温度对许氏平鲉性腺分化的影响及其机制
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摘要
本研究以许氏平鲉为实验对象,设置3组不同温度,即高温组(24℃)、对照组(20℃)和低温组(16℃),利用组织切片技术、酶联免疫法(ELISA)和实时荧光定量PCR(q RT-PCR)等技术探究温度对许氏平鲉性腺分化的影响及其潜在机制。结果显示,在40 dpb时,24℃下性腺发育最快,16℃下最慢,24℃、20℃和16℃下雌性率分别为70.0%、42.9%和33.3%。24℃和20℃下,E_2在较高水平持续时间较长,T水平在30～35 dpb时急剧降低,16℃下的E2水平迅速下降,35 dpb时T仍处于较高水平,说明在性腺分化期间,温度较高时,E_2水平较高,T水平较低,性腺偏雌性发育;反之,性腺偏雄性发育。在24℃下,35～40 dpb时的cyp19a1a mRNA的表达显著上调,可能与高温导致性腺分化偏雌性发育有关;在16℃下,30～50 dpb时ERβ2 mRNA的表达显著下调,说明ERβ2的表达被抑制可能与性腺偏雄性发育有关;24℃和20℃下,foxl2 mRNA的表达在25～35 dpb时处于较高水平,而低温组在30 dpb时表达水平开始上升,说明foxl2在性腺分化早期的高表达水平可能与卵巢分化的速率有关;在30～50 dpb,sox3、sox9和dmrt1的表达水平变化总趋势基本一致,说明三者之间的表达有一定的联系,可能与精巢的分化速率有关。
In this study, the black rockfish(Sebastes schlegelii) was selected as the research object. Three groups with different temperature treatments were set up, including high temperature group(24 ℃), control group(20 ℃)and low temperature group(16 ℃). Histology, ELISA and qRT-PCR methods were used to investigate the effect of different temperatures on gonadal differentiation and the potential mechanism. The results showed that at 40 dpb, the gonads developed fastest at 24 ℃, and slowest at 16 ℃. The female ratio were 70.0%, 42.9% and 33.3%,at 24 ℃, 20 ℃ and 16 ℃ respectively. At 24 ℃ and 20 ℃, high level of E_2 lasted for a long time and T level decreased sharply at 30-35 dpb. At 16 ℃, E_2 level dropped rapidly, and the T was still at a high level at 35 dpb. It indicated that at high temperature, the E_2 level was high, whereas T level was fairly low during gonad differentiation, and the gonad tended to be female; on the contrary, the gonad preferred to be male. At 24 ℃, the cyp19 a1 a mRNA expression was significantly up-regulated at 35-40 dpb, which may be associated with the partial female gonad development at high temperature. At 16 ℃, The ERβ2 mRNA expression was significantly decreased at 30-50 dpb. The inhibition of ERβ2 expression may be related to the partial male gonad development at low temperature. At 24 ℃ and 20 ℃, foxl2 mRNA expression was at relative high level at 25-35 dpb, and the expression level of low temperature treatment group began to increase at 30 dpb. It indicated that the high expression of foxl2 may be related to the rate of ovarian differentiation during the early stage of gonadal differentiation. At 30-50 dpb, the general trend of expression levels of sox3, sox9 and dmrt1 was consistent,indicating there is a connection between the expressions of the three genes, which might be related to the rate of testis differentiation.
In this study, the black rockfish(Sebastes schlegelii) was selected as the research object. Three groups with different temperature treatments were set up, including high temperature group(24 ℃), control group(20 ℃)and low temperature group(16 ℃). Histology, ELISA and qRT-PCR methods were used to investigate the effect of different temperatures on gonadal differentiation and the potential mechanism. The results showed that at 40 dpb, the gonads developed fastest at 24 ℃, and slowest at 16 ℃. The female ratio were 70.0%, 42.9% and 33.3%,at 24 ℃, 20 ℃ and 16 ℃ respectively. At 24 ℃ and 20 ℃, high level of E_2 lasted for a long time and T level decreased sharply at 30-35 dpb. At 16 ℃, E_2 level dropped rapidly, and the T was still at a high level at 35 dpb. It indicated that at high temperature, the E_2 level was high, whereas T level was fairly low during gonad differentiation, and the gonad tended to be female; on the contrary, the gonad preferred to be male. At 24 ℃, the cyp19 a1 a mRNA expression was significantly up-regulated at 35-40 dpb, which may be associated with the partial female gonad development at high temperature. At 16 ℃, The ERβ2 mRNA expression was significantly decreased at 30-50 dpb. The inhibition of ERβ2 expression may be related to the partial male gonad development at low temperature. At 24 ℃ and 20 ℃, foxl2 mRNA expression was at relative high level at 25-35 dpb, and the expression level of low temperature treatment group began to increase at 30 dpb. It indicated that the high expression of foxl2 may be related to the rate of ovarian differentiation during the early stage of gonadal differentiation. At 30-50 dpb, the general trend of expression levels of sox3, sox9 and dmrt1 was consistent,indicating there is a connection between the expressions of the three genes, which might be related to the rate of testis differentiation.
