苦豆子内生真菌诱导子促进宿主生物碱合成积累的生理防卫反应
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摘要
分别以4株苦豆子内生真菌菌液浓缩物为诱导子,研究不同诱导时间下,各诱导子对苦豆子组培苗中氧化苦参碱(OMA)含量以及防御酶苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)和过氧化氢酶(CAT)活性的影响。结果显示:4种苦豆子内生真菌的菌液浓缩物均可提高宿主中OMA的积累,其中诱导效果最佳的是内生真菌诱导子XKYKDF_(40),在0～15 d诱导期间宿主中OMA含量持续增长且始终高于对照,在诱导第15 d达到最高,为3.616 mg·g~(-1),是同时期对照组的2.09倍。4种内生真菌诱导子还可诱发宿主中生物碱合成关键酶PAL和3种抗氧化酶(POD、APX和CAT)活性升高。诱导子XKYKDF_(40)激发PAL活性的作用最佳,在诱导第6 d PAL活性达最大值,为61.92 U·g~(-1),是同时期对照组的1.42倍;与其他诱导子相比,诱导子XKZKDF_(27)能够明显诱发POD的活性,在诱导第12 d达到最大值,为62.30 U·mg~(-1),是同时期对照组的2.70倍;诱导子XKZKDF_(11)可诱导宿主中的APX和CAT活性大幅度提高,在诱导第12 d酶活性达到最高,分别为0.5538 U·mg~(-1)和19.82 U·mg~(-1),是同时期对照组的4.97倍和3.00倍。结果表明,苦豆子内生真菌诱导子的加入不仅激活了宿主组培苗的防御性应答反应,还促进了OMA的合成。关键字:
Four species of endophytic fungi were isolated from Sophora alopecuroides L., and their concentrated filtrates were prepared as the fungal elicitors. The effects of different endophytic fungal elicitors at different induction time on the content of oxymatrine(OMA) and the activities of phenylalanine(PAL), peroxidase(POD), ascorbate peroxidase(APX) and catalase(CAT) in the tissue culture seedlings of S. alopecuroides L. were investigated. The results showed that culture broth concentrates from 4 species of endophytic fungi of S. alopecuroides L. obviously improved the content of OMA in tissue culture seedlings of S. alopecuroides L. Among them, culture broth concentrates of the endophytic fungi XKGKDF_(40) was most effective with 0～15 d of induction, OMA content in tissue culture seedlings of S. alopecuroides L. continuously increased, compared with the control, and reached the maximum of 3.616 mg·g~(-1) on the 15 th day, which was 2.09 times that of the control group with the same induction time. Culture broth concentrates from 4 species of endophytic fungi of S. alopecuroides L. induced increase in activity of important PAL enzyme in alkaloid synthesis of tissue culture seedlings of S. alopecuroides L., and promoted the activities of antioxidant enzymes, such as POD, APX and CAT. The endophytic fungi XKGKDF_(40) was the most efficient elicitor on PAL activation, the enzyme activity reached a peak of 61.92 U·g~(-1) at 6 th day after induction, which was 1.42 times that of the control group at the same induction time; Compared with other elicitors, the elicitor XKZKDF_(27) could induce POD activity significantly, after the induction in 12 th day, POD activity reached the maximum of 62.30 U·mg~(-1), which is 2.70 times that of the control group at the same induction time; The elicitor XKZKDF_(11) also greatly improved the activities of APX and CAT in tissue culture seedlings of S. alopecuroides L., the enzyme activities reached the highest activity at the 12 th day of induction(0.5538 U·mg~(-1) and 19.82 U·mg~(-1)), which were 4.97 times and 3.00 times that of the control group at the same period respectively. The results indicated that the addition of endophytic fungal elicitors from S. alopecuroides L. not only activated the defensive response of the host tissue culture seedlings, but also promoted the synthesis of OMA of S. alopecuroides L..
Four species of endophytic fungi were isolated from Sophora alopecuroides L., and their concentrated filtrates were prepared as the fungal elicitors. The effects of different endophytic fungal elicitors at different induction time on the content of oxymatrine(OMA) and the activities of phenylalanine(PAL), peroxidase(POD), ascorbate peroxidase(APX) and catalase(CAT) in the tissue culture seedlings of S. alopecuroides L. were investigated. The results showed that culture broth concentrates from 4 species of endophytic fungi of S. alopecuroides L. obviously improved the content of OMA in tissue culture seedlings of S. alopecuroides L. Among them, culture broth concentrates of the endophytic fungi XKGKDF_(40) was most effective with 0～15 d of induction, OMA content in tissue culture seedlings of S. alopecuroides L. continuously increased, compared with the control, and reached the maximum of 3.616 mg·g~(-1) on the 15 th day, which was 2.09 times that of the control group with the same induction time. Culture broth concentrates from 4 species of endophytic fungi of S. alopecuroides L. induced increase in activity of important PAL enzyme in alkaloid synthesis of tissue culture seedlings of S. alopecuroides L., and promoted the activities of antioxidant enzymes, such as POD, APX and CAT. The endophytic fungi XKGKDF_(40) was the most efficient elicitor on PAL activation, the enzyme activity reached a peak of 61.92 U·g~(-1) at 6 th day after induction, which was 1.42 times that of the control group at the same induction time; Compared with other elicitors, the elicitor XKZKDF_(27) could induce POD activity significantly, after the induction in 12 th day, POD activity reached the maximum of 62.30 U·mg~(-1), which is 2.70 times that of the control group at the same induction time; The elicitor XKZKDF_(11) also greatly improved the activities of APX and CAT in tissue culture seedlings of S. alopecuroides L., the enzyme activities reached the highest activity at the 12 th day of induction(0.5538 U·mg~(-1) and 19.82 U·mg~(-1)), which were 4.97 times and 3.00 times that of the control group at the same period respectively. The results indicated that the addition of endophytic fungal elicitors from S. alopecuroides L. not only activated the defensive response of the host tissue culture seedlings, but also promoted the synthesis of OMA of S. alopecuroides L..
