金线莲内生促生真菌的筛选及其促生机制探讨
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摘要
分离野生金线莲内生真菌,考察它们系统发育地位;构建菌苗共同生长的体系,通过生长指标和有效成分变化评价菌株促生效应;监测金线莲中若干酶的活性变化,并对共生真菌促生机制进行探讨.结果显示,筛选的12株内生真菌分别归于10个种属,亲缘关系较远.其中多数内生真菌能够促进金线莲株高和黄酮含量,但降低了多糖和游离氨基酸含量.用熵权法对其中的cw-10和cw-4菌株进行评分,数值接近,但菌株cw-10对提高金线莲黄酮和多糖含量分别为菌株cw-4的1.38和1.60倍,因而选定cw-10为优良促生菌株.促生真菌对金线莲多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)活性没有太大影响,但显著提高了过氧化物酶(COD)、 4-香豆酸辅酶A连接酶(4CL)和查尔酮异构酶(CHI)活性.研究表明,促生真菌通过刺激黄酮合成酶活性增加金线莲黄酮的累积,其深入机制值得进一步探讨.
Endophytic fungi were isolated from wild A.roxburghii by plate culture and their phylogenetic tree was constructed based on molecular identification.Endophytic fungi were cultured with A.roxburghii,and the promoting effects of endophytic fungi were evaluated by plant growth indicators and medicinal components.Furthermore,enzymatic activities of A.roxburghii cultured with cw-4 and cw-10 strains were detected for analyzing promoting mechanism of the endophytic fungi.Results showed that the 12 selected endophytic fungi strains belonged to 10 different genera respectively.Most endophytic fungi improved stem length and flavonoid content of A.roxburghii,while decreased contents of polysaccharide and free amino acid.cw-4 and cw-10 strains were proposed as growth-promoting endophytic fungi for A.roxburghii simultaneously because of high evaluating values by entropy method.However,flavornoid and polysaccharide contents in A.roxburghii incubated with cw-10 strain were 1.38 and 1.60 times higher than cw-4 strain respectively.Finally,cw-10 was suggested to be the best growth-promoting endophytic fungi.And the two strains had not obvious effects on polyphenol oxidase(PPO) and phenylalanine ammonia-lyase(PAL) but increased activities of peroxidase(COD),4-coumaric acid CoAligase(4 CL) and chalcone isomerase(CHI).The study indicated that the growth-promoting endophytic fungi improved flavonoid content of A.roxburghii through stimulating activities of flavonoid synthetase,which needed to be studied profoundly.
Endophytic fungi were isolated from wild A.roxburghii by plate culture and their phylogenetic tree was constructed based on molecular identification.Endophytic fungi were cultured with A.roxburghii,and the promoting effects of endophytic fungi were evaluated by plant growth indicators and medicinal components.Furthermore,enzymatic activities of A.roxburghii cultured with cw-4 and cw-10 strains were detected for analyzing promoting mechanism of the endophytic fungi.Results showed that the 12 selected endophytic fungi strains belonged to 10 different genera respectively.Most endophytic fungi improved stem length and flavonoid content of A.roxburghii,while decreased contents of polysaccharide and free amino acid.cw-4 and cw-10 strains were proposed as growth-promoting endophytic fungi for A.roxburghii simultaneously because of high evaluating values by entropy method.However,flavornoid and polysaccharide contents in A.roxburghii incubated with cw-10 strain were 1.38 and 1.60 times higher than cw-4 strain respectively.Finally,cw-10 was suggested to be the best growth-promoting endophytic fungi.And the two strains had not obvious effects on polyphenol oxidase(PPO) and phenylalanine ammonia-lyase(PAL) but increased activities of peroxidase(COD),4-coumaric acid CoAligase(4 CL) and chalcone isomerase(CHI).The study indicated that the growth-promoting endophytic fungi improved flavonoid content of A.roxburghii through stimulating activities of flavonoid synthetase,which needed to be studied profoundly.
引文
[1]唐明娟,孟志霞,郭顺星.内生真菌对福建金线莲栽培及酶活性的影响 [J].中国药学杂志,2008,43(12):890-893.
