1912 生物工程学报 Chinese Journal of Biotechnology http://journals.im.ac.cn/cjbcn DOI: 10.13345/j.cjb.140279 张云茹等 / 可降解 TCE 的甲烷氧化菌 16S rdna 与 pmocab 基因簇序列分析 Decenber 25, 2014, 30(12): 1912 1923 2014 Chin J Biotech, All rights reserved 研究报告 可降解 TCE 的甲烷氧化菌 16S rdna 与 pmocab 基因簇序列分析 张云茹 1, 陈华清 1, 高艳辉 2, 邢志林 2, 赵天涛 2 1 400054 2 400054 张云茹, 陈华清, 高艳辉, 等. 可降解 TCE 的甲烷氧化菌 16S rdna 与 pmocab 基因簇序列分析. 生物工程学报, 2014, 30(12): 1900 1911. Zhang YR, Chen HQ, Gao YH, et al. Sequence analysis of 16S rdna and pmocab gene cluster of trichloroethylene-degrading methanotroph. Chin J Biotech, 2014, 30(12): 1900 1911. 摘要 : 甲烷氧化菌可高效催化甲烷和多种氯代烃降解, 开展甲烷单加氧酶的基因簇序列分析研究有助于深入了解催化机理, 并提升其在污染物生物降解领域中的应用价值 以甲烷为唯一碳源富集分离甲烷氧化菌, 取 5 种氯代烃作为共代谢基质考察其降解情况, 利用 MEGE5.05 软件构建基于 16S rdna 的系统发育树对所选菌株进行初步鉴定, 用半巢式 PCR 法分段扩增菌株的颗粒性甲烷单加氧酶 (pmmo) 基因簇并进行 T-A 克隆测序, 通过 ExPASy 计算 pmmo 三个亚基的理论分子量 筛选到了一株甲基孢囊菌 Methylocystis sp. JTC3, 在三氯乙烯 (TCE) 初始浓度为 15.64 μmol/l 条件下, 反应 5 d 后降解率可达 93.79% 经扩增 测序 拼接得到了 3 227 bp 的 pmocab 基因簇序列, 包括 771 bp 的 pmoc 基因 759 bp 的 pmoa 基因 1 260 bp 的 pmob 基因和 2 个非编码中间序列, 所对应 γ β α 亚基理论分子量分别为 29.1 kda 28.6 kda 和 45.6 kda 通过 Blast 比对发现 Methylocystis sp. JTC3 的 pmocab 基因簇序列与 Methylocystis sp. strain M 的 pmocab 一致性较高, 其中 pmoa 的序列相对保守 JTC3 菌株可高效降解 TCE, 对 pmocab 基因簇序列的详细分析可为 pmmo 活性中心特征 氯代烃类底物选择性等方面的深入研究提供信息数据基础 : 甲烷氧化菌, 三氯乙烯,pmoCAB 基因簇,16S rdna, 系统发育树 Received: May 20, 2014; Accepted: August 14, 2014 Supported by: National Natural Science Foundation of China (No. 51378522), Fundamental and Advanced Research Projects of Chongqing (No. cstc2013jcyja20009). Corresponding author: Tiantao Zhao. Tel/Fax: +86-23-62563221; E-mail: tiantaozhao@gmail.com (No. 51378522) (No. cstc2013jcyja20009)
张云茹等 / 可降解 TCE 的甲烷氧化菌 16S rdna 与 pmocab 基因簇序列分析 1913 Sequence analysis of 16S rdna and pmocab gene cluster of trichloroethylene-degrading methanotroph Yunru Zhang 1, Huaqing Chen 1, Yanhui Gao 2, Zhilin Xing 2, and Tiantao Zhao 2 1 School of Pharmacy and Bioengineering, Chongqing University of Technology, Chongqing 400054, China 2 School of Chemistry and Chemical Engineering, Chongqing University of Technology, Chongqing 400054, China Abstract: Methanotrophs could degrade methane and various chlorinated hydrocarbons. The analysis on methane monooxygenase gene cluster sequence would help to understand its catalytic mechanism and enhance the application in pollutants biodegradation. The methanotrophs was enriched and isolated with methane as the sole carbon source in the nitrate mineral salt medium. Then, five chlorinated hydrocarbons were selected as cometabolic substrates to study the biodegradation. The phylogenetic tree of 16S rdna using MEGE5.05 software was constructed to identify the methanotroph strain. The pmocab gene cluster encoding particulate methane monooxygenase (pmmo) was amplified by semi-nested PCR in segments. ExPASy was performed to analyze theoretical molecular weight of the three pmmo subunits. As a result, a strain of methanotroph was isolated. The