|Table of Contents|

Optimization of Hybridization Conditions and Specific Verification for 16S rRNA Oligonucleotide Probe of Endophytic Bacteria Strain XG32(PDF)

《南京师大学报（自然科学版）》[ISSN:1001-4616/CN:32-1239/N]

Issue:
2011年01期
Page:
89-95
Research Field:
生命科学
Publishing date:

Info

Title:
Optimization of Hybridization Conditions and Specific Verification for 16S rRNA Oligonucleotide Probe of Endophytic Bacteria Strain XG32
Author(s):
Liu Jia; Wang Meixia; Yan Shuzhen; Chen Shuanglin
Jiangsu Engineering and Technology Research Center for Microbiology,School of Life Sciences,Nanjing Normal University,Nanjing 210046,China
Keywords:
fluorescence in situ hybridization;  optim ization o f the hybr id ization conditions;  verification of spec ific probe
PACS:
Q938.1
DOI:
-
Abstract:
F luorescence in situ hybr id ization w as applied to investiga te the cloning and distr ibution of the endophy tic bacter ia stra in XG32 w ith the ro le of grow th-prom oting and d isease contro l in plan ts. Firstly, a spec ific probe w as designed theo re tica lly at the base on the m id- spec ific sequence o f the 16S rRNA of the stra in XG32 w ith the GenBank da tabase, BLAST prog ram, DNAc lub so ftwa re and Oligo softw are. Second ly, 12 stra ins wh ich sim ilar o r adjacen t to the stra in XG32 w ere used to ce rtify the spec ific ity of the probe by repeated hybrid ization in the basis o f the su itab le cond itions o f hybridization. The results ind ica ted that appropriate cond itions fo r hybridization o f the probe w ere 35% fo rmam ide and 1.2 mo l/L NaC l in hybr idiza tion bu ffer, 42℃ hybr id iza tion tem pera ture, 0.02 mol/L NaC l in w ash ing buffer. The hyb rid ization signal was clear betw een the probe XG32 and the stra in XG32. To sum up, the probe XG32 w as the specific probe o f the strain XG32.

References:

[ 1]刘维红, 闫淑珍, 刘五星, 等. ACC 脱氨酶活性细菌XG32 的鉴定[ J]. 南京师大学报: 自然科学版, 2007, 30( 2): 81- 83.
[ 2]刘维红, 闫淑珍, 杨启银, 等. ACC 脱氨酶活性细菌筛选及其对番茄初生苗生长的影响[ J] . 江苏农业科学, 2006( 2): 80-84.
[ 3]沈萍, 闫淑珍, 陈双林, 等. 具ACC脱氨酶活性的植物内生细菌对辣椒的促生作用和对疫霉病的防治作用[ J]. 植物保护学报, 2008( 1): 28-32.
[ 4]杨海莲, 孙晓璐, 宋未. 植物根际促生细菌和内生细菌的诱导抗病性的研究进展[ J]. 植物病理学报, 2000, 30( 2) : 106- 110.
[ 5]S turz A V, Chr istie B R, Now ak J. Ba teria l endophytes: po tential ro le in developing sustainable system s o f crop production [ J]. Cr itica lRev iew s in Plant Sc iences, 2000, 19( 1): 1-30.
[ 6]deW eger L A, van derB ijA J, Dekke rs L C, e t a .l Colonozation o f the rh izosphere o f crop plants by p lant-ben ific ia l Pseudom onas FEM S[ J] . M icrob io l Eco,l 1995, 17( 4): 221-227.
[ 7]QuadtH A, K loeppe r JW. Imm uno log ica l detec tion and loca liza tion of the cotton endophyte Enterobacte axbur iae JM 22 in different plant species[ J]. Can JM icrob ia ,l 1996, 42: 1 144-1 156.
[ 8]W hitesides S K, Spotts R A. Frequency, d istr ibution and charac teristics of endophy tic Pseudom onas sy ringae in pear trees[ J]. Phytopatho logy, 1991, 81( 4) : 453-457.
[ 9]吴蔼民, 顾本康, 傅正擎, 等. 内生菌73a在不同抗性品种棉花体内的定殖和消长动态研究[ J]. 植物病理学报, 2001, 31( 4) : 289-294.
[ 10]刘云霞, 张青文, 周明. 电镜免疫胶体金定位水稻内生细菌的研究[ J]. 农业生物技术学报, 1996, 4( 4): 344-358.
[ 11]冯娜, 郑慧芬, 何绍江, 等. 用g fp基因检测饭豆根瘤菌在饭豆根部的定殖动态[ J] . 华中农业大学学报, 2005, 24( 1): 49-51.
[ 12]Kutte r S, H artm ann A, Schm idM. Co lon ization o f barley ( H ordeum vu lgare) w ith Salm one lla enter ica and L ister ia spp. [ J]. FEM SM icrob io l E co ,l 2006( 56): 262-271.
[ 13]Lo Picco lo S, Ferra ro V, A lfonzo A, et a .l Presence of endophytic bacteria in V itis v inifera leaves as detected by fluorescence in situ hybr idiza tion[ J] . AnnM ic robio,l 2010( 60): 161-167.
[ 14]Da im s H, Ram sing N B, Schle iferK H, et a.l Cu ltiva tion- independent, sem iautom atic determ ination of absolute bacter ia l cell numbers in env ironm ental sam ples by fluorescence in situ hybridization[ J]. Appl Env ironM icrob io ,l 2001, 67( 12): 5 810- 5 818.
[ 15]Bond P L, E rhart R,W agnerM, et a.l Identification o f som e of them ajor g roups o f bacter ia in e fficient and non- efficient b iolog ica l phosphorus rem oval activated sludge system s[ J]. Appl Env ironM icrobio,l 1999, 65( 9): 4 077-4 084.
[ 16]余利岩, 徐平, 姚天爵. 新型A ctinobacter ia 荧光原位杂交( FISH )探针的设计和应用[ J] . 中国抗生素杂志, 2000, 25 ( 6): 401-406.
[ 17]H icks R E, Am ann R I, Stahl D A, et a .l Dual sta in ing of natural bacterioplankton w ith 4?? , 6-d iam idino-2-phenylindo le and fluo rescent o ligonucleotide probes targeting kingdom- leve l 16S rRNA sequences[ J]. App l Env ironM icrob io ,l 1992, 58( 7): 2 158-2 163.
[ 18]Amann R I, B inde r B J, O lson R J, et a.l Comb ination of 16S rRNA-targeted o ligonuc leo tide probes w ith flow cytom etry fo r analyzing m ix ed m icrob ia l popu la tions[ J]. Appl Env ironM icrobio,l 1990, 56( 6): 1 919-1 925.
[ 19]M anzW. In situ ana lys is of m icrob ia l biofilm s by rRNA-targeted oligonuc leotide prob ing [ J]. M e th Enzymo ,l 1999, 310: 79-91.
[ 20]Sam brook J, Fritsch E F, M aniatisT, et a.l M o lecular C loning: A LaboratoryM anua l[M ]. 2nd ed. New York: C old Spr ing H arbor Laboratory Press, 1989.
[ 21]Roche leau S, Greer CW, Law rence J R, et a .l D iffe rentia tion o f menthano saeta conc ilii andM ethanosarcina barkeri in anaerobicm esophilic g ranu la r Sludge by fluo rescent in situ hybridization and confocal scanning laserm icroscopy[ J]. Appl Env ironM ic robio,l 1999, 65( 5): 2 222-2 229.
[ 22]AndersonM L M, Young B D. Quantitative filter hybr idiza tion[M ] / / H arnes B D, H igg ins S J. Nucle icA cidH ybrid ization. Ox fo rd: IRL Press, 1985: 73-113.
[ 23]M o terA, U B G be.l Fluorescence in situ hybr id ization( FISH ) fo r direct v isua liza tion o fm icroorg an ism s[ J] . Journa l o fM -i c robiolog ica lM ethods, 2000, 41: 85-112

Memo

Memo:
-
Last Update: 2013-04-11