引文
[1]Yamamoto T.Sex differentiation[M]//Hoar W S,Randall D J.Fish physiology.New York:Academic Press,1969:117-175.
[2]宋卉,王树迎.鱼类原始生殖细胞的研究进展[J].动物医学进展,2004,25(5):22-23.Song H,Wang S Y.Progress in fish primordial germ cells[J].Progress in Veterinary Medicine,2004,25(5):22-23(in Chinese).
[3]彼尔索夫.鱼类的性别分化[M].卢浩泉,张天荫,译.北京:农业出版社,1982.ПерсовГМ.Sex Differentiation of fish[M].Lu H Q,Zhang T Y,trans.Beijing:Agriculture Press,1982(in Chinese).
[4]Brown E E,Baumann H,Conover D O.Temperature and photoperiod effects on sex determination in a fish[J].Journal of Experimental Marine Biology and Ecology,2014,461:39-43.
[5]Baroiller J F,D’Cotta H.Environment and sex determination in farmed fish[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology,2001,130(4):399-409.
[6]王念民,孙大江,曲秋芝,等.温度对鱼类性别分化和性别决定的影响[J].水产学杂志,2007,20(2):91-93.Wang N M,Sun D J,Qu Q Z,et al.Effects of temperature on the sex differentiation and determination of fishes[J].Chinese Journal of Fisheries,2007,20(2):91-93(in Chinese).
[7]Sfakianakis D G,Papadakis I E,Papadaki M,et al.Influence of rearing temperature during early life on sex differentiation,haemal lordosis and subsequent growth during the whole production cycle in European sea bass Dicentrarchus labrax[J].Aquaculture,2013,412-413:179-185.
[8]陈玉红,林丹军,尤永隆.泥鳅的性腺分化及温度对性腺分化的影响[J].中国水产科学,2007,14(1):74-82.Chen Y H,Lin D J,You Y L.Gonad differentiation in loach(Misgurnus anguillicaudatus)and the temperature effects[J].Journal of Fishery Sciences of China,2007,14(1):74-82(in Chinese).
[9]Goto-Kazeto R,Abe Y,Masai K,et al.Temperaturedependent sex differentiation in goldfish:establishing the temperature-sensitive period and effect of constant and fluctuating water temperatures[J].Aquaculture,2006,254(1-4):617-624.
[10]Pati?o R,Davis K B,Schoore J E,et al.Sex differentiation of channel catfish gonads:normal development and effects of temperature[J].Journal of Experimental Zoology,1996,276(3):209-218.
[11]岳敏娟,尤永隆,林丹军.温度对鲫鱼性腺分化的影响[J].动物学杂志,2009,44(1):9-16.Yue M J,You Y L,Lin D J.The influence of temperature on sex differentiation of Carassius auratus[J].Chinese Journal of Zoology,2009,44(1):9-16(in Chinese).
[12]Yamamoto E.Studies on sex-manipulation and production of cloned populations in hirame,Paralichthys olivaceus(Temminck et Schlegel)[J].Aquaculture,1999,173(1/2/3/4):235-246.
[13]邓思平,陈松林,田永胜,等.半滑舌鳎的性腺分化和温度对性别决定的影响[J].中国水产科学,2007,14(5):714-719.Deng S P,Chen S L,Tian Y S,et al.Gonadal differentiation and effects of temperature on sex determination in half-smooth tongue-sole,Cynoglossus semilaevis[J].Journal of Fishery Sciences of China,2007,14(5):714-719(in Chinese).