引文
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[7] 李培琴,于瑞婷,徐丹,等.内生真菌Berkleasmium sp.寡糖对盾叶薯蓣培养细胞和幼苗防御相关酶活性的影响(英文)[J].天然产物研究与开发,2015,27(4):585-591.
[8] 王梦亮,焦晋,邢婕,等.内生真菌ZPRa-R-1对红景天中关键信号分子及主要次生代谢物的影响[J].植物研究,2016,36(3):416-420.
[9] 尚博扬,杨萍,陈丽,等.苦豆子中生物碱成分的网络药理学研究[J].中国中药杂志,2018,43(1):160-167.
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[11] 周星辰.苦豆子内生真菌遗传多样性及其产喹诺里西啶生物碱菌株的筛选[D].银川:宁夏大学,2014.
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[17] 朱小文.不同提取条件对植物组织SOD、POD和CAT酶活性的影响[J].食品科技,2018,43(5):265-269.
[18] 崔晋龙,郭顺星,肖培根.内生菌与植物的互作关系及对药用植物的影响[J].药学学报,2017,52(2):214-221.
[19] 陶如,冯蕾,赵法兴,等.白花丹参毛状根诱导体系的建立与其内生真菌提高毛状根中丹酚酸含量的研究[J].时珍国医国药,2015,26(6):1469-1473.
[20] 张瑞芬,李培琴,周立刚.真菌诱导子对植物培养物生长和次生代谢产物合成影响之研究进展[J].中国农学通报,2008,24(9):260-264.
[21] 宁进,张秀英,程玉,等.真菌诱导子对毛葡萄愈伤组织细胞次生代谢的影响[J].中国南方果树,2015,44(2):33-38.
[22] 邱立友,戚元成,王明道,等.植物次生代谢物的自毒作用及其与连作障碍的关系[J].土壤,2010,42(1):1-7.
[23] 张长平,李春,元英进.真菌诱导子对悬浮培养南方红豆杉细胞次生代谢的影响[J].化工学报,2002,53(5):498-502.
[24] 徐晓梅,杨署光.苯丙氨酸解氨酶研究进展[J].安徽农业科学,2009,37(31):15115-15119.
[25] Polidoros A N,Scandalios J G.Role of hydrogen peroxide and different classes of antioxidants in the regulation of catalase and glutathione S-transferase gene expression in maize (Zea mays L.) [J].Physiologia Plantarum,1999,106(1):112-120.
[26] 刘零怡,赵丹莹,郑杨,等.植物在低温胁迫下的过氧化氢代谢及信号转导[J].园艺学报,2009,37(11):1701-1708.
[27] 刘淑宇,于新,陈发河,等.绿色木霉菌发酵液对杧果炭疽菌胞内抗性酶活性的影响[J].果树学报,2013,30(2):285-290.
[28] 张雨竹,董雪梅,郭春兰,等.桃色顶孢霉发酵液对大豆的促生及对抗氧化酶活性的影响[J].中国油料作物学报,2014,36(4):519-523.
[29] 薛鑫,张芊,吴金霞.植物体内活性氧的研究及其在植物抗逆方面的应用[J].生物技术通报,2013(10):6-11.
[30] 陶金华,濮雪莲,江曙.内生真菌诱导子对茅苍术细胞生长及苍术素积累的影响[J].中国中药杂志,2011,36(1):27-31.
[31] Maojun.Nitric oxide:a potential key point of the signaling network leading to plant secondary metabolite biosynthesis [J].Progress in Natural Science,2007,17(12):1397-1404.
[2] Lebrun-Garcia A,Bourque S,Binet M N,et al.Involvement of plasma membrane proteins in plant defenseresponses.Analysis of the cryptogein signal transduction in tobacco [J].Biochimie,1999,81(6):663-668.
[3] Singh G J,Oakey J S,Curtis W R.Interaction of methyl jasmonate,wounding and fungal elicitation during sesquiterpene induction in Hyoscyamus muticus in root cultures [J].Plant Cell Reports,1998,17(5):391-395.
