【目的】蜜蜂球囊菌Ascosphaera apis是一种专性侵染蜜蜂幼虫而导致白垩病的致死性真菌病原。基于前期已获得的高质量纳米孔(Nanopore)长读段测序数据,对蜜蜂球囊菌菌丝(AaM)和孢子(AaS)中的毒力因子相关全长转录本进行鉴定和分析,为毒...【目的】蜜蜂球囊菌Ascosphaera apis是一种专性侵染蜜蜂幼虫而导致白垩病的致死性真菌病原。基于前期已获得的高质量纳米孔(Nanopore)长读段测序数据,对蜜蜂球囊菌菌丝(AaM)和孢子(AaS)中的毒力因子相关全长转录本进行鉴定和分析,为毒力因子相关剪接异构体的功能研究提供参考信息和基础。【方法】利用BLAST工具,将AaM和AaS中的所有全长转录本比对Nr数据库,以鉴定蜜蜂球囊菌的毒力因子(几丁质酶、脂肪酶、水解酶和蛋白酶)相关的全长转录本。使用minimap2软件,将AaM和AaS中的全长转录本序列与蜜蜂球囊菌参考基因组注释的已知转录本序列进行比对,将比对到参考基因组的全长转录本进行归一化处理,再通过每百万计数法(Counts per million,CPM)计算毒力因子相关全长转录本的表达量。利用百迈克云平台的相关工具绘制转录本的表达量聚类热图。通过IGV浏览器对部分毒力因子相关全长转录本结构进行可视化。【结果】在AaM鉴定到毒力因子相关的367个基因及407个全长转录本,包括12条几丁质酶相关全长转录本,48条脂肪酶相关全长转录本,289条水解酶相关全长转录本,58条蛋白酶相关全长转录本。在AaS鉴定到毒力因子相关367个基因及400个全长转录本,包括14条几丁质酶相关全长转录本,63条脂肪酶相关全长转录本,267条水解酶相关全长转录本,56条蛋白酶相关全长转录本。另外,AaM和AaS特有的毒力因子(几丁质酶、脂肪酶)相关全长转录本分别有0条和17条,共有的毒力因子相关全长转录本有60条。进一步分析发现,蜜蜂球囊菌的部分毒力因子基因可通过可变剪接形成多条剪接异构体。【结论】共鉴定到蜜蜂球囊菌毒力因子(几丁质酶、脂肪酶、水解酶和蛋白酶)相关的367个基因和486条全长转录本;相比于蜜蜂球囊菌参考基因组注释的转录本,绝大多数毒力因子基因对应的全长�展开更多
Zea nicaraguensis, a wild relative of cultivated maize （Zea mays subsp, mays）, is considered to be a valuable germplasm to improve the waterlogging tolerance of cultivated maize. Use of reverse genetic-based gene cl...Zea nicaraguensis, a wild relative of cultivated maize （Zea mays subsp, mays）, is considered to be a valuable germplasm to improve the waterlogging tolerance of cultivated maize. Use of reverse genetic-based gene cloning and function verifi- cation to discover waterlogging tolerance genes in Z. nicaraguensis is currently impractical, because little gene sequence information for Z. nicaraguensis is available in public databases. In this study, Z. nicaraguensis seedlings were subjected to simulated waterlogging stress and total RNAs were isolated from roots stressed and non-stressed controls. In total, 80 mol L-1 Illumina 100-bp paired-end reads were generated. De novo assembly of the reads generated 81 002 final non-re- dundant contigs, from which 5 261 full-length transcripts were identified. Among these full-length transcripts, 3 169 had at least one Gene Ontology （GO） annotation, 2 354 received cluster of orthologous groups （COG） terms, and 1 992 were assigned a Kyoto encyclopedia of genes and genomes （KEGG） Orthology number. These sequence data represent a valuable resource for identification of Z. nicaraguensis genes involved in waterlogging response.展开更多
The number and variability of transcripts determine the diversity and complexity of the proteins in a species.To reveal the variations in transcript abundance and the related factors in Chinese fir(Cunninghamia lanceo...The number and variability of transcripts determine the diversity and complexity of the proteins in a species.To reveal the variations in transcript abundance and the related factors in Chinese fir(Cunninghamia lanceolata),we performed second-generation sequencing(RNA-seq)and third-generation sequencing(PacBio)analyses.The forms and rates of alternative splicing in Chinese fir were studied on the basis of the obtained transcripts.Furthermore,the number of full-length transcripts(isoforms)produced by alternative splicing and the variation patterns in‘Long 15'were analyzed.The