phylogenetic analysis indicated that the strain belongs to a species of Methylocystis, and it was named as Methylocystis sp. JTC3. The degradation rate of trichloroethylene (TCE) reached 93.79% when its initial concentration was 15.64 μmol/l after 5 days. We obtained the pmocab gene cluster of 3 227 bp including pmoc gene of 771 bp, pmoa gene of 759 bp, pmob gene of 1 260 bp and two noncoding sequences in the middle by semi-nested PCR, T-A cloning and sequencing. The theoretical molecular weight of their corresponding gamma, beta and alpha subunit were 29.1 kda, 28.6 kda and 45.6 kda respectively analyzed using ExPASy tool. The pmocab gene cluster of JTC3 was highly identical with that of Methylocystis sp. strain M analyzed by Blast, and pmoa sequences is more conservative than pmoc and pmob. Finally, Methylocystis sp. JTC3 could degrade TCE efficiently. And the detailed analysis of pmocab from Methylocystis sp. JTC3 laid a solid foundation to further study its active sites features and its selectivity to chlorinated hydrocarbon. Keywords: methanotroph, trichloroethylene, pmocab gene cluster, 16S rdna, phylogenetic tree (Trichloroethylene TCE) [1-2] [3-4] TCE (Methane monooxygenase MMO) [5-6] MMO (Solution methane monooxygenase smmo) (Particulate methane monooxygenase pmmo) [7-8] smmo pmmo [9-11] Methylosinus trichosporium OB3b Methylococcus capsulatus Bath smmo [12-13] smmo (MMOH) (MMOR) (MMOB) cjb@im.ac.cn
1914 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech December 25, 2014 Vol.30 No.12 orfy pmmo [14-16] pmmo α β γ 1 1 1 (αβγ) 3 pmmo pmmo 3 pmocab γ β α Basu [17] Methylococcus capsulatus Bath pmmo α (47 kda) β (27 kda) γ (23 kda) 63 kda (pmmor) 3 (pmmoh) pmmo smmo MMOR MMOB pmmo (αβγ) 3 [18] [12] pmmo pmmo [13,19] [20] GenBank GenBank pmocab 9 7 ( pmocab) 5 pmocab GenBank MMO pmocab TCE JTC3 TCE 16S rdna pmocab 1 材料与方法 1.1 材料 NMS [21] 1 18 a 13.2% 0.47% 9.96% ph 7.73 LA Taq DL 2 000 bp DNA marker 500 bp DNA ladder (Dye plus) ( ) :Eppendorf 5332 PCR BIORAD SC-6 000A ( ECD ) 1.2 方法 1.2.1 4 mm 2 mm 100 g 500 ml 100 ml 30 2 10 NMS 4 5 http://journals.im.ac.cn/cjbcn
张云茹等 / 可降解 TCE 的甲烷氧化菌 16S rdna 与 pmocab 基因簇序列分析 1915 表 1 引物及其核苷酸序列 Table 1 Primers and their nucleotide sequences Primers Nucleotide sequences (5 3 ) F27 [22] R1429 [22] A189gc [23] Mb661 [23] PU 5 PU 3-1 PU 3-2 PU 3-3 PD 5-1 PD 5-2 PD 5-3 PD 3 AGAGTTTGATCATGGCTCAG TACGGTTACCTTGTTACGACTT GGNGACTGGGACTTCTGG CCGGMGCAACGTCYTTACC ATGAGCTCVACGACTRRCACRGC HGC GGAAGACGGAAGTTGACCCAC AGTTGACGTAGCGGTTGATCCAC ATCATAGCAGACGGGAACACGAG CTCGTGTTCCCGTCTGCTATGAT TCACGGCGGTTGTCGGTTCGCT AATACATCCGCATGGTCGAGC AGTWTCGCTTAGGGCATGTC 1.2.2 DNA T-A TIANGEN DP302 DNA PCR TIANGEN DP209 DNA T-A pmd 19-T Vector Cloning Kit 1.2.3 JTC3 JTC3 1 ml 20 ml NMS 100 ml 20 ml 30 170 r/min 2 3 d 20 ml 1 ml [24] JTC3 TCE 3 TCE (0 70 μmol/l) TCE TCE TCE 1.2.4 GDX-104 2 m 35 ml/min 10 ml/min ( ) 120 ( ) 90 200 0.1 ml 0.00 na ( ) TCD TCX-01 1 m 40 ml/min ( ) 120 ( ) 90 120 1.2.5 JTC3 16S rdna JTC3 DNA F27/R1429 16S rdna 16S rdna MEGA 5.05 (NJ) NCBI (National center for biotechnology information) Blast (Basic local alignment search tool) JTC3 PCR 94 5 min 94 35 s 55 30 s 72 35 s 30 72 5 min 1.2.6 JTC3 pmocab JTC3 A189gc/Mb661 pmoa cjb@im.ac.cn
