Quantitative real-time PCR (qPCR) was applied to rapid screening of positive plasmid clones. Insert-specific primer pairs were used in qPCR colony screening, and false positive colonies could easily be distinguished f...Quantitative real-time PCR (qPCR) was applied to rapid screening of positive plasmid clones. Insert-specific primer pairs were used in qPCR colony screening, and false positive colonies could easily be distinguished from true positive ones by comparing their Ct values. In addition, qPCR is particularly suitable when amplicon is small (<150 bp). This method is sensitive, simple and fast, obviates the need for gel electrophoresis, and is a cost-effective alternative to the traditional PCR approach.展开更多
本文从间日疟患者血液中分离出间日疟原虫,将其基因组 DNA 片段克隆到载体 pUC12质粒中,构建了间日疟原虫 DNA 文库。利用质粒的遗传标志初步筛选重组克隆;用^(32)P 标记的间日疟原虫DNA,人白细胞 DNA 和恶性疟原虫 DNA 为探针分别作菌...本文从间日疟患者血液中分离出间日疟原虫,将其基因组 DNA 片段克隆到载体 pUC12质粒中,构建了间日疟原虫 DNA 文库。利用质粒的遗传标志初步筛选重组克隆;用^(32)P 标记的间日疟原虫DNA,人白细胞 DNA 和恶性疟原虫 DNA 为探针分别作菌落杂交,差异筛选出4株间日疟原虫特异的重组克隆,命名为 pVA1—4。然后酶切鉴定其插入片段,并用 Southern blot 分析,表明重组质粒 pVAl 含有间日疟原虫特异性 DNA 片段。展开更多
Mycoviruses have been found to infect more than 12 species of Penicillium, but have not been isolated from Penicillium italicum(P. italicum). In this study, we isolated and characterized a new double-stranded RNA(ds R...Mycoviruses have been found to infect more than 12 species of Penicillium, but have not been isolated from Penicillium italicum(P. italicum). In this study, we isolated and characterized a new double-stranded RNA(ds RNA) virus, designated Penicillium italicum chrysovirus 1(Pi CV1), from the citrus pathogen P. italicum HSPi-YN1. Viral genome sequencing and molecular characterization indicated that Pi CV1 was highly homologous to the previously described Penicillium chrysogenum virus. We further constructed the mutant HSPi-YN1 Dpks P defective in the polyketide synthase gene(pks P), which is involved in pigment biosynthesis, and these mutants formed albino(white) colonies. Then we applied hyphal anastomosis method to horizontally transmit Pi CV1 from the white virus-donors(i.e., HSPi-YN1 mutants) to wild-type recipients(i.e., P.italicum strains HSPi-CQ54, HSPi-HB4, and HSPi-HN1), and the desirable Pi CV1-infected isogenic recipients, a certain part of blue wild-type strains, can be eventually selected and confirmed by viral genomic ds RNA profile analysis. This bluewhite colony screening would be an easier method to select virus-infected P. italicum recipients, according to distinguishable color phenotypes between blue virus-recipients and white virus-donors. In summary, the current work newly isolated and characterized Pi CV1, verified its horizontal transmission among dually cultured P. italicum isolates, and based on these, established an effective and simplified approach to screen Pi CV1-infected isogenic recipients.展开更多
利用快速扩展随机树算法(Rapidly-exploring random tree,RRT)进行路径规划时,在狭窄复杂区域与空旷障碍区域融合环境下,存在随机性大、搜索时间长、路径曲折等问题。为此,提出了一种基于蚁群的环境分区目标偏置RRT算法。首先,采用分环...利用快速扩展随机树算法(Rapidly-exploring random tree,RRT)进行路径规划时,在狭窄复杂区域与空旷障碍区域融合环境下,存在随机性大、搜索时间长、路径曲折等问题。为此,提出了一种基于蚁群的环境分区目标偏置RRT算法。首先,采用分环境的随机概率采样并结合人工势场的目标偏向扩展策略,以提高算法收敛速度,增强算法搜索能力。其次,为解决规划路径曲折且冗余点多的问题,提出改进蚁群寻优路径,并结合跳点筛选策略及三次B样条以消除冗余点平滑最终路径。最后,改进后的算法与A*算法、目标偏向RRT算法进行了对比分析。仿真结果表明:改进后的算法节点耗费量降低了54.8%,时间平均缩短了75.88%,从而验证了算法的有效性。展开更多
文摘Quantitative real-time PCR (qPCR) was applied to rapid screening of positive plasmid clones. Insert-specific primer pairs were used in qPCR colony screening, and false positive colonies could easily be distinguished from true positive ones by comparing their Ct values. In addition, qPCR is particularly suitable when amplicon is small (<150 bp). This method is sensitive, simple and fast, obviates the need for gel electrophoresis, and is a cost-effective alternative to the traditional PCR approach.
