目的:利用ITS2序列对市售火把花根药材及其混伪品进行DNA分子鉴定,以确保其临床用药的安全和有效。方法:对收集到的火把花根药材及其混伪品样品进行DNA提取,ITS2引物的PCR扩增与双向测序;采用CodonCode Aligner V 6.0.2软件对测序峰图...目的:利用ITS2序列对市售火把花根药材及其混伪品进行DNA分子鉴定,以确保其临床用药的安全和有效。方法:对收集到的火把花根药材及其混伪品样品进行DNA提取,ITS2引物的PCR扩增与双向测序;采用CodonCode Aligner V 6.0.2软件对测序峰图进行校对拼接,经隐马尔夫模型的HMMer注释,并预测其二级结构;使用MEGA6.0计算遗传距离,构建NJ系统发育树。结果:火把花根药材及其混伪品的ITS2序列长度均在210~224 bp之间,其种间K2P遗传距离为0.012~0.690,火把花根与雷公藤、东北雷公藤、苦皮藤、南蛇藤及金樱根ITS2条形码变异位点分别为5、7、27、26和88个,其差异较为明显,经与GenBank数据库进行比对的结果表明,ITS2序列及其二级结构能准确区分火把花根药材及其混伪品。结论:DNA条形码ITS2序列可作为火把花根药材真伪鉴别的有效手段,也为后期火把花根片中成药的质量控制及品质评价提供了技术保障。展开更多
Identification of powdery mildew pathogens on melon（Cucumis melo） is important for melon breeding and diseaseresistant germplasm selection. In this study, a powdery mildew pathogen that infected melon plants in Heil...Identification of powdery mildew pathogens on melon（Cucumis melo） is important for melon breeding and diseaseresistant germplasm selection. In this study, a powdery mildew pathogen that infected melon plants in Heilongjiang Province, China, was investigated in terms of host identification, morphological characteristics and phylogenetic relationships. The morphological characteristics of the pathogen were observed at five phases in the life cycle： germinating conidia, primary germ tube, hyphae, conidiophores, and colonization. The conidia were elliptical, colorless, catenulate, and the average length was 29.07 μm and average width was 17.82 μm. One ascus and eight ascospores were produced. DNA was extracted from 0.01 g conidiophores from a strain of powdery mildew pathogen that infected melon. ITS ribosomal DNA region（524 bp） was amplified with the universal ITS1 and ITS4 primers. The nucleotide sequence showed 100% similarity with ITS sequences for three Podosphaera fusca strains obtained from the GenBank database. The identity of the pathogen was confirmed as Sphaerotheca fuliginea. International standard differential hosts were used to identify S. fuliginea strain as 2F race. These results supported the notion that Podosphaera fusca was a synonym of S. fuliginea.展开更多
A specialized test of two-hybrid library type three-frame cDNA yeast for Muskmelon Fusarium oxysporum using the switching mechanism at the 5'end of RNA template(SMART)technology was constructed to screen for inter...A specialized test of two-hybrid library type three-frame cDNA yeast for Muskmelon Fusarium oxysporum using the switching mechanism at the 5'end of RNA template(SMART)technology was constructed to screen for interaction protein genes for wilt disease and to further research the molecular mechanisms of Fusarium oxysporum pathogenesis to explain the interactions between plant and pathogen.A 500-bp cDNA was purified and extracted using SMART and LD-PCR technology to synthesize ds cDNA and was then homogenized and purified to remove the fragments.After processing,the ds cDNA was connected to three types of reading frame pGADT7-SfiI carriers,and the three connection products in E.coli Electrocell were used to build the primary cDNA library.The titer of three ORF cDNA primary library storage capacities was 2.6×10^6,1.8×10^6 and 3×10^6 cfu;the PCR identification of the ORF 1 and 2 gene recombination rate was 94%,the ORF 3 gene recombination rate was 100%,and the insert length distribution was 0.5-4.0 kb as a single band.To reach the quality requirements for library construction,three kinds of reading frame cDNA primary libraries were mixed and amplified,and the plasmid was transformed into the Y187 yeast strain.The titer of the Y187 yeast library was determined to be 3.5×107 cfu?mL-1,and the base of the yeast library was approximately 1 600 000 cfu.The results showed that the construction of muskmelon Fusarium-specific two-hybrid library type three-frame cDNA yeast had a higher reservoir capacity and recombination rate and met the yeast two-hybrid screening requirements.展开更多
The melon varieties MR-1 and M4-7 were used as the parent materials to produce hybrids.A total of 170 F2 individuals were used as the mapping population to construct a genetic linkage map.In this study,240 cleaved amp...The melon varieties MR-1 and M4-7 were used as the parent materials to produce hybrids.A total of 170 F2 individuals were used as the mapping population to construct a genetic linkage map.In this study,240 cleaved amplified polymorphism sequence(CAPS)molecular markers were developed,and 105 of which showed polymorphism between parents.The map contained 105 markers grouped into 12 linkage groups,spanning a total length of 1998.85 cM,with an average length of 19.04 cM between markers.The composite interval mapping(CIM)method was used to detect QTLs related with melon stigma and ovary traits:stigma longitudinal diameter(STLD),stigma transverse diameter(STTD),stigma weight(STW),ovary longitudinal diameter(OLD),and ovary transverse diameter(OTD).A total of nine QTLs were identified:six for stigma size and three for ovary size.A total of six loci were detected for stigma size,which included two loci for stigma longitudinal diameter,two loci for stigma transverse diameter,and two loci for stigma weight.The major effective locus regulating stigma transverse diameter was qSTTD-9-1.A total of three loci were identified for ovary size traits,including one locus for ovary longitudinal diameter and two loci for ovary transverse diameter.展开更多
