为建立草莓果实总RNA的提取方法,针对草莓成熟果实中富含多糖、多酚和色素等次级代谢物质,总RNA提取难度大的特点,比较改良的EASYspin植物RNA提取试剂盒法和改良的CTAB法提取草莓果实中总RNA的质量。通过琼脂糖凝胶电泳和核酸蛋白测定仪...为建立草莓果实总RNA的提取方法,针对草莓成熟果实中富含多糖、多酚和色素等次级代谢物质,总RNA提取难度大的特点,比较改良的EASYspin植物RNA提取试剂盒法和改良的CTAB法提取草莓果实中总RNA的质量。通过琼脂糖凝胶电泳和核酸蛋白测定仪(Nano Drop 2000)检测2种方法提取总RNA的浓度、纯度及完整性等。改良的EASYspin植物RNA提取试剂盒法和改良的CTAB法都能够完成草莓果实总RNA的提取,电泳检测在28S和18S处呈2条清晰的条带,OD260/OD280值和OD260/OD230值均在2.0左右;改良的EASYspin植物RNA提取试剂盒法成本较高,且提取总RNA质量劣于改良的CTAB法,但是EASYspin植物RNA提取试剂盒法操作简便,安全可靠,节省时间。通过RT-PCR验证,2种方法所获得的草莓果实总RNA质量较好,可达到分子生物学试验对RNA质量的要求。展开更多
紫肉甘薯块根富含多糖、多酚、淀粉、蛋白质和色素等物质,会影响紫肉甘薯块根总RNA的提取。本研究对五种不同的RNA提取方法进行比较,分析各自的优缺点,并对所得总RNA的浓度和纯度进行测定。研究结果表明,改良CTAB法和华越洋试剂盒提取...紫肉甘薯块根富含多糖、多酚、淀粉、蛋白质和色素等物质,会影响紫肉甘薯块根总RNA的提取。本研究对五种不同的RNA提取方法进行比较,分析各自的优缺点,并对所得总RNA的浓度和纯度进行测定。研究结果表明,改良CTAB法和华越洋试剂盒提取法均能提取出浓度、纯度较高的RNA,且RNA可用于反转录,并扩增出Ib Tub A基因的特异条带,能够进行后续实验分析,其中华越洋试剂盒提取法操作更加快捷和方便。另外三种提取方法都存在一些弊端,所提取RNA质量略差,不能达到后续实验的要求。增加去色素环节可以有效去除色素,三个不同色素差异品种的提取结果较为一致。展开更多
[ Objective] The aim of this study was to investigate whether HSP70 can be used as a stress monitoring indicator in Cypnnus carpio breeding. [Method] Based on HSP70 sequence of Cyprinus carpio （AY120894）, one pair o...[ Objective] The aim of this study was to investigate whether HSP70 can be used as a stress monitoring indicator in Cypnnus carpio breeding. [Method] Based on HSP70 sequence of Cyprinus carpio （AY120894）, one pair of primers was designed and synthesized, while the total RNA of liver tissues in Cyprinus carpio was extracted. Some cDNA fragments of Cyprinus carpio HSP70 were cloned by RT-PCR, and its differential expression in various tissues such as heart, intestine, mucus, gonad, swim bladder, gill and fin in Cyprinus carpio was also studied. [Result] The cDNA sequence of 480 bp was obtained from Cypdnus carpio HSPTO gene by RT-PCR amplification. Homology comparison between the deduced amino acid sequence after sequencing and that of other types of fish showed that the homology among Cyprinus carpio, Danio rerio, Ohcorhynehus mylciss, Paralichthys olivaceus, Xiphophoorus maculates and Carassius auratus was 96%, 98%, 98%, 96%, 98% and 96% respectively. The expression of HSP70 was detected in eight tissues of Cypnnus carpio. The expression was the highest in heart, followed by swim bladder and fin, but there was no significant difference between them （ P 〉 0.05 ）. There was no significant difference among the ex- pression in three tissues of intestine, mucus and fat （ P〉0.05）, but their expression was significantly higher than those in gonad and gill （ P〈 0.05）. [ Conclusion] HSPTO gene expression is a suitable criterion for monitoring the stress degree, stress capacity and healthy conditions in Cyprinus carpio breeding.展开更多
