巴西橡胶树染色体制片方法的改良及FISH信号检测
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摘要
本研究以巴西橡胶树热研7-33-97品种幼叶为材料,通过改良配制纤维素酶与果胶酶混合酶的溶剂、酶解时间等参数,对酶解去壁低渗方法进行了相应改良。结果表明,在37℃下,采用磷酸盐缓冲液(NaH2PO4,31.21 g/L; Na2HPO4, 71.64 g/L; Na Cl, 8 g/L; pH=5.5)作为溶剂,配制终浓度为5%纤维素酶、4%果胶酶的酶混合液时,酶解时间为1.0 h,大大缩短了酶解时间。用该法制备的中期标本,染色体形态分散、清晰、细胞膨大良好,荧光原位杂交信号检测后,信号正常。本研究可为橡胶树细胞遗传学、基因定位提供细胞学方法。
In this study, young leaves of Brazilian rubber tree(Hevea brasiliensis) of Reyan 7-33-97 variety were used as materials. The method of enzymatic hydrolysis and low permeability for cell wall removal was modified by improving the solvent and time of enzymatic hydrolysis of mixed cellulase and pectinase. The results showed that enzymolysis time was 1.0 h by using final concentration of 5% cellulase and 4% pectinase enzyme mixture prepared at 37 ℃, using phosphate buffer(NaH2 PO4, 31.21 g/L; Na2 HPO4, 71.64 g/L; NaCl, 8 g/L; pH=5.5) as the solvent, this modification greatly shortened the time of enzymatic hydrolysis. The metaphase samples prepared by this method showed that the chromosome morphology was dispersive, clear and the cells were well expanded. The signal of the fluorescence in situ hybridization was well detected. This study can provide a kind of cytological approach for studies of cytogenetics and gene localization of rubber tree.
In this study, young leaves of Brazilian rubber tree(Hevea brasiliensis) of Reyan 7-33-97 variety were used as materials. The method of enzymatic hydrolysis and low permeability for cell wall removal was modified by improving the solvent and time of enzymatic hydrolysis of mixed cellulase and pectinase. The results showed that enzymolysis time was 1.0 h by using final concentration of 5% cellulase and 4% pectinase enzyme mixture prepared at 37 ℃, using phosphate buffer(NaH2 PO4, 31.21 g/L; Na2 HPO4, 71.64 g/L; NaCl, 8 g/L; pH=5.5) as the solvent, this modification greatly shortened the time of enzymatic hydrolysis. The metaphase samples prepared by this method showed that the chromosome morphology was dispersive, clear and the cells were well expanded. The signal of the fluorescence in situ hybridization was well detected. This study can provide a kind of cytological approach for studies of cytogenetics and gene localization of rubber tree.
引文
Ahirwar R.,Sharma J.G.,Nahar P.,and Kumar S.,2017,Immobilization studies of cellulase on three engineered polymer surfaces,Biocatalysis and Agricultural Biotechnology,11:248-251
Bauman J.G.,Wiegant J.,Borst P.,and van Duijn P.,1980,Anew method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochromelabelled RNA,Exp.Cell Res.,128(2):485-490
Chen R.Y.,Song W.Q.,and Li X.L.,1982,Wall degradation hypotonic method of preparing chromosome samples in plant and its significance in the cytogenetics,Yichuan Xuebao(Journal of Genetics and Genomics),9(2):151-159(陈瑞阳,宋文芹,李秀兰,1982,植物染色体标本制备的去壁,低渗法及其在细胞遗传学中的意义,遗传学报,9(2):151-159)
Chen X.J.,2014a,Microdissection of the chromosome 1,5 and9 and construction of their DNA libraries in Hevea brasiliensis Reyan7-33-97,Tesis for M.S.,Hainan University,Supervisor:Wang Y.,pp.71(陈学俊,2014,巴西橡胶树热研7-33-97第1,5和9号染色体的分离及文库的构建,硕士学位论文,海南大学,导师:王英,pp.71)
