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1 43 6 Vol143,No ACTA PEDOLOGICA SINICA Nov., g (, ) CONSTRUCTION OF TYPICAL RED SOIL2DERIVED METAGENOMIC L IBRARY AND FUNCTIONAL ANALYSIS IN JIANGXI PROVINCE Huang Tingting Cui Zhongli Zhang Lu Cao Hui g Li Shunpeng ( Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Nanjing Agricultural University, Nanjing , China) ;DNA ; ; S15413 ; Q93811 A, , [1 ], [2 ], (metagenomics),, [3,4 ], 42, [5 9 ] [10,11 ], DNA, TA,, 1 111,, 1718, mm, 4 6, m, 5 8 [12 ], ph 4141, 1132 g kg - 1, 0167 g kg - 1, g kg - 1, 0 5 cm 5 15 cm cm( a, b, c),,, 4 mm, - 70 DNA 1 3 ( ) ( KZCX32SW2427) ( ) g,e2mail : edu. cn : (1982 ),,,, : ; :

2 ( :cfu g - 1 ) (cm) a b c :a,b,c a 0 5 cm, b 5 15 cm, c cm 112 puc 18 (Amp + ) DH5 ; Taq TA pmd182t Vector Takara, (Amp) CTAB ( ) SDS( ) Tris ( ) EDTA ; K Merk ; 113 DNA DNA Blending method [13 ] 260 nm DNA, 230 nm 280 nm DNA 16S rdna DNA DNA, [14 ] BamHI DNA, DNA 2 6 kb DNA 45 min, Taq da : 10 Taq 5 l, Mg 2 + (25 mmol L - 1 ) 4 l, dntp (25 mmol L - 1 ) 5 l, DNA 10 l, Taq (5 U l - 1 ) 015 l, 50 l :94 3 min ;72 45 min ;10 10 min Inoue DH5 [14 ] ; T (pmd218,takara) 16, DH5, h ; LB,, 30 % DNA ;,, BLAST BioEdit ClustalX (ORF) 116 GenBank, GeneBank H3, H5, H10, H222, H223, H227, X6, Q41, (Accession number) : DQ185049, DQ185050, DQ185051, DQ185052, DQ185053,DQ195083,DQ195084,DQ DNA 2 DNA 23 kb, DNA g, DNA PCR

3 6 : 1039, DNA [15,16 ] A 260 / A 230 A 260 / A 280 DNA [17 ] DNA ( 2), 16S rdna, DNA 0 5 cm, 5 15 cm DNA 2 DNA a,b,c : DNA ;M DNA/ Hind Marker 2 DNA A 260 / A 230 A 260 / A 280 DNA g g - 1 a b c , DNA, TA,, DNA DNA( 2 DNA ), DNA,,, DNA TA [18 ], BamH DNA, 2 6 kb, Taq PCR da, da, T, 1g , 80 % ( 4), DNA DNA, DNA G + C %, DNA ( Pst, EcoR, BamH Hind ) DNA, BamH ( 3) 3 DNA 1 : Pst ;2 : EcoR ;3 : Hind ;4 : BamH ; 5 : DNA ;6 : DNA/ Hind Marker 4 1,21 : DNA/ Hind Marker ;2 10, : 16 ; 11,12 : puc ,, BLAST 10, 2 DNA, 8, BioEdit ClustalX (Opening reading frame,

4 ORF), ( 3) 3 ORF ORF 1) S 2) E 3) H222 DQ Frame 1, 239aa Frame21, 98aa Frame22, 140aa IcmF, H223 DQ Frame 1, 117aa H227 DQ Frame 1, 169aa WD Frame21, 74aa Frame22, 129aa DNA I H3 DQ Frame 1, 87aa, PDZ C Frame21, 87aa ATP DNA H5 DQ Frame 1, 74aa LacOPZ H10 DQ Frame 1, 131aa Frame21, 131aa X6 DQ Q41 DQ Frame 3, 68 aa ATP cassette,sub2family B (MDR/ TAP) ) ORF, frame 1, 2 ORF, frame21, - 2 ORF 3 3 ORF 2) S (bits score),, 3) E ( E value),e ; S E 3,, DNA, H2-3, 29 % 55 % 9 ( 5),,, (S) ( H) (D),, 5 H : H223 ;2 : Bradyrhizobium japonicum USDA 110 ;3 : Magnetospirillum magnetotacticum ;4 : Paracoccus denitrificans ;5 : Rhodospirillum rubrum ;6 : Rhizobium sp ; 7 : Neisseria meningitidis Z2491 ;8 : Neisseria gonorrhoeae FA 1090 ;9 : Thiobacillus denitrificans ATCC ;10 : Mesorhizobium sp1 BNC1 H222 ORF ( ) (

