样品预处理方法对小麦和玉米叶子中内源激素质量分数的影响
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摘要
高效液相色谱法已成为应用较为广泛的一种测定植物内源激素的方法,然而,植物体内其他内含物的存在常常干扰和影响检测结果,因而对植物材料预处理方法也提出了更高的要求。本试验以小麦和玉米叶片为材料,对目前几种常用的预处理方法进行比较。从高效液相色谱的测定结果来看,不同的预处理方法显著影响小麦叶子中IAA、GA和6-BA以及玉米叶子中IAA、GA、ZT、6-BA和ABA的检测质量分数。其中,仅用甲醇提取就进行高效液相色谱检测,得到激素质量分数不高;用甲醇提取、石油醚脱色后,加入PVP或PVPP去除杂质,经乙酸乙酯萃取,最后检测的激素质量分数也较低;而将样品用甲醇提取、经石油醚脱色和乙酸乙酯萃取、再过C18小柱的预处理过程,得到的各种激素质量分数最高。主效应分析结果表明,植物材料和处理方法对5种内源激素质量分数的影响均达到显著水平(P<0.05),且植物材料的影响效应大于处理方法。因此,检测不同植物中的不同种类内源激素需要不同的方法;对小麦和玉米叶片,最后一种预处理过程是一种较为适宜的方法,其测定结果可能会更为准确地反映内源激素的质量分数。
The high performance liquid chromatography(HPLC) currently is widely used for determinating endogenous hormones in plants, however, other compounds in the plants often interfere and affect the detection results. Thereby, a higher requirement is needed for the pretreatment of plant materials. In this experiment, influences of several pre-treatment methods on detection results of endogenous hormone contents in wheat and maize leaves were compared. The result of HPLC showed that the contents of auxin(IAA), ghibelline(GA) and 6-benzaminoadenine(6-BA) in wheat leaves and IAA, GA, zeatin(ZT), 6-BA and abscisic acid(ABA) in maize leaves were significantly affected by different pre-treatment methods. Among them, the samples were only extracted with methanol for HPLC detection, and the hormone content was not high and the impurities seriously affected the results;after the samples were extracted with methanol, decolorized with petroleum ether, the impurities were removed by using PVP or PVPP, and then it was extracted by using ethyl acetate, finally, the detected contents of various hormones were still low; however, through the procedure of methanol extracts of samples were decolorized with petroleum ether and extracted using ethyl acetate, and then, pre-treated with C18 small columns, the detected contents of various hormones were the highest.The results of the main effect analysis showed that the effects of plant materials and treatment methods on the contents of 5 endogenous hormones reached a significant level(P<0.05), and the effects of plant materials were greater than those of the treatment methods. Therefore, different methods are needed to detect endogenous hormones of different kinds in different plants, so the last pre-treatment process is a suitable method for wheat and maize leaves, and the determination results may more accurately reflect the actual content of endogenous hormones.
The high performance liquid chromatography(HPLC) currently is widely used for determinating endogenous hormones in plants, however, other compounds in the plants often interfere and affect the detection results. Thereby, a higher requirement is needed for the pretreatment of plant materials. In this experiment, influences of several pre-treatment methods on detection results of endogenous hormone contents in wheat and maize leaves were compared. The result of HPLC showed that the contents of auxin(IAA), ghibelline(GA) and 6-benzaminoadenine(6-BA) in wheat leaves and IAA, GA, zeatin(ZT), 6-BA and abscisic acid(ABA) in maize leaves were significantly affected by different pre-treatment methods. Among them, the samples were only extracted with methanol for HPLC detection, and the hormone content was not high and the impurities seriously affected the results;after the samples were extracted with methanol, decolorized with petroleum ether, the impurities were removed by using PVP or PVPP, and then it was extracted by using ethyl acetate, finally, the detected contents of various hormones were still low; however, through the procedure of methanol extracts of samples were decolorized with petroleum ether and extracted using ethyl acetate, and then, pre-treated with C18 small columns, the detected contents of various hormones were the highest.The results of the main effect analysis showed that the effects of plant materials and treatment methods on the contents of 5 endogenous hormones reached a significant level(P<0.05), and the effects of plant materials were greater than those of the treatment methods. Therefore, different methods are needed to detect endogenous hormones of different kinds in different plants, so the last pre-treatment process is a suitable method for wheat and maize leaves, and the determination results may more accurately reflect the actual content of endogenous hormones.
引文
[1] 谢君,张义正.植物内源激素的反相高效液相色谱法测定[J].分析测试学报,2001,20(1):60-62.XIE J,ZHANG Y ZH.Determination of plant endogenous hormones by reversed-phase high performance liquid chromatography [J].Journal of Analysis and Testing,2001,20(1):60-62.