Chen X.J.,Wang Y.,Gao J.J.,Gao H.Q.,and Zhuang N.S.,2014,Microdissection and microcloning of the ninth chromosome in rubber tree(Hevea brasliensis),Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),22(8):966-972(陈学俊,王英,高佳佳,高和琼,庄南生,2014,巴西橡胶树第9染色体的显微分离及微克隆文库的构建,农业生物技术学报,22(8):966-972)
Fouzia B.,Muhammad I.,Zahid A.,Khizar H.B.,and Akash R.,2017,Improved catalytic functionalities of purified pristine and chitosan-immobilized polygalacturonase and pectin lyase,Chemical Engineering Research and Design,128:146-154
Gao H.Q.,Wang Y.,Jin G.,Qiu H.Y.,and Zhuang N.S.,2009,Optimization of chromosome preparations of Hevea leaves,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),30(5):565-569(高和琼,王英,金鸽,邱海燕,庄南生,2009,橡胶树叶片染色体制片方法的优化,热带作物学报,30(5):565-569)
Huang Y.,2007,The hydrolysis mechanics of cellulose and influencing factor,Shandong Huagong(ShanDong Chemical Industry),9(5):29-31(黄翊,2007,纤维素酶水解机理及影响因素,山东化工,9(5):29-31)
Kong D.J.,Liang D.D.,and He S.P.,1980,Preparation of chromosome specimon of root-tips of Hevea Brasiliensis by cell suspension method,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),1(1):105-106(孔德蹇,梁达德,何世屏,1980,橡胶树根尖染色体观察的细胞悬液制片法,热带作物学报,1(1):105-106)
Li Y.K.,2003,General situation and developmental current on natural rubber production of the world,Redai Nongye Keji(Tropical Agricultural Science&Technology),26(S):37-45(李一鲲,2003,世界主要天然橡胶生产国生产概况及发展趋势和认识,热带农业科技,26(S):37-45)
Lichter P.,Cremer T.,Borden J.,Manuelidis L.,and Ward D.C.,1988,Delineation of individual human chromosome in metaphase and interphase cells by in the situ hybridization using recombinant DNA libraries,Human Genetics,80(3):224-234
Nederlof P.M.,van der Flier S.,Wiegant J.,Raap A.K.,Tanke H.J.,Ploem J.S.,and van der Ploeg M.,1990,Multiple fluorescence in situ hybridization,Cytometry,11(1):126-131
Pardue M.L.,and Gall J.G.,1969,Molecular hybridization of radioactive DNA to the DNA of cytological preparations,Proc.Natl.Acad.Sci.USA,64(2):600-604
Torres S.,Sayago J.E.,Ordo觡ez R.M.,and Isla M.I.,2011,A colorimetric method to quantify endo-polygalacturonase activity,Enzyme Microb.Technol.,48(2):123-128
Wang Y.,Gao H.Q.,Zhuang N.S.,Huang D.Y.,and Ma S.,2008,Karyotype analysis on Saccharum spont YC NO.11 and Badila,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),24(3):52-57(王英,高和琼,庄南生,黄东益,马帅,2008,崖城割手密11号与拔地拉核型比较分析,中国农学通报,24(3):52-57)
Zhang M.,Chen Y.,Wu Y.N.,and Chen Z.B.,2014,Application of fluorescence in situ hybridization,Harbin Shifan Daxue Ziran Kexue Xuebao(Natural Sciences Journal of Harbin Normal University),30(6):90-93(张淼,陈瑛,吴忆宁,陈兆波,2014,荧光原位杂交技术的应用进展,哈尔滨师范大学自然科学学报,30(6):90-93)
Bauman J.G.,Wiegant J.,Borst P.,and van Duijn P.,1980,Anew method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochromelabelled RNA,Exp.Cell Res.,128(2):485-490
