2008, 16 (6): 586592

doi: 10.3724/SP.J.1003.2008.08118
http: //www.biodiversity-science.net

Biodiversity Science

16S rDNA-RFLP

*
(,

310006)

: , ,

DNA, 16S rDNA, PCR-RFLP

23 kbDNAPCR-RFLP74,
77.3%16S rDNA, 22.7%
, 7412
, (37.8%), (16.2%)(12.2%)(10.8%)(10.8%)
(8.1%), 4
, 13.5%5.4%12.2%6.8%,

: , , 16S rDNA, RFLP

Bacteria diversity in paddy field soil by 16S rDNA-RFLP analysis in

Ningxia
Jianping Zhang, Naiyuan Dong, Haobin Yu, Yongjun Zhou, Yongliang Lu, Ruimei Geng, Liuqing Yu*
State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006

Abstract: Rice is one of the most important crops in the Ningxia region of China, and rice planting helps to
maintain ecosystem balance and prevent land desertification. Soil microbial diversity provides basic functions for rice field soil ecosystems. To better understand bacterial diversity and community composition in
Ningxia paddy soil, the total bacterial DNA was extracted from paddy soil collected from a typical rice field
of Ningxia using the culture independent method. A 16S ribosomal DNA (16S rDNA) clone library of soil
bacteria was constructed. The 16S rDNA fragments were analyzed by PCR-RFLP. Further sequencing and
cluster analysis were conducted to elucidate the bacterial diversity. Over 23 kb DNA fragments were obtained
from the paddy soil and 74 MspI restriction endonuclease types were detected by PCR-RFLP analysis. Sequence analysis revealed that 77.3% of clone sequences were similar to those of uncultured bacteria in the
environment, while only 22.7% clone sequences were most closely related to those of cultured bacteria in
GenBank, suggesting great potential for undeveloped bacterial resources was available in paddy fields. Our
phylogenetic analysis found that the sequenced clones fell into 12 major lineages within the domain bacteria.
Among them, members of the Proteobacteria were the dominant group, accounting for 37.8%, including
-Proteobacteria (13.5%), -Proteobacteria (12.2%), -Proteobacteria (6.8%) and -Proteobacteria (5.4%),
followed by Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi and Planctomycetes division with
16.2%, 12.2%, 10.8%, 10.8%, 8.1%, respectively. Firmicutes, Gemmatimonadetes, and Verrucomicrobia
were less well represented. Our study revealed an extensive diversity of soil bacteria in a paddy field in

: 2008-05-21; : 2008-08-25
: (2006RG013) 863 (2006AA10A214)(Y306180)
* Author for correspondence. E-mail: liuqyu53@yahoo.com.cn

: 16S rDNA-RFLP

587

Ningxia.
Key words: paddy rice field, bacterial diversity, 16S rDNA, RFLP analysis

, 16S rDNAPCR-RFLP

DGGE/TGGE(/)

, CNSP

(Collins et al., 1992; Kent &

Triplett, 2002),

PCR-RFLP

(Graff & Stubner, 2002)(,

2004)(, 2003)

(Gundula et al., 2005)(Werner et al., 2000;

Chin et al., 2004),

1
1.1

(2007)

; (2007)

9.27, 200 mm,

, ,

; (2006)

200710, 500 m2

1.0 kg, 310 cm,

, ,

, 4 mm

70,

DNA

1.2

DNA Omega ; LA-Taq

DNAMspIDNA

, ,

pMD19-T VectorIPTGX-GalTakara

1% (Amann et al., 1995;

; KSigma;

Torsvik & Ovreas, 2002)

DNA

10

, 10

1.3

DNA

(Rosello & Amann, 2001)

10 g(), 13 mL DNA

16S rDNA

(100 mM Tris-HCl (pH8.0), 100 mM EDTA

588

Biodiversity Science

16

(pH8.0), 100 mM(pH8.0), 1.5 M NaCl, 1%

L, , 10010 min,

CTAB), K(200 g/mL)

12,000 rpm, 410 min, PCR

225 r/min3730 min;

16S rDNA: pMD19-T Vector

5 mL TE(1 mg/mL),

BacBEST RVSP6W,

2 h; 7.5 mL 20% (w/v) SDS, 652 h,

10PCR(plus

1015 min, 6,000 g10

Mg2+) 5 L, dNTPs (25 mM) 4 L, BacBEST

min, 100 mL

RV(5 M) 2 L, SP6W(5 M) 2 L, LA-Taq

4 mL DNA1 mL 20%

DNA2.5 U, 5 L, ddH2O

SDS, 10 s, 6510 min; 6,000 g

50 L: 9410 min; 94

10 min; ,

1 min, 601 min, 721 min, 30;