1916 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech December 25, 2014 Vol.30 No.12 PCR 94 5 min 94 1 min 59 45 s 68 40 s 35 68 5 min GenBank pmocab JTC3 pmoa Primer Premier 5.0 6 ( 1 1) PCR pmob pmoc T-A pmoa pmocab PCR [25] PCR 2 94 5 min 94 35 s 55 30 s 72 35 s 30 72 5 min 图 1 pmocab 基因簇构成及引物设计位置示意图 Fig. 1 Constitute map of pmocab gene clusters and location of primer designed where the black and gray regions are primer sites and noncoding regions respectively. The digital sites of 1 to 8 are PU 5, PU 3-1, PU 3-2, PU 3-3, PD 5-1, PD 5-2, PD 5-3, PD 3 respectively. 表 2 pmoc 和 pmob 基因片段三轮半巢式 PCR 所用引物及模板 Table 2 Primers and templates used in semi-nested PCR Semi- nested PCR pmoc-terminal fragment pmob-terminal fragment The first round Template Genome DNA Primer PU 5 /PU3-3 PD 5-1/PD3 The second round Template PCR product of first round diluted 20 times Primer PU 5 /PU 3-2 PD 5-2/PD 3 The third round Template PCR product of second round diluted 20 times Primer PU 5 /PU 3-1 PD 5-3/PD 3 Blast JTC3 pmocab DNAMAN 6.0.3.99 MMO pmocab ExPASy Compute pi/mw pmmo 2 结果与分析 2.1 甲烷氧化菌的分离纯化 JTC3 NMS 1 1.5 mm 0.5 μm 2.2 甲烷氧化菌 JTC3 降解氯代烃 JTC3 1,2-1,2- TCE 5 5 d 2 2A http://journals.im.ac.cn/cjbcn
张云茹等 / 可降解 TCE 的甲烷氧化菌 16S rdna 与 pmocab 基因簇序列分析 1917 TCE 4 Oldenhuis [26] TCE TCE 5 d 0.29 μmol/l 2B 5 d TCE 5 d (15.64±0.27) μmol/l (0.97±0.041) μmol/l 93.79% 5 d TCE 0.20 250.38 μmol/l V max 120 [27] Methylocystis sp. M TCE 7.6 μmol/l ( 1 ppm) 6 d TCE 7 TCE 91% [28] Methylocystis sp. M JTC3 TCE TCE Oldenhuis [26] TCE TCE Choi [29] TCE pmoa TCE pmoa TCE JTC3 3 TCE (12.55 20.76 μmol/l) JTC3 TCE 60.76 μmol/l 20 h TCE JTC3 pmmo 图 2 甲烷氧化菌 JTC3 降解 5 种氯代烃效果及 TCE 浓度变化 Fig. 2 Degradation of chlorinated hydrocarbons (A) by Methylocystis sp. JTC3 and variation of TCE concentration over time (B). 图 3 不同 TCE 浓度对甲烷氧化的影响 Fig. 3 Effect of TCE concentration on methane oxidation. cjb@im.ac.cn
1918 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech December 25, 2014 Vol.30 No.12 2.3 甲烷氧化菌 JTC3 菌株的 16S rdna 序列分析及其分类鉴定 PCR 1 500 bp 16S rdna ( 4) 1 487 bp 16S rdna ( 5) Methylocystis sp. M 98% Blast JTC3 α / / Methylocystis sp. JTC3 5 4 α 6 γ 95% JTC3 α Methylocystis sp. M 99% NCBI Blast JTC3 16S rdna 92% EB-1 IMET 10484 WI 14M 99% 95% 16S rdna 图 4 甲烷氧化菌 JTC3 的 16S rdna Fig. 4 PCR products electrophoresis of 16S rdna from Methylocystis sp. JTC3. 1 4: 16S rdna of Methylocystis sp. JTC3. 图 5 Fig. 5 基于 16S rdna 的 Methylocystis sp. JTC3 系统发育树 Phylogenetic analysis of the 16S rdna from Methylocystis sp. JTC3. http://journals.im.ac.cn/cjbcn