文摘目的:利用ITS2序列对市售火把花根药材及其混伪品进行DNA分子鉴定,以确保其临床用药的安全和有效。方法:对收集到的火把花根药材及其混伪品样品进行DNA提取,ITS2引物的PCR扩增与双向测序;采用CodonCode Aligner V 6.0.2软件对测序峰图进行校对拼接,经隐马尔夫模型的HMMer注释,并预测其二级结构;使用MEGA6.0计算遗传距离,构建NJ系统发育树。结果:火把花根药材及其混伪品的ITS2序列长度均在210~224 bp之间,其种间K2P遗传距离为0.012~0.690,火把花根与雷公藤、东北雷公藤、苦皮藤、南蛇藤及金樱根ITS2条形码变异位点分别为5、7、27、26和88个,其差异较为明显,经与GenBank数据库进行比对的结果表明,ITS2序列及其二级结构能准确区分火把花根药材及其混伪品。结论:DNA条形码ITS2序列可作为火把花根药材真伪鉴别的有效手段,也为后期火把花根片中成药的质量控制及品质评价提供了技术保障。
基金Supported by the Earmarked Fund for Modern Agro-industry Technology Research System(CARS-26-02)the National Natural Science Foundation(31000917)Heilongjiang Excellent Young Funding(JC200712)
文摘Identification of powdery mildew pathogens on melon（Cucumis melo） is important for melon breeding and diseaseresistant germplasm selection. In this study, a powdery mildew pathogen that infected melon plants in Heilongjiang Province, China, was investigated in terms of host identification, morphological characteristics and phylogenetic relationships. The morphological characteristics of the pathogen were observed at five phases in the life cycle： germinating conidia, primary germ tube, hyphae, conidiophores, and colonization. The conidia were elliptical, colorless, catenulate, and the average length was 29.07 μm and average width was 17.82 μm. One ascus and eight ascospores were produced. DNA was extracted from 0.01 g conidiophores from a strain of powdery mildew pathogen that infected melon. ITS ribosomal DNA region（524 bp） was amplified with the universal ITS1 and ITS4 primers. The nucleotide sequence showed 100% similarity with ITS sequences for three Podosphaera fusca strains obtained from the GenBank database. The identity of the pathogen was confirmed as Sphaerotheca fuliginea. International standard differential hosts were used to identify S. fuliginea strain as 2F race. These results supported the notion that Podosphaera fusca was a synonym of S. fuliginea.
基金Supported by the National Nature Science Foundation of China(31372088)the "Academic Backbone" Project of Northeast Agricultural University(15XG05)China Agriculture Research System(CARS-26-02)
文摘A specialized test of two-hybrid library type three-frame cDNA yeast for Muskmelon Fusarium oxysporum using the switching mechanism at the 5'end of RNA template(SMART)technology was constructed to screen for interaction protein genes for wilt disease and to further research the molecular mechanisms of Fusarium oxysporum pathogenesis to explain the interactions between plant and pathogen.A 500-bp cDNA was purified and extracted using SMART and LD-PCR technology to synthesize ds cDNA and was then homogenized and purified to remove the fragments.After processing,the ds cDNA was connected to three types of reading frame pGADT7-SfiI carriers,and the three connection products in E.coli Electrocell were used to build the primary cDNA library.The titer of three ORF cDNA primary library storage capacities was 2.6×10^6,1.8×10^6 and 3×10^6 cfu;the PCR identification of the ORF 1 and 2 gene recombination rate was 94%,the ORF 3 gene recombination rate was 100%,and the insert length distribution was 0.5-4.0 kb as a single band.To reach the quality requirements for library construction,three kinds of reading frame cDNA primary libraries were mixed and amplified,and the plasmid was transformed into the Y187 yeast strain.The titer of the Y187 yeast library was determined to be 3.5×107 cfu?mL-1,and the base of the yeast library was approximately 1 600 000 cfu.The results showed that the construction of muskmelon Fusarium-specific two-hybrid library type three-frame cDNA yeast had a higher reservoir capacity and recombination rate and met the yeast two-hybrid screening requirements.
基金Supported by the National Natural Science Foundation of China(31772333)China Agriculture Research System of MOF and MARA(CARS-25)
文摘The melon varieties MR-1 and M4-7 were used as the parent materials to produce hybrids.A total of 170 F2 individuals were used as the mapping population to construct a genetic linkage map.In this study,240 cleaved amplified polymorphism sequence(CAPS)molecular markers were developed,and 105 of which showed polymorphism between parents.The map contained 105 markers grouped into 12 linkage groups,spanning a total length of 1998.85 cM,with an average length of 19.04 cM between markers.The composite interval mapping(CIM)method was used to detect QTLs related with melon stigma and ovary traits:stigma longitudinal diameter(STLD),stigma transverse diameter(STTD),stigma weight(STW),ovary longitudinal diameter(OLD),and ovary transverse diameter(OTD).A total of nine QTLs were identified:six for stigma size and three for ovary size.A total of six loci were detected for stigma size,which included two loci for stigma longitudinal diameter,two loci for stigma transverse diameter,and two loci for stigma weight.The major effective locus regulating stigma transverse diameter was qSTTD-9-1.A total of three loci were identified for ovary size traits,including one locus for ovary longitudinal diameter and two loci for ovary transverse diameter.