[2]陈雨薇,唐军荣,段雪甜,等.金线莲组培苗菌根化研究 [J].中华中医药杂志,2016,31(9):3778-3780.
[3]关璟,王春兰,郭顺星,等.真菌对福建金线莲总黄酮含量的影响 [J].中国药学杂志,2008,43(13):1031-1032.
[4]陈晓梅,郭顺星,王春兰.四种内生真菌对金线莲无菌苗生长及多糖含量的影响 [J].中国药学杂志,2005,39(1):18-21.
[5]周新,李宏杰.黄酮类化学物的生物活性及临床应用进展 [J].中国新药杂志,2007,46(5):350-354.
[6]DURBIN M L,LEARN G H,HULTLEY C A,et al.Evolution of the chalone synthase gene family in the genus Ipomoca [J].Proceedings of the National Academy of Sciences of the United States of America,1995,92(8):3338-3342.
[7]DOXIN R A,PAIVA N L.Stress-induced phenylpropanoid metabolism [J].Plant Cell,1995,7(7):1085-1097.
[8]苏李维,李胜,马绍英,等.葡萄抗寒性综合评价方法的建立 [J].草业学报,2015,24(3):70-79.
[9]唐忠厚,魏猛,陈晓光,等.不同肉色甘薯块根主要营养品质特征与综合评价 [J].中国农业科学,2014,47(9):1705-1714.
[10]李橙,杨志新,刘树庆,等.河北省主产区葡萄品质综合评价方法的比较分析 [J].安徽农业科学,2011,39(17):10229-10234.
[11]杨禹伟,陈华,姜波,等.一种加工番茄品质的多性状评价方法 [J].中国农业大学学报,2017,22(1):131-137.
[12]于雪梅.金线莲与内生真菌相互作用机理研究 [D].北京:中国协和医科大学,2000.
[13]唐楠楠,陶佳青,陈常理,等.台湾金线莲与浙江金线莲多糖含量及抗氧化活性比较研究 [J].西北植物学报,2016,36(3):521-526.
[14]关璟,王春兰,郭顺兴,等.福建金线莲总黄酮提取工艺的研究 [J].中国药学杂志,2008,43(21):1615-1617.
[15]李合生,孙群,赵世杰,等.植物生理生化实验原理和技术 [M].北京:高等教育出版社,2000:193-194.
[16]LISTER C E,LANCASTER J E.Developmental changes in enzymes of flavonoid biosynthesis in the skins of red and green apple cultivars [J].Journal of the Science Food & Agriculture,1996,71(3):313-320.
[17]KNOBLOCK K H,HAHLBROCK K.Isoenzymes of p-coumarate:CoA ligase from cell suspension cultures of Glycine max [J].European Journal of Biochemistry,1975,52(2):311-320.
[18]WILLIAM N,DE T M,KAUFFMANN S,et al.Identification and characterization of maize pathogensis-related proteins.Four maize PR proteins are chitinases [J].Plant Molecular Biology,1988,11(4):529-538.
[19] 张龙翔.生化实验方法和技术 [M].北京:人民教育出版社,1981.
[20]胡陶,李潞滨,杨凯,等.中国兰属植物菌根真菌的分离与鉴定 [J].北京林业大学学报,2000,30(3):132-135.
[21]STEFFENS J C.Comparison of polyphenol oxidase expression in glandular trichomes of solanum and lycopersicon species [J].Plant Physiology,1992,100(4):1885-1890.
[22]TARTOURA K A H.Effect of abscisic acid on endogenous IAA,auxin protector levels and peroxidase activity during adventitious root initiation in Vigna radiata,cuttings [J].Acta Physiologiae Plantarum,2001,23(2):149-156.
[23]黄运峰,杨小波.兰科菌根研究综述 [J].热带亚热带植物学报,2008,16(3):283-288.
[24]MASUHARA G,KIMURA S,KATSUYA K.Seasonal changes in the mycorrhizae of Bletilla striata(Orchideae) [J].Trasactions of the Mycological Society of Japan,1988,29(1):25-31.
[25]SATAFFORD H A.Flavonid evolution:an enzymic approach [J].Plant Physiology,1991(96):680-685.