张云茹等 / 可降解 TCE 的甲烷氧化菌 16S rdna 与 pmocab 基因簇序列分析 1919 2.4 JTC3 菌株的 pmocab 基因簇分段扩增及序列分析 JTC3 pmocab pmoa PCR 6A 500 bp DNA 489 bp GenBank pmoa (AM849782.1) 99% pmoa PCR JTC3 pmoc pmob 6B GenBank pmocab JTC3 pmocab pmoc pmob PCR 6B pmoc 1 000 bp 1 500 bp DNA ( 1 340 bp) pmob 1 500 bp DNA ( 1 570 bp) PCR pmoc pmob 2.5 pmocab 基因簇序列分析 pmoc pomb pmoa pmocab GenBank pmocab KJ499432 pmoa KF742674 pmob KF742675 pmoc KF742673 pmocab 3 226 bp 1 771 bp pmoc 1 087 1 845 bp 759 bp pmoa 1 967 3 226 bp 1 260 bp pmob Blast JTC3 7 JTC3 Methylocystis sp. M pmoc pmoa pmob 3 99% 99% 100% 100% JTC3 pmmo M pmmo JTC3 16S rdna M JTC3 JTC3 TCE M 图 6 Methylocystis sp. JTC3 的 pmocab 基因簇分段扩增电泳图 Fig. 6 Segmentation amplification of pmocab cluster from Methylocystis sp. JTC3. (A) pmoa gene segment. 1 3: pmoa gene segment. (B) The third time of semi-nested PCR. 1:pomC segment; 2:pmoB segment. cjb@im.ac.cn
1920 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech December 25, 2014 Vol.30 No.12 图 7 JTC3 菌株与 3 株甲烷氧化菌的 pmmo 序列一致性比较 Fig. 7 Comparision of sequences of pmmo genes from Methylocystis sp. JTC3 with several known methanetrophs. Methylocysti sp. SC2 Methylocystis sp. GSC357 JTC3 pmmo 85% 4 3 pmoa 94% 90% pmocab 16S rdna [30] pmocab 3 pmoa [31-32] GenBank pmoa 450 520 bp pmoa ATG TAA 759 bp pmoa ExPASy Methylocystis sp. JTC3 pmmo α β γ 6.56 6.96 5.05 45.6 kda 28.6 kda 29.1 kda Semrau pmocab pmmo α β γ 3 46 kda 28 kda 29 kda [33] pmmo α β γ, Methylococcus capsulatus Bath pmmo 3 47 kda 27 kda 23 kda [17] Methylosinus trichosporium OB3b pmmo 3 41 kda 26 kda 25 kda [13] pmmo 3 pmmo 3 结论与展望 Methylocystis sp. JTC3 TCE TCE 16S rdna pmocab 3 226 bp Methylocystis sp. M pmocab SC2 GSC357 Methylocystis sp. JTC3 Methylocystis sp. http://journals.im.ac.cn/cjbcn
张云茹等 / 可降解 TCE 的甲烷氧化菌 16S rdna 与 pmocab 基因簇序列分析 1921 M pmmo pmoc γ Methylocystis sp. M 2 (D/S 5 T/A131) Methylocystis sp. TJC3 5 1 131 Methylocystis sp. M 5 131 2 2 JTC3 TCE M M TCE smmo [34] smmo TCE pmmo TCE [35] JTC3 TCE 10 μmol/l pmmo smmo TCE pmmo JTC3 TCE pmmo pmmo pmocab JTC3 REFERENCES [1] Choi SA, Lee EH, Cho KS. Effect of trichloroethylene and tetrachloroethylene on methane oxidation and community structure of methanotrophic consortium J Environ Sci Health, 2013, 48(13): 1723 1731. [2] Wei CH, Zhang XX, Ren Y, et al. Pollution control of persistent organic pollutants, in water system: adsorption/enrichment, biodegradation and process analysis. Environ Chem, 2011, 30(1): 300 309 (in Chinese).,,,. :.., 2011, 30(1): 300 309. [3] Semrau JD, DiSpirito AA, Yoon S. Methanotrophs and copper. FEMS Microbiol Lett, 2010, 34(4): 496 531. [4] Wei SZ. Methanotrophs and their applications in environment treatment: a review. Chin J Appl Ecol, 2012, 23(8): 2309 2318 (in Chinese)..., 2012, 23(8): 2309 2318. [5] Semrau JD. Bioremediation via methanotrophy: over view of recent findings and suggestions for future research. Front Microbiol, 2011, 2(209): 1 7. [6] Han B, Su T, Li X, et al. Research progresses of methanotrophs and methane monooxygenase. Chin J Biotech, 2008, 24(9): 1511 1519 (in Chinese).,,,.., 2008, 