【目的】比较手工法和试剂盒法提取土壤RNA对原核微生物多样性的影响,评价两种方法研究土壤宏转录组的技术偏好性。【方法】针对黑龙江海伦砂质黑壤土、江苏滨海黏心夹砂土、江西鹰潭第四纪红黏土不同母质发育形成的三种水稻土,采用手...【目的】比较手工法和试剂盒法提取土壤RNA对原核微生物多样性的影响,评价两种方法研究土壤宏转录组的技术偏好性。【方法】针对黑龙江海伦砂质黑壤土、江苏滨海黏心夹砂土、江西鹰潭第四纪红黏土不同母质发育形成的三种水稻土,采用手工和试剂盒法分别获得微生物总RNA,利用高通量测序原核微生物16S r RNA多样性,通过紫外分光和琼脂糖凝胶电泳分析RNA质量,比较手工和试剂盒法提取RNA对水稻土原核微生物群落的影响规律。【结果】三种水稻土中试剂盒提取RNA的纯度皆高于手工法,但RNA提取总量不一致。试剂盒提取黑龙江和江苏水稻土的RNA总量高于手工法,而手工法提取的江西水稻土RNA总量高于试剂盒法。高通量测序发现土壤类型而不是RNA提取方法决定了原核微生物多样性指数和群落结构。3个水稻土共检测到27门和409属,两种RNA提取方法偏好性提取了19门和181属,这些门和属占水稻土微生物总丰度平均值分别为40.4%和44.4%。与手工提取RNA相比,试剂盒法发现11个微生物门的丰度显著偏高(P<0.05),但仅有Armatimonadetes门在三种水稻土同时存在专一偏好性;手工法提取也发现11个微生物门的丰度显著高于试剂盒法,并且仅有Firmicutes门在三种水稻土中同时存在专一偏好性。在微生物属水平,三种水稻土中均发现试剂盒法偏好性提取了2属,而手工法偏好性提取了5属。进一步针对72个优势微生物属(丰度>0.1%且同时存在于三种水稻土),发现其占所有微生物丰度80%强,且48属的变化规律与RNA提取方法无关。例如,手工法提取水稻土好氧甲烷氧化菌的规律为:黑龙江(1.68%)>江西(0.90%)>江苏(0.59%),而试剂盒法也得到一致结果,黑龙江(0.52%)>江西(0.18%)>江苏(0.13%)。【结论】两种RNA提取方法本身的特异偏好性较小。三种水稻土27门409属中,仅有2门7属可能存在方法本身的专一偏好性,即在三种水稻土中,这些微生物均被试剂盒法或手工法特异性偏好提取,占所有原核微生物门和属比例仅为7%和1%左右。此外,针对同一水稻土,手工和试剂盒法提取RNA的总量、纯度、微生物相对丰度明显不同,在原核微生物门和属的分类水平,约70%的门和22%的属的丰度具有统计显著性差异,但两种RNA提http://journals.im.ac.cn/actamicro取方法均能反映优势微生物类群在三种水稻土中的变异规律,土壤类型对原核微生物多样性的影响远大于RNA提取方法本身的偏好性。未来原核微生物宏转录组研究中,应优先考虑科学问题及其实验处理可能导致的微生物组及其转录差异,结合微生物RNA提取特点,最大限度地发挥宏转录组技术优势。展开更多
文摘为建立草莓果实总RNA的提取方法,针对草莓成熟果实中富含多糖、多酚和色素等次级代谢物质,总RNA提取难度大的特点,比较改良的EASYspin植物RNA提取试剂盒法和改良的CTAB法提取草莓果实中总RNA的质量。通过琼脂糖凝胶电泳和核酸蛋白测定仪(Nano Drop 2000)检测2种方法提取总RNA的浓度、纯度及完整性等。改良的EASYspin植物RNA提取试剂盒法和改良的CTAB法都能够完成草莓果实总RNA的提取,电泳检测在28S和18S处呈2条清晰的条带,OD260/OD280值和OD260/OD230值均在2.0左右;改良的EASYspin植物RNA提取试剂盒法成本较高,且提取总RNA质量劣于改良的CTAB法,但是EASYspin植物RNA提取试剂盒法操作简便,安全可靠,节省时间。通过RT-PCR验证,2种方法所获得的草莓果实总RNA质量较好,可达到分子生物学试验对RNA质量的要求。
文摘紫肉甘薯块根富含多糖、多酚、淀粉、蛋白质和色素等物质,会影响紫肉甘薯块根总RNA的提取。本研究对五种不同的RNA提取方法进行比较,分析各自的优缺点,并对所得总RNA的浓度和纯度进行测定。研究结果表明,改良CTAB法和华越洋试剂盒提取法均能提取出浓度、纯度较高的RNA,且RNA可用于反转录,并扩增出Ib Tub A基因的特异条带,能够进行后续实验分析,其中华越洋试剂盒提取法操作更加快捷和方便。另外三种提取方法都存在一些弊端,所提取RNA质量略差,不能达到后续实验的要求。增加去色素环节可以有效去除色素,三个不同色素差异品种的提取结果较为一致。