transcript diversity in Chinese fir was largely caused by six alternative splicing forms,of which intron retention was the most common(47.98%of all alternative splicing events),followed by alternative 3′splice site(24.29%).The third-generation sequencing analysis detected 61613 isoforms in‘Long 15',with each gene producing 1-10 isoforms.Only 0.06%of the genes produced more than 10 isoforms.Transcript abundance was similar among Chinese fir varieties,but 615 more transcripts were detected in‘Long 15'than in clone 1339,implying that‘Long 15'synthesized more protein types in vivo than 1339.This difference may explain why‘Long 15'grows better and is more adaptable to environmental conditions than 1339.An examination of Chinese fir clone Kai6 detected more transcripts after fertilization than following a nutrient stress treatment.Moreover,transcript polymorphism was greater after fertilization than in response to nutrient stress.This finding may be useful for improving fertilizer applications to enhance Chinese fir growth and development.Sequences and alternative splicing forms are critical for research on the Chinese fir transcriptome and the potential biological consequences of alternative splicing.展开更多
目的研究犬贾第虫病毒全长cDNA体外RNA转录体的感染性,为进一步研究犬贾第虫病毒的特性、深入阐明病毒的致病机理奠定基础。方法根据已知的GCV(长春株)基因序列设计一套特异性重叠引物,利用RT-PCR技术,构建感染性犬贾第虫病毒基因组全长...目的研究犬贾第虫病毒全长cDNA体外RNA转录体的感染性,为进一步研究犬贾第虫病毒的特性、深入阐明病毒的致病机理奠定基础。方法根据已知的GCV(长春株)基因序列设计一套特异性重叠引物,利用RT-PCR技术,构建感染性犬贾第虫病毒基因组全长cDNA;用T7 RiboMAXTM Express Large Scale RNA Production System制备体外转录体,电穿孔方法感染无病毒株犬贾第虫滋养体,通过提取总核酸和超薄切片电镜观察感染情况。结果成功构建犬贾第虫病毒基因组全长cDNA,体外转录体电穿孔后72 h,提取的总核酸中发现6.2 kb的dsRNA带,电镜观察到完整的病毒粒子。结论该转录体具有感染性,可以在无病毒株犬贾第虫滋养体内复制表达,为进一步研究GCV奠定基础。展开更多
文摘【目的】蜜蜂球囊菌Ascosphaera apis是一种专性侵染蜜蜂幼虫而导致白垩病的致死性真菌病原。基于前期已获得的高质量纳米孔(Nanopore)长读段测序数据,对蜜蜂球囊菌菌丝(AaM)和孢子(AaS)中的毒力因子相关全长转录本进行鉴定和分析,为毒力因子相关剪接异构体的功能研究提供参考信息和基础。【方法】利用BLAST工具,将AaM和AaS中的所有全长转录本比对Nr数据库,以鉴定蜜蜂球囊菌的毒力因子(几丁质酶、脂肪酶、水解酶和蛋白酶)相关的全长转录本。使用minimap2软件,将AaM和AaS中的全长转录本序列与蜜蜂球囊菌参考基因组注释的已知转录本序列进行比对,将比对到参考基因组的全长转录本进行归一化处理,再通过每百万计数法(Counts per million,CPM)计算毒力因子相关全长转录本的表达量。利用百迈克云平台的相关工具绘制转录本的表达量聚类热图。通过IGV浏览器对部分毒力因子相关全长转录本结构进行可视化。【结果】在AaM鉴定到毒力因子相关的367个基因及407个全长转录本,包括12条几丁质酶相关全长转录本,48条脂肪酶相关全长转录本,289条水解酶相关全长转录本,58条蛋白酶相关全长转录本。在AaS鉴定到毒力因子相关367个基因及400个全长转录本,包括14条几丁质酶相关全长转录本,63条脂肪酶相关全长转录本,267条水解酶相关全长转录本,56条蛋白酶相关全长转录本。另外,AaM和AaS特有的毒力因子(几丁质酶、脂肪酶)相关全长转录本分别有0条和17条,共有的毒力因子相关全长转录本有60条。进一步分析发现,蜜蜂球囊菌的部分毒力因子基因可通过可变剪接形成多条剪接异构体。【结论】共鉴定到蜜蜂球囊菌毒力因子(几丁质酶、脂肪酶、水解酶和蛋白酶)相关的367个基因和486条全长转录本;相比于蜜蜂球囊菌参考基因组注释的转录本,绝大多数毒力因子基因对应的全长�
基金supported by the Basic Research Program of China (973 Program, 2014CB138705)the National Natural Science Foundation of China (31371639)the Sichuan Youth Science and Technology Foundation of China (12ZB091)