24(9): 1511 1519. [7] Cao YB, Yin B, Niu YB, et al. Research progresses of methanotrophs. Environ sci, 2012, 6: 24 26 (in Chinese).,,,.., 2012, 6: 24 26. [8] Semrau JD, DiSpirito AA, Vuilleumier S. Facultative methanotrophy: false leads, true results, and suggestions for future research. FEMS Microbiol Lett, 2011, 323(1): 1 12. [9] Jagadevan S, Semrau JD. Priority pollutant degradation by the facultative methanotroph, Methylocystis strain SB2. Appl Microbiol Biotechnol, 2013, 97(11): 5089 5096. cjb@im.ac.cn
1922 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech December 25, 2014 Vol.30 No.12 [10] Speitel Jr GE, Thompson RC, Weissman D. Biodegradation kinetics of Methylosinus trichosporium OB3b at low concentrations of chloroform in the presence and absence of enzyme competition by methane. Water Res, 1993, 27(1): 15 24. [11] Van Hylckama VJ, De Koning W, Janssen DB. Transformation kinetics of chlorinated ethenes by Methylosinus trichosporium OB3b and detection of unstable epoxides by on-line gas chromatography. Appl Microbiol Biotechnol, 1996, 62(9): 3304 3312. [12] Culpepper MA, Rosenzweig AC. Architecture and active site of particulate methane monooxygenase. Crit Rev Biochem Mol Biol, 2012, 47(6): 483 492. [13] Lieberman RL, Rosenzweig AC. Biological methane oxidation: regulation, biochemistry, and active site structure of particulate methane monooxygenase. Crit Rev Biochem Mol Biol, 2004, 39(3): 147 164. [14] Culpepper MA, Cutsail Ⅲ GE, Hoffman BM, et al. Evidence for oxygen binding at the active site of particulate methane monooxygenase. J Am Chem Soc, 2012, 134(18): 7640 7643. [15] Han B, Su T, Yang C, et al. Heterologous expression of particulate methane monooxygenase in different host cells. Chin J Biotech, 2009, 25(8): 1151 1159 (in Chinese).,,,.., 2009, 25(8): 1151 1159. [16] Yoon S, Im J, Bandow N, et al. Constitutive expression of pmmo by Methylocystis strain SB2 when grown on multi-carbon substrates: implications for biodegradation of chlorinated ethenes. Environ Microbiol Rep, 2011, 3(2): 182 188. [17] Basu P, Katterle B, Andersson K, et al. The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein. Biochem J, 2003, 369(2): 417 427. [18] Smith SM, Rawat S, Telser J, et al. Crystal structure and characterization of particulate methane monooxygenase from Methylocystis species strain M. Biochemistry, 2011, 50(47): 10231 10240. [19] Horz HP, Yimga MT, Liesack W. Detection of methanotroph diversity on roots of submerged rice plants by molecular retrieval ofpmoa, mmox, mxaf, and 16S rrna and ribosomal DNA, including pmoa-based terminal restriction fragment length polymorphism profiling. Appl Environ Microbiol, 2001, 67(9): 4177 4185. [20] Zhao T, Li H, Cheng C. 16S rrna two level structure analysis in the classification and identification of microorganisms. Food Ferm Indus, 2011, 37(12): 105 108 (in Chinese).,,. 