[14]路畅,苏利娜,朱邦科.鱼类性别决定及分化相关基因研究进展[J].湖北农业科学,2014,53(13):2981-2986.Lu C,Su L N,Zhu B K.Advances on the genes related with sex determination and differentiation of fish[J].Hubei Agricultural Sciences,2014,53(13):2981-2986(in Chinese).
[15]文爱韵,尤锋,徐永立,等.鱼类性别决定与分化相关基因研究进展[J].海洋科学,2008,32(1):74-80.Wen A Y,You F,Xu Y L,et al.Research progress on the study of sex determination and differentiation relateds to gene of fish[J].Marine Sciences,2008,32(1):74-80(in Chinese).
[16]游鑫,邓闵,刘全圣,等.黄颡鱼性腺分化及温度对性别分化的影响[J].华中农业大学学报,2016,35(1):106-113.You X,Deng M,Liu Q S,et al.Gonadal differentiation and effects of temperature on sex determination in yellow catfish,Pseudobagrus fulvidraco Richardson[J].Journal of Huazhong Agricultural University,2016,35(1):106-113(in Chinese).
[17]Omoto N,Koya Y,Chin B,et al.Gonadal sex differentiation and effect of rearing temperature on sex ratio in black rockfish(Sebastes schlegeli)[J].Ichthyological Research,2010,57(2):133-138.
[18]Pankhurst N W,Munday P L.Effects of climate change on fish reproduction and early life history stages[J].Marine and Freshwater Research,2011,62(9):1015-1026.
[19]Nakamura M,Bhandari R K,Higa M.The role estrogens play in sex differentiation and sex changes of fish[J].Fish Physiology and Biochemistry,2003,28(1/2/3/4):113-117.
[20]游鑫.温度对黄颡鱼性腺分化、性激素及早期生长的影响[D].武汉:华中农业大学,2015.You X.The effect of temperature on sex differentiation,sexsteroidhormones and growth in yellow catfish,Pseudobagrus fulvidraco Richardson[D].Wuhan:Huazhong Agricultural University,2015(in Chinese).
[21]Blázquez M,Felip A,Zanuy S,et al.Critical period of androgen-inducible sex differentiation in a teleost fish,the European sea bass[J].Journal of Fish Biology,2001,58(2):342-358.
[22]Lee C H,Na O S,Yeo I K,et al.Effects of sex steroid hormones and high temperature on sex differentiation in black rockfish,Sebastes schlegeli[J].Korean Journal of Fisheries and Aquatic Sciences,2000,33(5):373-377.
[23]Luzio A,Monteiro S M,Garcia-Santos S,et al.Zebrafish sex differentiation and gonad development after exposure to 17α-ethinylestradiol,fadrozole and their binary mixture:a stereological study[J].Aquatic Toxicology,2015,166:83-95.
[24]Guiguen Y,Fostier A,Piferrer F,et al.Ovarian aromatase and estrogens:a pivotal role for gonadal sex differentiation and sex change in fish[J].General and Comparative Endocrinology,2010,165(3):352-366.
[25]Díaz N,Piferrer F.Lasting effects of early exposure to temperature on the gonadal transcriptome at the time of sex differentiation in the European sea bass,a fish with mixed genetic and environmental sex determination[J].BMC Genomics,2015,16(1):679.
[26]Wang Y Y,Sun L X,Zhu J J,et al.Epigenetic control of cyp19a1a expression is critical for high temperature induced Nile tilapia masculinization[J].Journal of Thermal Biology,2017,69:76-84.
[27]王厚鹏.稀有鮈鲫ER基因的克隆及内分泌干扰物对其表达的影响[D].杨凌:西北农林科技大学,2012.Wang H P.Molecular cloning of estrogen receptor genes and their expression upon endocrine disrupting chemicals exposure in rare minnow[D].Yangling:Northwest A&F University,2012(in Chinese).
[28]Li C G,Wang H,Chen H J,et al.Differential expression analysis of genes involved in high-temperature induced sex differentiation in Nile tilapia[J].Comparative Biochemistry and Physiology-Part B:Biochemistry and Molecular Biology,2014,177-178:36-45.
[29]Sharma P,Tang S,Mayer G D,et al.Effects of thyroid endocrine manipulation on sex-related gene expression and population sex ratios in zebrafish[J].General and Comparative Endocrinology,2016,235:38-47.