[2]陈雨薇,唐军荣,段雪甜,等.金线莲组培苗菌根化研究 [J].中华中医药杂志,2016,31(9):3778-3780.
[3]关璟,王春兰,郭顺星,等.真菌对福建金线莲总黄酮含量的影响 [J].中国药学杂志,2008,43(13):1031-1032.
[4]陈晓梅,郭顺星,王春兰.四种内生真菌对金线莲无菌苗生长及多糖含量的影响 [J].中国药学杂志,2005,39(1):18-21.
[5]周新,李宏杰.黄酮类化学物的生物活性及临床应用进展 [J].中国新药杂志,2007,46(5):350-354.
[6]DURBIN M L,LEARN G H,HULTLEY C A,et al.Evolution of the chalone synthase gene family in the genus Ipomoca [J].Proceedings of the National Academy of Sciences of the United States of America,1995,92(8):3338-3342.
[7]DOXIN R A,PAIVA N L.Stress-induced phenylpropanoid metabolism [J].Plant Cell,1995,7(7):1085-1097.
[8]苏李维,李胜,马绍英,等.葡萄抗寒性综合评价方法的建立 [J].草业学报,2015,24(3):70-79.
[9]唐忠厚,魏猛,陈晓光,等.不同肉色甘薯块根主要营养品质特征与综合评价 [J].中国农业科学,2014,47(9):1705-1714.
[10]李橙,杨志新,刘树庆,等.河北省主产区葡萄品质综合评价方法的比较分析 [J].安徽农业科学,2011,39(17):10229-10234.
[11]杨禹伟,陈华,姜波,等.一种加工番茄品质的多性状评价方法 [J].中国农业大学学报,2017,22(1):131-137.
[12]于雪梅.金线莲与内生真菌相互作用机理研究 [D].北京:中国协和医科大学,2000.
[13]唐楠楠,陶佳青,陈常理,等.台湾金线莲与浙江金线莲多糖含量及抗氧化活性比较研究 [J].西北植物学报,2016,36(3):521-526.
[14]关璟,王春兰,郭顺兴,等.福建金线莲总黄酮提取工艺的研究 [J].中国药学杂志,2008,43(21):1615-1617.
[15]李合生,孙群,赵世杰,等.植物生理生化实验原理和技术 [M].北京:高等教育出版社,2000:193-194.
[16]LISTER C E,LANCASTER J E.Developmental changes in enzymes of flavonoid biosynthesis in the skins of red and green apple cultivars [J].Journal of the Science Food & Agriculture,1996,71(3):313-320.
[17]KNOBLOCK K H,HAHLBROCK K.Isoenzymes of p-coumarate:CoA ligase from cell suspension cultures of Glycine max [J].European Journal of Biochemistry,1975,52(2):311-320.
[18]WILLIAM N,DE T M,KAUFFMANN S,et al.Identification and characterization of maize pathogensis-related proteins.Four maize PR proteins are chitinases [J].Plant Molecular Biology,1988,11(4):529-538.
[19] 张龙翔.生化实验方法和技术 [M].北京:人民教育出版社,1981.
[20]胡陶,李潞滨,杨凯,等.中国兰属植物菌根真菌的分离与鉴定 [J].北京林业大学学报,2000,30(3):132-135.
[21]STEFFENS J C.Comparison of polyphenol oxidase expression in glandular trichomes of solanum and lycopersicon species [J].Plant Physiology,1992,100(4):1885-1890.
[22]TARTOURA K A H.Effect of abscisic acid on endogenous IAA,auxin protector levels and peroxidase activity during adventitious root initiation in Vigna radiata,cuttings [J].Acta Physiologiae Plantarum,2001,23(2):149-156.
[23]黄运峰,杨小波.兰科菌根研究综述 [J].热带亚热带植物学报,2008,16(3):283-288.
[24]MASUHARA G,KIMURA S,KATSUYA K.Seasonal changes in the mycorrhizae of Bletilla striata(Orchideae) [J].Trasactions of the Mycological Society of Japan,1988,29(1):25-31.
[25]SATAFFORD H A.Flavonid evolution:an enzymic approach [J].Plant Physiology,1991(96):680-685.