喀拉库勒湖两株侵染同一宿主黄杆菌低温噬菌体的分离及生物学特性研究  

Isolation and Characterization of Two Cold-Active Bacteriophages That Infect the Same Host Flavobacteria in Karakul Lake

作　　者：李明源 王继莲 张甜 周茜 美合热阿依·木台力甫 LI Mingyuan;WANG Jilian;ZHANG Tian;ZHOU Qian;Mihray·Mutallip(Collage of Life and Geography Sciences,Kashi University/Key Laboratory of Ecology and Biological Resources in Yarkand Oasis of Education of Xinjiang Uygur Autonomous Region,Kashi Xinjiang 844006,China)

机构地区：[1]喀什大学生命与地理科学学院/叶尔羌绿洲生态与生物资源研究自治区高校重点实验室,新疆喀什844006

出　　处：《西南大学学报（自然科学版）》2021年第12期38-48,共11页Journal of Southwest University(Natural Science Edition)

基　　金：新疆维吾尔自治区自然科学基金项目(2016D01B011);新疆维吾尔自治区高校科研计划项目(XJEDU2018Y038).

摘　　要：低温噬菌体作为高原湖泊中的主要生物类群,在调控原核菌群结构及C,N等元素循环方面意义重大.从喀拉库勒湖中分离纯化低温噬菌体并对其生物学特性进行研究,采用双层平板法分离纯化低温噬菌体,利用透射电镜观察噬菌体粒子形态;结合16S rDNA序列比对及系统发育分析确定宿主菌分类地位;对噬菌体最佳感染复数、一步生长曲线和吸附速率等基本生物学特性进行比较,分析理化因素对噬菌体的影响;采用PEG沉淀法浓缩噬菌体颗粒;酚-氯仿法提取噬菌体基因组并进行酶切.结果表明:从喀拉库勒湖分离到两株能侵染同一宿主黄杆菌Flavobacterium sp.KHhb03的噬菌体KH-sph01和KH-sph02,其均能在4℃侵染宿主并增殖,从形态上分别归属为长尾噬菌体科(Siphoviridae)和肌尾噬菌体科(Myoviridae).KH-sph01和KH-sph02均对热敏感,在pH值为7~8范围内最稳定,对氯仿、表面活性剂等不敏感,对蛋白酶K敏感;最佳感染复数分别为10和0.1,吸附速率均较强,裂解量分别为3.16个和10.22个,基因组均为dsDNA,初步估算基因组大小分别为60~65 kb和40~45 kb.得出噬菌体KH-sph01和KH-sph02能侵染同一宿主菌Flavobacterium sp.KHhb03,但生物学特性各异.As the main biological group in plateau lakes,the cold-active bacteriophages are of great significance in regulating the structure of bacterial flora and in the circulation of elements such as C and N.The cold-active bacteriophages were isolated from Karakul Lake,and their biological characteristics were studied.Bacteriophages were isolated and purified with the double-layer plate method.The morphology of the phages was observed with transmissionelectron microscopy.The 16S rDNA sequence alignment and phylogenetic analysis were combined to determine the host bacteria classification status.The basic biological characteristics,such as the optimal phage infection complex number,one-step growth curve and adsorption rate,were compared.The effects of temperature,pH and chemical reagents on the phages were analyzed.The phage particles were concentrated by PEG precipitation.The genome of the phages was extracted with chloroform extraction.Restriction fragment length polymorphism(RFLP)analysis of the genomic DNA,constituent proteins and physiological analysis of the bacteriophages were carried out to characterize the phages.Two lytic cold-active bacteriophages,designated as KH-sph01 and KH-sph02,were isolated from the Karakul Lake.Their host strain KHhb03 was identified as a member of the genus Flavobacterium.They were morphologically classified as Siphoviridae and Myoviridae.Physiological analysis results showed that both cold-active bacteriophages had infection activity at 4℃,and were sensitive to heat,insensitive to chloroform and detergents,but sensitive to protease K.They were most stable at a pH in the range of 7-8,and their optimal infection complex was 10 and 0.1,and their cracking volume was 3.16 and 10.22,respectively.The genome of KH-sph01 and KH-sph02 consisted of double-stranded DNA and were approximately 60-65 kb and 40-45 kb in size,respectively.Kh-sph01 and Kh-sph02 can infect the same host bacteria Flavobacterium sp.KHhb03,but are different inbiological characteristics.

关 键 词：低温噬菌体 喀拉库勒湖 黄杆菌属 生物学特性 

分 类 号：Q939.48[生物学—微生物学]