[30]Yoshiura Y,Senthilkumaran B,Watanabe M,et al.Synergistic expression of Ad4BP/SF-1 and cytochrome P-450 aromatase(ovarian type)in the ovary of Nile tilapia,Oreochromis niloticus,during vitellogenesis suggests transcriptional interaction[J].Biology of Reproduction,2003,68(5):1545-1553.
[31]Wang D D,Zhang G R,Wei K J,et al.Molecular identification and expression of the Foxl2 gene during gonadal sex differentiation in northern snakehead Channa argus[J].Fish Physiology and Biochemistry,2015,41(6):1419-1433.
[32]Hua J J,Han J,Guo Y Y,et al.The progestin levonorgestrel affects sex differentiation in zebrafish at environmentally relevant concentrations[J].Aquatic Toxicology,2015,166:1-9.
[33]田佳,陈芸,王艺磊,等.鱼类性别决定的影响因素[J].生命科学,2010,22(10):971-977.Tian J,Chen Y,Wang Y L,et al.Influencing factors of sex determination in fish[J].Chinese Bulletin of Life Sciences,2010,22(10):971-977(in Chinese).
[34]Shin H S,An K W,Park M S,et al.Quantitative mRNAexpression of sox3 and DMRT1 during sex reversal,and expression profiles after GnRHa administration in black porgy,Acanthopagrus schlegeli[J].Comparative Biochemistry and Physiology-Part B:Biochemistry and Molecular Biology,2009,154(1):150-156.
[35]Poonlaphdecha S,Pepey E,Canonne M,et al.Temperature induced-masculinisation in the Nile tilapia causes rapid up-regulation of both dmrt1 and amh expressions[J].General and Comparative Endocrinology,2013,193:234-242.
[36]马丽曼.许氏平鲉性别相关基因Sox3、Sox9和Dmrt1的研究[D].青岛:中国海洋大学,2014.Ma L M.Study on sex related genes Sox3,Sox9 and Dmrt1 in black rockfish,Sebastes schlegelii[D].Qingdao:Ocean University of China,2014(in Chinese).
[37]闫浩,梁利群,常玉梅,等.遗传和温度因素对鱼类性别分化相关基因表达及性别比例的影响[J].大连海洋大学学报,2017,32(1):111-118.Yan H,Liang L Q,Chang Y M,et al.Effects of inheritance and temperature on sex determination and differentiation related genes and sex ratio in fish:a review[J].Journal of Dalian Ocean University,2017,32(1):111-118(in Chinese).
[2]宋卉,王树迎.鱼类原始生殖细胞的研究进展[J].动物医学进展,2004,25(5):22-23.Song H,Wang S Y.Progress in fish primordial germ cells[J].Progress in Veterinary Medicine,2004,25(5):22-23(in Chinese).
[3]彼尔索夫.鱼类的性别分化[M].卢浩泉,张天荫,译.北京:农业出版社,1982.ПерсовГМ.Sex Differentiation of fish[M].Lu H Q,Zhang T Y,trans.Beijing:Agriculture Press,1982(in Chinese).
[4]Brown E E,Baumann H,Conover D O.Temperature and photoperiod effects on sex determination in a fish[J].Journal of Experimental Marine Biology and Ecology,2014,461:39-43.
[5]Baroiller J F,D’Cotta H.Environment and sex determination in farmed fish[J].Comparative Biochemistry and Physiology Part C:Toxicology&Pharmacology,2001,130(4):399-409.
[6]王念民,孙大江,曲秋芝,等.温度对鱼类性别分化和性别决定的影响[J].水产学杂志,2007,20(2):91-93.Wang N M,Sun D J,Qu Q Z,et al.Effects of temperature on the sex differentiation and determination of fishes[J].Chinese Journal of Fisheries,2007,20(2):91-93(in Chinese).
[7]Sfakianakis D G,Papadakis I E,Papadaki M,et al.Influence of rearing temperature during early life on sex differentiation,haemal lordosis and subsequent growth during the whole production cycle in European sea bass Dicentrarchus labrax[J].Aquaculture,2013,412-413:179-185.