7210 min5 L PCREB

(25: 24: 1)

1%1.5 kb

, 15,000 g 15 min,

PCR5 L, 1 UMspI

100 mL, 2, 0.6

371 h, EDTA, EB

DNA 10 min, 4 12,000 g

2%,

20 min, , 70%2;

PCR,

, DNA500 L TEDNA

OmegaDNA

1.7

DNA

16S rDNARarefaction
aRarefactWinRarefaction,

, EB

16S rDNA

1.8

1.4

16S rDNARDP-II(Ribosomal Da-

DNA16S rDNA
27F(5-AGAGTTTGATCCT-

tabase Project) , CHECK-

GGCTCAG-3) 1492R (5-TACCTTGTTACGA-

CHIMERA ; BLAST

CTT-3)16S rDNA,

(http://blast.ncbi.nlm.nih.gov/Blast.cgi)

1.5 kbPCR50 L, 10PCR

2+

(plus Mg ) 5 L; dNTPs (25 mM) 4 L;

16S rDNAClustalX Version 1.83

27F1492R(5 M) 2 L; LA-Taq DNA2.5

, MEGA3.1

U; 5 L, DNADNA

, Neighbor-Joining

1050100: 94

10 min; 941 min, 601 min, 72

1 min, 30; 7210 minPCR
EB1%
1.5

16S rDNA
16S rDNA

16S rDNAGenBank
EU589248EU589321

2
2.1

, pMD19-T Vector System

, JM109

4.751074.901062.45105 cfu/g

(Ampicillin)/IPTG/X-GalLB

9.07%, pH6.4, 19.7 g/kg,

, 371624 h,

1.35 g/kg

4, PCR

2.2

1.6 16S rDNAPCRRFLP

, 100

DNA16S rDNA

: 16S rDNA-RFLP

589

DNA, 23 kbDNADNA
,
DNA DNA 4.68 g/g ,
A260/A2801.87DNA
16S rDNA, :
10DNA1.5 kb
, ; 50100
DNA1.5 kb
, 50PCR
16S
rDNA
2.3

16S rDNARFLP
RFLP,

1 16S rDNARarefaction

Fig. 1 Rarefaction curve for bacterial 16S rDNA clone library

derived from paddy soil in Ningxia

pMD19-T
E. coli JM109, 403

Blast, 48%

PCR, PCR

RFLPMspI, 1.5 kb16S rDNA

9799%, 46.7% 9097%,

5.3%8590%77.3%

, 74RFLP

16S rDNA

22.7%

2.4

16S rDNARarefaction

16S rDNARFLP,

2, 74

192

12, (Proteobac-

16S rDNA, 16S rDNA

teria) , 37.8%,

RFLP(OTU)16S rDNA

(Acidobacteria) 16.2%, (Actinobacteria)

Rarefaction(1), 162

12.2%, (Bacteroidetes) 10.8%,

, RFLP

(Chloroflexi)10.8%, (Planctomycetes)

8.1%, (Firmicutes)

RFLP

(Gemmatimonadetes) (Verrucomic-

, 16S rDNA

robia), 4.1%

, , 4, : 13.5%5.4%

KempAller (2004)

12.2%6.8%

22516S rDNA

, 16S rDNA,

OTU
, PCR
,

DNA

16S rDNA(Richard et al., 2001),

DNA

2.5

16S rDNA

NCBI

, DNA,

590

Biodiversity Science

16

2 16S rDNAGenBank
Fig. 2 A phylogenetic tree of soil bacteria based on 16S rDNA sequences. Sequences without GenBank access number are those
obtained in this study.

: 16S rDNA-RFLP

591

PCR

(Timothy et al., 1995)

(, 2007)pH

DNA,

6.4, ,

DNA, ,

DNA, PCRDNA

16.2%, (15.2%)(12.2%)

, DNA

,
16S rDNA: 77.3%

(Zhou et al., 2003)

(Schabereiter-

16S

Gurtner et al., 2002),

rDNA ,

, ,

,
(, 2000)

4
12,
,

( , 2006; ,
2007)(Zhou et
al., 1997)
, 2(Agrobacterium
sp., AY174112) (Rhizobium sp.,
AY822761) 99% 98%,
Agrobacterium Rhizobium,
,
(Katrin et al., 1999),
,
2
(Lysobacter sp.,
DQ462461, 98%) (Pantoea agglomerans, AY691543, 99%)

(2005)
, CPA-2 (Pantoea agglomerans
CPA-2)
(Costa et al., 2002)

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