Chen R.Y.,Song W.Q.,and Li X.L.,1982,Wall degradation hypotonic method of preparing chromosome samples in plant and its significance in the cytogenetics,Yichuan Xuebao(Journal of Genetics and Genomics),9(2):151-159(陈瑞阳,宋文芹,李秀兰,1982,植物染色体标本制备的去壁,低渗法及其在细胞遗传学中的意义,遗传学报,9(2):151-159)
Chen X.J.,2014a,Microdissection of the chromosome 1,5 and9 and construction of their DNA libraries in Hevea brasiliensis Reyan7-33-97,Tesis for M.S.,Hainan University,Supervisor:Wang Y.,pp.71(陈学俊,2014,巴西橡胶树热研7-33-97第1,5和9号染色体的分离及文库的构建,硕士学位论文,海南大学,导师:王英,pp.71)
Chen X.J.,Wang Y.,Gao J.J.,Gao H.Q.,and Zhuang N.S.,2014,Microdissection and microcloning of the ninth chromosome in rubber tree(Hevea brasliensis),Nongye Shengwu Jishu Xuebao(Journal of Agricultural Biotechnology),22(8):966-972(陈学俊,王英,高佳佳,高和琼,庄南生,2014,巴西橡胶树第9染色体的显微分离及微克隆文库的构建,农业生物技术学报,22(8):966-972)
Fouzia B.,Muhammad I.,Zahid A.,Khizar H.B.,and Akash R.,2017,Improved catalytic functionalities of purified pristine and chitosan-immobilized polygalacturonase and pectin lyase,Chemical Engineering Research and Design,128:146-154
Gao H.Q.,Wang Y.,Jin G.,Qiu H.Y.,and Zhuang N.S.,2009,Optimization of chromosome preparations of Hevea leaves,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),30(5):565-569(高和琼,王英,金鸽,邱海燕,庄南生,2009,橡胶树叶片染色体制片方法的优化,热带作物学报,30(5):565-569)
Huang Y.,2007,The hydrolysis mechanics of cellulose and influencing factor,Shandong Huagong(ShanDong Chemical Industry),9(5):29-31(黄翊,2007,纤维素酶水解机理及影响因素,山东化工,9(5):29-31)
Kong D.J.,Liang D.D.,and He S.P.,1980,Preparation of chromosome specimon of root-tips of Hevea Brasiliensis by cell suspension method,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),1(1):105-106(孔德蹇,梁达德,何世屏,1980,橡胶树根尖染色体观察的细胞悬液制片法,热带作物学报,1(1):105-106)
Li Y.K.,2003,General situation and developmental current on natural rubber production of the world,Redai Nongye Keji(Tropical Agricultural Science&Technology),26(S):37-45(李一鲲,2003,世界主要天然橡胶生产国生产概况及发展趋势和认识,热带农业科技,26(S):37-45)
Lichter P.,Cremer T.,Borden J.,Manuelidis L.,and Ward D.C.,1988,Delineation of individual human chromosome in metaphase and interphase cells by in the situ hybridization using recombinant DNA libraries,Human Genetics,80(3):224-234
Nederlof P.M.,van der Flier S.,Wiegant J.,Raap A.K.,Tanke H.J.,Ploem J.S.,and van der Ploeg M.,1990,Multiple fluorescence in situ hybridization,Cytometry,11(1):126-131
Pardue M.L.,and Gall J.G.,1969,Molecular hybridization of radioactive DNA to the DNA of cytological preparations,Proc.Natl.Acad.Sci.USA,64(2):600-604
Torres S.,Sayago J.E.,Ordo觡ez R.M.,and Isla M.I.,2011,A colorimetric method to quantify endo-polygalacturonase activity,Enzyme Microb.Technol.,48(2):123-128
Wang Y.,Gao H.Q.,Zhuang N.S.,Huang D.Y.,and Ma S.,2008,Karyotype analysis on Saccharum spont YC NO.11 and Badila,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),24(3):52-57(王英,高和琼,庄南生,黄东益,马帅,2008,崖城割手密11号与拔地拉核型比较分析,中国农学通报,24(3):52-57)
Zhang M.,Chen Y.,Wu Y.N.,and Chen Z.B.,2014,Application of fluorescence in situ hybridization,Harbin Shifan Daxue Ziran Kexue Xuebao(Natural Sciences Journal of Harbin Normal University),30(6):90-93(张淼,陈瑛,吴忆宁,陈兆波,2014,荧光原位杂交技术的应用进展,哈尔滨师范大学自然科学学报,30(6):90-93)