦草耐高温功能基因之间的互作机制提供技术支撑。以高温处理不同时段的多年...酵母双杂交技术是研究蛋白质之间相互作用的常用手段之一。构建高温处理条件下多年生黑麦草叶片酵母双杂交cDNA文库,能为后续利用酵母双杂交技术探究多年生黑麦草耐高温功能基因之间的互作机制提供技术支撑。以高温处理不同时段的多年生黑麦草叶片为材料,分别提取总RNA后进行等量混合,使用SMART(Switching Mechanism At 5'end of RNA Transcript)技术合成cDNA,再通过胞内同源重组在酵母菌株Y187中构建了高温处理条件下多年生黑麦草叶片全长cDNA文库。所构文库转化效率为1.15×10^(5) CFU/μg,文库滴度为1.43×10^(7) CFU/mL,重组率为100%,说明本文库质量合格,可以用于后续酵母文库筛选。展开更多
基金supported by the Rural & Environment Science & Analytical Services (RESAS) Division of the Scottish Government through project JHI-B1-1the Biotechnology and Biological Sciences Research Council (BBSRC) through awards BB/ S015663/1+2 种基金BB/X009068/1Research Leaders 2025 fellowship funded by European Union’s Horizon 2020 research and innovation programme under Marie Sklodowska-Curie grant agreement no. 754380the Research/Scientific Computing teams at The James Hutton Institute and NIAB for providing computational resources and technical support for the “UK’s Crop Diversity Bioinformatics HPC” (BBSRC grant BB/ S019669/1)。
文摘An accurate assessment of host and pathogen gene expression during infection is critical for understanding the molecular aspects of host-pathogen interactions.Often,pathogen-derived transcripts are difficult to ascertain at early infection stages owing to the unfavourable transcript representation compared to the host genes.In this study,we compare two sequencing techniques,RNAseq and enrichment sequencing(RenSeq and PenSeq)of cDNA,to investigate gene expression patterns in the doubled monoploid potato(DM)infected with the late blight pathogen Phytophthora infestans.Our results reveal distinct advantages of cDNA RenSeq and PenSeq over traditional RNAseq in terms of target gene representation and transcriptional quantification at early infection stages.Throughout the infection time course,cDNA enrichment sequencing enables transcriptomic analyses for more targeted host and pathogen genes.For highly expressed genes that were sampled in parallel by both cDNA enrichment and RNAseq,a high level of concordance in expression profiles is observed,indicative of at least semi-quantitative gene expression representation following enrichment.
文摘酵母双杂交技术是研究蛋白质之间相互作用的常用手段之一。构建高温处理条件下多年生黑麦草叶片酵母双杂交cDNA文库,能为后续利用酵母双杂交技术探究多年生黑麦草耐高温功能基因之间的互作机制提供技术支撑。以高温处理不同时段的多年生黑麦草叶片为材料,分别提取总RNA后进行等量混合,使用SMART(Switching Mechanism At 5'end of RNA Transcript)技术合成cDNA,再通过胞内同源重组在酵母菌株Y187中构建了高温处理条件下多年生黑麦草叶片全长cDNA文库。所构文库转化效率为1.15×10^(5) CFU/μg,文库滴度为1.43×10^(7) CFU/mL,重组率为100%,说明本文库质量合格,可以用于后续酵母文库筛选。