[4] Chong T M,Abdullah M A,Lai O M,et al.Effective elicitation factors in Morinda elliptica cell suspension culture [J].Process Biochemistry,2005,40(11):3397-3405.
[5] Zhao J.Elicitor signal transduction leading to biosynthesis of plant defensive secondary metabolites [M].USA:Global Science Books Ltd,2006:344-357.
[6] 肖佳雷,来永才,李炜,等.内生真菌与植物的防御机制研究进展[J].Botanical Research,2013,2(1):13-17.
[7] 李培琴,于瑞婷,徐丹,等.内生真菌Berkleasmium sp.寡糖对盾叶薯蓣培养细胞和幼苗防御相关酶活性的影响(英文)[J].天然产物研究与开发,2015,27(4):585-591.
[8] 王梦亮,焦晋,邢婕,等.内生真菌ZPRa-R-1对红景天中关键信号分子及主要次生代谢物的影响[J].植物研究,2016,36(3):416-420.
[9] 尚博扬,杨萍,陈丽,等.苦豆子中生物碱成分的网络药理学研究[J].中国中药杂志,2018,43(1):160-167.
[10] 胡会平,刘建慧,孙鑫,等.苦参碱及衍生物的生物活性和合成研究进展[J].鲁东大学学报(自然科学版),2015,31(4):339-345.
[11] 周星辰.苦豆子内生真菌遗传多样性及其产喹诺里西啶生物碱菌株的筛选[D].银川:宁夏大学,2014.
[12] 高媛,孙牧笛,徐全智,等.苦豆子内生真菌对宿主培养物生长及喹诺里西啶类生物碱合成的影响[J].干旱地区农业研究,2017,35(3):212-218.
[13] Farmer E E.Effects of fungal elicitor on lignin biosynthesis in cell suspension cultures of soybean[J].Plant Physiology,1985,78(2):338-342.
[14] 宛国伟,董娟娥,梁宗锁,等.培养条件对离体丹参根苯丙氨酸解氨酶和多酚氧化酶活性的影响[J].西北植物学报,2007,27(12):2471-2477.
[15] 魏池泉.云南白芸豆过氧化物酶的提取及其酶学性质的研究[D].昆明:云南师范大学,2008.
[16] 孙云.茶叶抗坏血酸过氧化物酶(APX)的生理学与分子生物学研究[D].福州:福建农林大学,2009.
[17] 朱小文.不同提取条件对植物组织SOD、POD和CAT酶活性的影响[J].食品科技,2018,43(5):265-269.
[18] 崔晋龙,郭顺星,肖培根.内生菌与植物的互作关系及对药用植物的影响[J].药学学报,2017,52(2):214-221.
[19] 陶如,冯蕾,赵法兴,等.白花丹参毛状根诱导体系的建立与其内生真菌提高毛状根中丹酚酸含量的研究[J].时珍国医国药,2015,26(6):1469-1473.
[20] 张瑞芬,李培琴,周立刚.真菌诱导子对植物培养物生长和次生代谢产物合成影响之研究进展[J].中国农学通报,2008,24(9):260-264.
[21] 宁进,张秀英,程玉,等.真菌诱导子对毛葡萄愈伤组织细胞次生代谢的影响[J].中国南方果树,2015,44(2):33-38.
[22] 邱立友,戚元成,王明道,等.植物次生代谢物的自毒作用及其与连作障碍的关系[J].土壤,2010,42(1):1-7.
[23] 张长平,李春,元英进.真菌诱导子对悬浮培养南方红豆杉细胞次生代谢的影响[J].化工学报,2002,53(5):498-502.
[24] 徐晓梅,杨署光.苯丙氨酸解氨酶研究进展[J].安徽农业科学,2009,37(31):15115-15119.
[25] Polidoros A N,Scandalios J G.Role of hydrogen peroxide and different classes of antioxidants in the regulation of catalase and glutathione S-transferase gene expression in maize (Zea mays L.) [J].Physiologia Plantarum,1999,106(1):112-120.
[26] 刘零怡,赵丹莹,郑杨,等.植物在低温胁迫下的过氧化氢代谢及信号转导[J].园艺学报,2009,37(11):1701-1708.
[27] 刘淑宇,于新,陈发河,等.绿色木霉菌发酵液对杧果炭疽菌胞内抗性酶活性的影响[J].果树学报,2013,30(2):285-290.
[28] 张雨竹,董雪梅,郭春兰,等.桃色顶孢霉发酵液对大豆的促生及对抗氧化酶活性的影响[J].中国油料作物学报,2014,36(4):519-523.
[29] 薛鑫,张芊,吴金霞.植物体内活性氧的研究及其在植物抗逆方面的应用[J].生物技术通报,2013(10):6-11.
[30] 陶金华,濮雪莲,江曙.内生真菌诱导子对茅苍术细胞生长及苍术素积累的影响[J].中国中药杂志,2011,36(1):27-31.
[31] Maojun.Nitric oxide:a potential key point of the signaling network leading to plant secondary metabolite biosynthesis [J].Progress in Natural Science,2007,17(12):1397-1404.
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