[2] 邹焱,苏以荣.打顶及施用植物生长调节剂对烟草内源激素的影响[J].烟草科技,2008,13(10):50-52.ZOU Y,SU Y R.Effects of topping and application of plant growth regulators on endogenous hormones in tobacco [J].Tobacco Science and Technology,2008,13(10):50-52.
[3] 付晓陆,汪少敏,徐敏.液相色谱梯度洗脱法检测石蒜组织中4种内源激素的方法[J].浙江农业科学,2012,7(1):1020-1022.FU X L,WANG SH M,XU M.Determination of 4 endogenous hormones in lycoris chinensis tissues by gradient elution with liquid chromatography [J].Zhejiang Agricultural Science,2012,7(1):1020-1022.
[4] 赵莉,潘远智,朱峤.GA3、IBA和6-BA对香水百合开花特性及内源激素的影响[J].湖北农业科学,2012,51(7):1385-1389.ZHAO L,PAN Y ZH,ZHU Q.Effects of GA3,IBA and 6-ba on flowering characteristics and endogenous hormones of lilium fragrances [J].Hubei Agricultural Science,2012,51(7):1385-1389.
[5] 张玉琼,仲延龙,高翠云,等.高效液相色谱法分离和测定小麦中的5种内源激素[J].色谱,2013,31(8):800-803.ZHANG Y Q,ZHONG Y L,GAO C Y,et al.Separation and determination of 5 endogenous hormones in wheat by high performance liquid [J].Chromatography,2013,31(8):800-803.
[6] 未晓巍,勾畅,徐民泽,等.高效液相色谱法测定玉米内源激素方法的改进研究[J].玉米科学,2013(3):144-148.WEI X W,GOU CH,XU M Z,et al.Improvement of HPLC method for determination of endogenous hormones in maize [J].Maize Science,2013(3):144-148.
[7] 陈昆松,陈青俊,张上隆.本地早柑桔果实贮藏中内源ABA、IAA变化与枯水的关系[J].园艺学报,1997,24(3):291-292.CHEN K S,CHEN Q J,ZHANG SH L.Relationship between endogenous ABA and IAA changes and dry water in local citrus fruits [J].Acta Horticulturae Sinica,1997,24(3):291-292.
[8] 哈基姆,张上隆,陈大明,等.CPPU、GA3处理对藤稔葡萄生物学性状及其生理基础效应的研究[J].中国农业科学,1998,31(1):92-94.HA J M,ZHANG SH L,CHEN D M,et al.Study on the biological characters and physiological basic effects of CPPU and GA3 treatment on minoru tengica [J].Scientia Agricultura Sinica,1998,31(1):92-94.
[9] 陈昆松,李方,张上隆,等.ABA和IAA对猕猴桃果实成熟进程的调控[J].园艺学报,1999,26(2):81-86.CHEN K S,LI F,ZHANG SH L,et al.ABA and IAA regulation of kiwi fruit ripening process [J].Acta Horticultura Sinica,1999,26(2):81-86.
[10] 郑春霞,王强.HPLC分离和测定香梨三种植物激素[J].新疆农业大学学报,2000,23(1):54-56.ZHENG CH X,WANG Q.HPLC was used to isolate and determine three plant hormones of pear [J].Journal of Xinjiang Agricultural University,2000,23(1):54-56.
[11] 张占畅,孙志红,郑小能,等.高效液相色谱法测定库尔勒香梨幼果中的4种内源激素[J].新疆农业科学,2017,54(5):886-892.ZHANG ZH CH,SUN ZH H,ZHENG X N,et al.Determination of 4 endogenous hormones in young fruit of korla pear by HPLC [J].Xinjiang Agricultural Science,2017,54(5):886-892.
[12] 吴敏,陈昆松,贾惠娟,等.桃果实采后软化过程中内源IAA、ABA和乙烯的变化[J].果树学报,2003,20(3):157-160.WU M,CHEN K S,JIA H J,et al.Changes of endogenous IAA,ABA and ethylene in peach fruits during postharvest softening [J].Journal of Fruit Trees,2003,20(3):157-160.
[13] 陈波浪,郑春霞,盛建东,等.HPLC分离和测定棉花中3种植物激素[J].新疆农业大学学报,2006,29(1):28-30.CHEN B L,ZHENG CH X,SHENG J D,et al.Three plant hormones in cotton were isolated and determined by HPLC [J].Journal of Xinjiang Agricultural University,2006,29(1):28-30.