5 6 : 1041 ) ( 6),,, ( Acidobacteria group) ( Solibacteres) 81 %, 40 % 50 % 16S rdna,, 6 H222 ORF 1 : Streptomyces coelicolor A3(2) ;2 : Streptomyces avermitilis MA24680 ;3 : Nocardioides sp1 JS614 ;4 : Bifidobacterium longum DJO10A ;5 :Lactobacillus casei ATCC 334 ;6 : Desulfitobacterium hafniense DCB22 ;7 : Bacillus halodurans C2125 ;8 :H222 ;9 : Solibacter usitatus Ellin6076 ;10 : Thiobacillus denitrificans ATCC DNA ( ) ( ) DNA DNA,, PCR ; DNA DNA,, [13 ] DNA, DNA,, DNA 10 kb,,, PCR da dt,, T,,,, [19 ],,,,,,

6 , [ 1 ] Whitman W B, Coleman D C, Wiebe WJ. Prokaryotes : The unseen majority. Proc. Natl. Acad. Sci., 1998, 95 : [ 2 ] Torsvik V, Ovreas L. Microbial diversity and function in soil : From gene to ecosystems. Current Opinion in Microbiology, 2002, 5 : [ 3 ],Tibbits J,Adams M.., 2004, 41 (1) : [ 4 ] Handelsman J. Metagenomics : Application of genomics to uncultured microorganisms. Microbiology and Biology Reviews, 2004, 68 : [ 5 ] Yun J, Kang S, Park S, et al. Characterization of a novel amylolytic enzyme encoded by a gene from a soil2derived metagenomic library. Appl. Environ. Microbiol., 2004, 70 (12) : [ 6 ] Courtois S, Cappellano C M, Ball M, et al. Recombinant environ2 mental libraries provide access to microbial diversity for drug discov2 ery from natural products. Appl. Environ. Microbiol., 2003, 69 (1) : [ 7 ] Henne A, Daniel R, Schmitz R A, et al. Construction of environ2 mental DNA libraries in Escherichia coli and screening for the pres2 ence of genes conferring utilization of 42hydroxybutyrate. Appl. Envi2 ron. Microbiol., 1999, 65 (9) : [ 8 ] Rondon M R, August P R, Bettermann A D, et al. Cloning the soil metagenome : A strategy for accessing the genetic and functional di2 versity of uncultured microorganisms. Appl. Environ. Microbiol., 2000, 66 (6) : [ 9 ] Tyson G W, Chapman J, Hugenholtz P, et al. Community structure and metabolism through reconstruction of microbial genomes from the environment. Nature, 2004, 428 : [10 ],,,. DNA., 2001, 41 (2) : [11 ],,,. DNA., 2004, 10 (6) : [12 ],,.. :, [13 ],,,. DNA., 2004, 36 (6) : [14 ] J, D W.. 3. :, 2002 [15 ] Liu B R, Jia G M, Chen J, et al. A review of methods for studying microbial diversity in soils. Pedosphere, 2006, 16 (1) : [16 ] Burgmann H, Pesaro M, Widmer F, et al. A strategy for optimizing quality and quantity of DNA extracted from soil. J. Microbiol. Meth2 ods, 2001, 45 (1) : 7 20 [17 ] Zhou J Z, Bruns M A, Tiedje J M. DNA recovery from soils of di2 verse composition. Applied and Environmental Microbiology, 1996, 62 (2) : [ 18 ] Wilkinson D E, Jeanicke T, Cowan D A. Efficient molecular cloning of environmental DNA from geothermal sediments. Biotechnology Let2 ters, 2002, 24 : [19 ].. :,

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr PCR 龙平, 崔占虎, 李虔全, 徐建平, 张春红, 周立社 * *, 李旻辉 ( 包头医学院, 内蒙古包头 014060) DNA 30 28 DNA trnl-trnf PCR PCR 136 bp 323 bp 10% PCR PCR Astragalus membranaceus Fisch. Bge. var. mongho- licus Bge. Hsiao A. membranaceus