16S rrna., 2011, 37(12): 105 108. [21] Zhao TT, Zhang LJ, Zhang YR, et al. Characterization of Methylocystis strain JTA1 isolated from aged refuse and its tolerance to chloroform. J Environ Sci, 2013, 25(4): 770 775. [22] Luo MF, Wu H, Wang L, et al. Growth characteristics of a methane-utilizing mixed consortia MY9. Chin J Proc Eng, 2009, 9(1): 113 117 (in Chinese).,,,. MY9., 2009, 9(1): 113 117. [23] Kolb S, Knief C, Stubner S. Quantitative detection of methanotrophsin soil by novel pmoa-targeted real-time PCR assays Appl Environ Microbiol, 2003, 69(5): 2423 2429. [24] Robbins GA, Wang S, Stuart JD. Using the static headspace method to determine Henry's law constants. Analy Chem, 1993, 65(21): 3113 3118. [25] Chen SX, Wang XW, Fang YL. Rapid characterization of recombination clone by PCR screening of individual bacterial colonies. Microbiol China, 2006, 33(3): 52 56 (in Chinese).,,. PCR http://journals.im.ac.cn/cjbcn
张云茹等 / 可降解 TCE 的甲烷氧化菌 16S rdna 与 pmocab 基因簇序列分析 1923., 2006, 33(3): 52 56. [26] Oldenhuis R, Oedzes JY, Van der Waarde JJ, et al. Kinetics of chlorinated hydrocarbon degradation by Methylosinus trichosporium OB3b and toxicity of trichloroethylene. Appl Microbiol Biotechnol, 1991, 57(1): 7 14. [27] Xing ZH, Zhang LJ, Zhao TT. Recent research, analysis of kinetics and prospect on degradation of chlorinated hydrocarbons via obligate and facultative methanotrophs. Chin J Biotech, 2014, 30(4): 531 544 (in Chinese)..., 2014, 30(4): 531 544. [28] Uchiyama H, Nakajima T, Yagi O, et al. Aerobic degradation of trichloroethylene by a new type Ⅱ methane-utilizing bacterium, strain M. Agri biol chem, 1989, 53(11): 2903 2907. [29] Choi SA, Lee EH, Cho KS. Effect of trichloroethylene and tetrachloroethylene on methane oxidation and community structure of methanotrophic consortium. J Environ Sci Health, 2013, 48(13): 1723 1731. [30] Knief C, Vanitchung S, Harvey NW, et al. Diversity of methanotrophic bacteria in tropical upland soils under different land uses. Appl Environ Microbiol, 2005, 71(7): 3826 3831. [31] Dam B, Dam S, Blom J, et al. Genome analysis coupled with physiological studies reveals a diverse nitrogen metabolism in Methylocystis sp. strain SC2. PLoS ONE, 2013, 8(10): e74767. [32] Iguchi H, Yurimoto H, Sakai Y. Soluble and particulate methane monooxygenase gene clusters of the type I methanotroph Methylovulum miyakonense HT12. FEMS Microbiol Lett, 2010, 312(1): 71 76. [33] Semrau JD Chistoserdov A, Lebron J, et al. Particulate methane monooxygenase genes in methanotrophs. J Bacteriol, 1995, 177(11): 3071 3079. [34] Shigematsu T, Hanada S, Eguchi M, et al. Soluble methane monooxygenase gene clusters from trichlorethylene-degrading Methylomonas sp. strains and detection of methanotrophs during in situ bioremediation. Appl Environ Microbiol, 1999, 65(12): 5198 5206. [35] Baker PW, Futamata H, Harayama S, et al. Molecular diversity of pmmo and smmo in a TCE-contaminated aquifer during bioremediation. FEMS Microbiol Ecol, 2001, 38(2/3): 161 167. ( ) cjb@im.ac.cn