文摘本文从间日疟患者血液中分离出间日疟原虫,将其基因组 DNA 片段克隆到载体 pUC12质粒中,构建了间日疟原虫 DNA 文库。利用质粒的遗传标志初步筛选重组克隆;用^(32)P 标记的间日疟原虫DNA,人白细胞 DNA 和恶性疟原虫 DNA 为探针分别作菌落杂交,差异筛选出4株间日疟原虫特异的重组克隆,命名为 pVA1—4。然后酶切鉴定其插入片段,并用 Southern blot 分析,表明重组质粒 pVAl 含有间日疟原虫特异性 DNA 片段。
基金supported by the National Natural Science Foundations of China (No. 31371893)the Natural Science Fund of Hubei Province (No. 2018CFB676)the Project of Hubei Key Laboratory of Genetic Regulation and Integrative Biology (Grant No. GRIB20184)
文摘Mycoviruses have been found to infect more than 12 species of Penicillium, but have not been isolated from Penicillium italicum(P. italicum). In this study, we isolated and characterized a new double-stranded RNA(ds RNA) virus, designated Penicillium italicum chrysovirus 1(Pi CV1), from the citrus pathogen P. italicum HSPi-YN1. Viral genome sequencing and molecular characterization indicated that Pi CV1 was highly homologous to the previously described Penicillium chrysogenum virus. We further constructed the mutant HSPi-YN1 Dpks P defective in the polyketide synthase gene(pks P), which is involved in pigment biosynthesis, and these mutants formed albino(white) colonies. Then we applied hyphal anastomosis method to horizontally transmit Pi CV1 from the white virus-donors(i.e., HSPi-YN1 mutants) to wild-type recipients(i.e., P.italicum strains HSPi-CQ54, HSPi-HB4, and HSPi-HN1), and the desirable Pi CV1-infected isogenic recipients, a certain part of blue wild-type strains, can be eventually selected and confirmed by viral genomic ds RNA profile analysis. This bluewhite colony screening would be an easier method to select virus-infected P. italicum recipients, according to distinguishable color phenotypes between blue virus-recipients and white virus-donors. In summary, the current work newly isolated and characterized Pi CV1, verified its horizontal transmission among dually cultured P. italicum isolates, and based on these, established an effective and simplified approach to screen Pi CV1-infected isogenic recipients.
基金Natural Science Foundation of Shaanxi Province(No.2019JM-286)。
文摘利用快速扩展随机树算法(Rapidly-exploring random tree,RRT)进行路径规划时,在狭窄复杂区域与空旷障碍区域融合环境下,存在随机性大、搜索时间长、路径曲折等问题。为此,提出了一种基于蚁群的环境分区目标偏置RRT算法。首先,采用分环境的随机概率采样并结合人工势场的目标偏向扩展策略,以提高算法收敛速度,增强算法搜索能力。其次,为解决规划路径曲折且冗余点多的问题,提出改进蚁群寻优路径,并结合跳点筛选策略及三次B样条以消除冗余点平滑最终路径。最后,改进后的算法与A*算法、目标偏向RRT算法进行了对比分析。仿真结果表明:改进后的算法节点耗费量降低了54.8%,时间平均缩短了75.88%,从而验证了算法的有效性。