[8]陈玉红,林丹军,尤永隆.泥鳅的性腺分化及温度对性腺分化的影响[J].中国水产科学,2007,14(1):74-82.Chen Y H,Lin D J,You Y L.Gonad differentiation in loach(Misgurnus anguillicaudatus)and the temperature effects[J].Journal of Fishery Sciences of China,2007,14(1):74-82(in Chinese).
[9]Goto-Kazeto R,Abe Y,Masai K,et al.Temperaturedependent sex differentiation in goldfish:establishing the temperature-sensitive period and effect of constant and fluctuating water temperatures[J].Aquaculture,2006,254(1-4):617-624.
[10]Pati?o R,Davis K B,Schoore J E,et al.Sex differentiation of channel catfish gonads:normal development and effects of temperature[J].Journal of Experimental Zoology,1996,276(3):209-218.
[11]岳敏娟,尤永隆,林丹军.温度对鲫鱼性腺分化的影响[J].动物学杂志,2009,44(1):9-16.Yue M J,You Y L,Lin D J.The influence of temperature on sex differentiation of Carassius auratus[J].Chinese Journal of Zoology,2009,44(1):9-16(in Chinese).
[12]Yamamoto E.Studies on sex-manipulation and production of cloned populations in hirame,Paralichthys olivaceus(Temminck et Schlegel)[J].Aquaculture,1999,173(1/2/3/4):235-246.
[13]邓思平,陈松林,田永胜,等.半滑舌鳎的性腺分化和温度对性别决定的影响[J].中国水产科学,2007,14(5):714-719.Deng S P,Chen S L,Tian Y S,et al.Gonadal differentiation and effects of temperature on sex determination in half-smooth tongue-sole,Cynoglossus semilaevis[J].Journal of Fishery Sciences of China,2007,14(5):714-719(in Chinese).
[14]路畅,苏利娜,朱邦科.鱼类性别决定及分化相关基因研究进展[J].湖北农业科学,2014,53(13):2981-2986.Lu C,Su L N,Zhu B K.Advances on the genes related with sex determination and differentiation of fish[J].Hubei Agricultural Sciences,2014,53(13):2981-2986(in Chinese).
[15]文爱韵,尤锋,徐永立,等.鱼类性别决定与分化相关基因研究进展[J].海洋科学,2008,32(1):74-80.Wen A Y,You F,Xu Y L,et al.Research progress on the study of sex determination and differentiation relateds to gene of fish[J].Marine Sciences,2008,32(1):74-80(in Chinese).
[16]游鑫,邓闵,刘全圣,等.黄颡鱼性腺分化及温度对性别分化的影响[J].华中农业大学学报,2016,35(1):106-113.You X,Deng M,Liu Q S,et al.Gonadal differentiation and effects of temperature on sex determination in yellow catfish,Pseudobagrus fulvidraco Richardson[J].Journal of Huazhong Agricultural University,2016,35(1):106-113(in Chinese).
[17]Omoto N,Koya Y,Chin B,et al.Gonadal sex differentiation and effect of rearing temperature on sex ratio in black rockfish(Sebastes schlegeli)[J].Ichthyological Research,2010,57(2):133-138.
[18]Pankhurst N W,Munday P L.Effects of climate change on fish reproduction and early life history stages[J].Marine and Freshwater Research,2011,62(9):1015-1026.
[19]Nakamura M,Bhandari R K,Higa M.The role estrogens play in sex differentiation and sex changes of fish[J].Fish Physiology and Biochemistry,2003,28(1/2/3/4):113-117.
[20]游鑫.温度对黄颡鱼性腺分化、性激素及早期生长的影响[D].武汉:华中农业大学,2015.You X.The effect of temperature on sex differentiation,sexsteroidhormones and growth in yellow catfish,Pseudobagrus fulvidraco Richardson[D].Wuhan:Huazhong Agricultural University,2015(in Chinese).
[21]Blázquez M,Felip A,Zanuy S,et al.Critical period of androgen-inducible sex differentiation in a teleost fish,the European sea bass[J].Journal of Fish Biology,2001,58(2):342-358.
[22]Lee C H,Na O S,Yeo I K,et al.Effects of sex steroid hormones and high temperature on sex differentiation in black rockfish,Sebastes schlegeli[J].Korean Journal of Fisheries and Aquatic Sciences,2000,33(5):373-377.