文摘Zea nicaraguensis, a wild relative of cultivated maize （Zea mays subsp, mays）, is considered to be a valuable germplasm to improve the waterlogging tolerance of cultivated maize. Use of reverse genetic-based gene cloning and function verifi- cation to discover waterlogging tolerance genes in Z. nicaraguensis is currently impractical, because little gene sequence information for Z. nicaraguensis is available in public databases. In this study, Z. nicaraguensis seedlings were subjected to simulated waterlogging stress and total RNAs were isolated from roots stressed and non-stressed controls. In total, 80 mol L-1 Illumina 100-bp paired-end reads were generated. De novo assembly of the reads generated 81 002 final non-re- dundant contigs, from which 5 261 full-length transcripts were identified. Among these full-length transcripts, 3 169 had at least one Gene Ontology （GO） annotation, 2 354 received cluster of orthologous groups （COG） terms, and 1 992 were assigned a Kyoto encyclopedia of genes and genomes （KEGG） Orthology number. These sequence data represent a valuable resource for identification of Z. nicaraguensis genes involved in waterlogging response.
基金Supported by the Fundamental Research Funds of the Central Public Welfare Research Institutes of The Chinese Academy of Forestry(CAFYbb2017ZA001-1-2)the"14^(th)Five-year"Forest Variety Breeding Project of Zhejiang Province(2021C010010808)。
文摘The number and variability of transcripts determine the diversity and complexity of the proteins in a species.To reveal the variations in transcript abundance and the related factors in Chinese fir(Cunninghamia lanceolata),we performed second-generation sequencing(RNA-seq)and third-generation sequencing(PacBio)analyses.The forms and rates of alternative splicing in Chinese fir were studied on the basis of the obtained transcripts.Furthermore,the number of full-length transcripts(isoforms)produced by alternative splicing and the variation patterns in‘Long 15'were analyzed.The transcript diversity in Chinese fir was largely caused by six alternative splicing forms,of which intron retention was the most common(47.98%of all alternative splicing events),followed by alternative 3′splice site(24.29%).The third-generation sequencing analysis detected 61613 isoforms in‘Long 15',with each gene producing 1-10 isoforms.Only 0.06%of the genes produced more than 10 isoforms.Transcript abundance was similar among Chinese fir varieties,but 615 more transcripts were detected in‘Long 15'than in clone 1339,implying that‘Long 15'synthesized more protein types in vivo than 1339.This difference may explain why‘Long 15'grows better and is more adaptable to environmental conditions than 1339.An examination of Chinese fir clone Kai6 detected more transcripts after fertilization than following a nutrient stress treatment.Moreover,transcript polymorphism was greater after fertilization than in response to nutrient stress.This finding may be useful for improving fertilizer applications to enhance Chinese fir growth and development.Sequences and alternative splicing forms are critical for research on the Chinese fir transcriptome and the potential biological consequences of alternative splicing.
文摘目的研究犬贾第虫病毒全长cDNA体外RNA转录体的感染性,为进一步研究犬贾第虫病毒的特性、深入阐明病毒的致病机理奠定基础。方法根据已知的GCV(长春株)基因序列设计一套特异性重叠引物,利用RT-PCR技术,构建感染性犬贾第虫病毒基因组全长cDNA;用T7 RiboMAXTM Express Large Scale RNA Production System制备体外转录体,电穿孔方法感染无病毒株犬贾第虫滋养体,通过提取总核酸和超薄切片电镜观察感染情况。结果成功构建犬贾第虫病毒基因组全长cDNA,体外转录体电穿孔后72 h,提取的总核酸中发现6.2 kb的dsRNA带,电镜观察到完整的病毒粒子。结论该转录体具有感染性,可以在无病毒株犬贾第虫滋养体内复制表达,为进一步研究GCV奠定基础。