文摘[ Objective] The aim of this study was to investigate whether HSP70 can be used as a stress monitoring indicator in Cypnnus carpio breeding. [Method] Based on HSP70 sequence of Cyprinus carpio （AY120894）, one pair of primers was designed and synthesized, while the total RNA of liver tissues in Cyprinus carpio was extracted. Some cDNA fragments of Cyprinus carpio HSP70 were cloned by RT-PCR, and its differential expression in various tissues such as heart, intestine, mucus, gonad, swim bladder, gill and fin in Cyprinus carpio was also studied. [Result] The cDNA sequence of 480 bp was obtained from Cypdnus carpio HSPTO gene by RT-PCR amplification. Homology comparison between the deduced amino acid sequence after sequencing and that of other types of fish showed that the homology among Cyprinus carpio, Danio rerio, Ohcorhynehus mylciss, Paralichthys olivaceus, Xiphophoorus maculates and Carassius auratus was 96%, 98%, 98%, 96%, 98% and 96% respectively. The expression of HSP70 was detected in eight tissues of Cypnnus carpio. The expression was the highest in heart, followed by swim bladder and fin, but there was no significant difference between them （ P 〉 0.05 ）. There was no significant difference among the ex- pression in three tissues of intestine, mucus and fat （ P〉0.05）, but their expression was significantly higher than those in gonad and gill （ P〈 0.05）. [ Conclusion] HSPTO gene expression is a suitable criterion for monitoring the stress degree, stress capacity and healthy conditions in Cyprinus carpio breeding.
文摘【目的】比较手工法和试剂盒法提取土壤RNA对原核微生物多样性的影响,评价两种方法研究土壤宏转录组的技术偏好性。【方法】针对黑龙江海伦砂质黑壤土、江苏滨海黏心夹砂土、江西鹰潭第四纪红黏土不同母质发育形成的三种水稻土,采用手工和试剂盒法分别获得微生物总RNA,利用高通量测序原核微生物16S r RNA多样性,通过紫外分光和琼脂糖凝胶电泳分析RNA质量,比较手工和试剂盒法提取RNA对水稻土原核微生物群落的影响规律。【结果】三种水稻土中试剂盒提取RNA的纯度皆高于手工法,但RNA提取总量不一致。试剂盒提取黑龙江和江苏水稻土的RNA总量高于手工法,而手工法提取的江西水稻土RNA总量高于试剂盒法。高通量测序发现土壤类型而不是RNA提取方法决定了原核微生物多样性指数和群落结构。3个水稻土共检测到27门和409属,两种RNA提取方法偏好性提取了19门和181属,这些门和属占水稻土微生物总丰度平均值分别为40.4%和44.4%。与手工提取RNA相比,试剂盒法发现11个微生物门的丰度显著偏高(P<0.05),但仅有Armatimonadetes门在三种水稻土同时存在专一偏好性;手工法提取也发现11个微生物门的丰度显著高于试剂盒法,并且仅有Firmicutes门在三种水稻土中同时存在专一偏好性。在微生物属水平,三种水稻土中均发现试剂盒法偏好性提取了2属,而手工法偏好性提取了5属。进一步针对72个优势微生物属(丰度>0.1%且同时存在于三种水稻土),发现其占所有微生物丰度80%强,且48属的变化规律与RNA提取方法无关。例如,手工法提取水稻土好氧甲烷氧化菌的规律为:黑龙江(1.68%)>江西(0.90%)>江苏(0.59%),而试剂盒法也得到一致结果,黑龙江(0.52%)>江西(0.18%)>江苏(0.13%)。【结论】两种RNA提取方法本身的特异偏好性较小。三种水稻土27门409属中,仅有2门7属可能存在方法本身的专一偏好性,即在三种水稻土中,这些微生物均被试剂盒法或手工法特异性偏好提取,占所有原核微生物门和属比例仅为7%和1%左右。此外,针对同一水稻土,手工和试剂盒法提取RNA的总量、纯度、微生物相对丰度明显不同,在原核微生物门和属的分类水平,约70%的门和22%的属的丰度具有统计显著性差异,但两种RNA提http://journals.im.ac.cn/actamicro取方法均能反映优势微生物类群在三种水稻土中的变异规律,土壤类型对原核微生物多样性的影响远大于RNA提取方法本身的偏好性。未来原核微生物宏转录组研究中,应优先考虑科学问题及其实验处理可能导致的微生物组及其转录差异,结合微生物RNA提取特点,最大限度地发挥宏转录组技术优势。