[23]Luzio A,Monteiro S M,Garcia-Santos S,et al.Zebrafish sex differentiation and gonad development after exposure to 17α-ethinylestradiol,fadrozole and their binary mixture:a stereological study[J].Aquatic Toxicology,2015,166:83-95.
[24]Guiguen Y,Fostier A,Piferrer F,et al.Ovarian aromatase and estrogens:a pivotal role for gonadal sex differentiation and sex change in fish[J].General and Comparative Endocrinology,2010,165(3):352-366.
[25]Díaz N,Piferrer F.Lasting effects of early exposure to temperature on the gonadal transcriptome at the time of sex differentiation in the European sea bass,a fish with mixed genetic and environmental sex determination[J].BMC Genomics,2015,16(1):679.
[26]Wang Y Y,Sun L X,Zhu J J,et al.Epigenetic control of cyp19a1a expression is critical for high temperature induced Nile tilapia masculinization[J].Journal of Thermal Biology,2017,69:76-84.
[27]王厚鹏.稀有鮈鲫ER基因的克隆及内分泌干扰物对其表达的影响[D].杨凌:西北农林科技大学,2012.Wang H P.Molecular cloning of estrogen receptor genes and their expression upon endocrine disrupting chemicals exposure in rare minnow[D].Yangling:Northwest A&F University,2012(in Chinese).
[28]Li C G,Wang H,Chen H J,et al.Differential expression analysis of genes involved in high-temperature induced sex differentiation in Nile tilapia[J].Comparative Biochemistry and Physiology-Part B:Biochemistry and Molecular Biology,2014,177-178:36-45.
[29]Sharma P,Tang S,Mayer G D,et al.Effects of thyroid endocrine manipulation on sex-related gene expression and population sex ratios in zebrafish[J].General and Comparative Endocrinology,2016,235:38-47.
[30]Yoshiura Y,Senthilkumaran B,Watanabe M,et al.Synergistic expression of Ad4BP/SF-1 and cytochrome P-450 aromatase(ovarian type)in the ovary of Nile tilapia,Oreochromis niloticus,during vitellogenesis suggests transcriptional interaction[J].Biology of Reproduction,2003,68(5):1545-1553.
[31]Wang D D,Zhang G R,Wei K J,et al.Molecular identification and expression of the Foxl2 gene during gonadal sex differentiation in northern snakehead Channa argus[J].Fish Physiology and Biochemistry,2015,41(6):1419-1433.
[32]Hua J J,Han J,Guo Y Y,et al.The progestin levonorgestrel affects sex differentiation in zebrafish at environmentally relevant concentrations[J].Aquatic Toxicology,2015,166:1-9.
[33]田佳,陈芸,王艺磊,等.鱼类性别决定的影响因素[J].生命科学,2010,22(10):971-977.Tian J,Chen Y,Wang Y L,et al.Influencing factors of sex determination in fish[J].Chinese Bulletin of Life Sciences,2010,22(10):971-977(in Chinese).
[34]Shin H S,An K W,Park M S,et al.Quantitative mRNAexpression of sox3 and DMRT1 during sex reversal,and expression profiles after GnRHa administration in black porgy,Acanthopagrus schlegeli[J].Comparative Biochemistry and Physiology-Part B:Biochemistry and Molecular Biology,2009,154(1):150-156.
[35]Poonlaphdecha S,Pepey E,Canonne M,et al.Temperature induced-masculinisation in the Nile tilapia causes rapid up-regulation of both dmrt1 and amh expressions[J].General and Comparative Endocrinology,2013,193:234-242.
[36]马丽曼.许氏平鲉性别相关基因Sox3、Sox9和Dmrt1的研究[D].青岛:中国海洋大学,2014.Ma L M.Study on sex related genes Sox3,Sox9 and Dmrt1 in black rockfish,Sebastes schlegelii[D].Qingdao:Ocean University of China,2014(in Chinese).
[37]闫浩,梁利群,常玉梅,等.遗传和温度因素对鱼类性别分化相关基因表达及性别比例的影响[J].大连海洋大学学报,2017,32(1):111-118.Yan H,Liang L Q,Chang Y M,et al.Effects of inheritance and temperature on sex determination and differentiation related genes and sex ratio in fish:a review[J].Journal of Dalian Ocean University,2017,32(1):111-118(in Chinese).
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