[14] 郭敏敏,王清连,胡根海.利用高效液相色谱法分离和测定棉花组织培养过程中4种内源激素[J].生物技术通讯,2009,20(2):213-216.GUO M M,WANG Q L,HU G H.HPLC was used to isolate and determine 4 endogenous hormones in cotton tissue culture [J].Biotechnology Communications,2009,20(2):213-216.
[15] 谢君.高效液相色谱测定多种植物内源激素方法研究[J].四川农业大学学报,1997,15(3):297-299.XIE J.Determination of various plant endogenous hormones by HPLC [J].Journal of Sichuan Agricultural University,1997,15(3):297-299.
[16] 朱智甲,郝振芳,张晓惠.高效液相色谱紫外检测法测定小麦幼苗中的内源激素[J].光谱实验室,1999,16(3):281-283.ZHU ZH J,HAO ZH F,ZHANG X H.Determination of endogenous hormones in wheat seedlings by HPLC ultraviolet detection [J].Spectroscopic Laboratory,1999,16(3):281-283.
[17] 陈洁,张永中,张谦,等.低磷胁迫下不同基因型玉米内源激素的动态变化[J].玉米科学,2013,21(5):6-12.CHEN J,ZHANG Y ZH,ZHANG Q,et al.Dynamic changes of endogenous hormones in maize of different genotypes under phosphorus stress [J].Maize Science,2013,21(5):6-12.
[18] 杨途熙,魏安智,郑元,等.高效液相色谱法同时分离测定仁用杏花芽中8种植物激素[J].分析化学,2007,35(9):1359-1361.YANG T X,WEI A ZH,ZHENG Y,et al.Simultaneous separation and determination of 8 plant hormones in flower bud of apricot kernel by HPLC [J].Analytical Chemistry,2007,35(9):1359-1361.
[19] 曾庆钱,陈厚彬,鲁才浩,等.HPLC测定荔枝不同器官中内源激素流程的优化[J].果树学报,2006,23(1):145-148.ZENG Q Q,CHEN H B,LU C H,et al.Optimization of endogenous hormone flow in litchi organs by HPLC [J].Acta Fruticosa Sinica,2006,23(1):145-148.
[20] 于玉梅,刘春香,朱妍妍,等.高效液相色谱法在黄瓜果实内源激素测定上的应用及改进[J].山东农业科学,2008,32(7):97-99.YU Y M,LIU CH X,ZHU Y Y,et al.Application and improvement of HPLC in determination of endogenous hormones in cucumber fruits [J].Shandong Agricultural Science,2008,32(7):97-99.
[21] 符继红,孙晓红,王吉德,等.植物激素定量分析方法研究进展[J].科学通报,2010,55(33):3163-3176.FU J H,SUN X H,WANG J D,et al.Advances in quantitative analysis of plant hormones [J].Chinese Science Bulletin,2010,55(33):3163-3176.
[2] 邹焱,苏以荣.打顶及施用植物生长调节剂对烟草内源激素的影响[J].烟草科技,2008,13(10):50-52.ZOU Y,SU Y R.Effects of topping and application of plant growth regulators on endogenous hormones in tobacco [J].Tobacco Science and Technology,2008,13(10):50-52.
[3] 付晓陆,汪少敏,徐敏.液相色谱梯度洗脱法检测石蒜组织中4种内源激素的方法[J].浙江农业科学,2012,7(1):1020-1022.FU X L,WANG SH M,XU M.Determination of 4 endogenous hormones in lycoris chinensis tissues by gradient elution with liquid chromatography [J].Zhejiang Agricultural Science,2012,7(1):1020-1022.
[4] 赵莉,潘远智,朱峤.GA3、IBA和6-BA对香水百合开花特性及内源激素的影响[J].湖北农业科学,2012,51(7):1385-1389.ZHAO L,PAN Y ZH,ZHU Q.Effects of GA3,IBA and 6-ba on flowering characteristics and endogenous hormones of lilium fragrances [J].Hubei Agricultural Science,2012,51(7):1385-1389.
[5] 张玉琼,仲延龙,高翠云,等.高效液相色谱法分离和测定小麦中的5种内源激素[J].色谱,2013,31(8):800-803.ZHANG Y Q,ZHONG Y L,GAO C Y,et al.Separation and determination of 5 endogenous hormones in wheat by high performance liquid [J].Chromatography,2013,31(8):800-803.
[6] 未晓巍,勾畅,徐民泽,等.高效液相色谱法测定玉米内源激素方法的改进研究[J].玉米科学,2013(3):144-148.WEI X W,GOU CH,XU M Z,et al.Improvement of HPLC method for determination of endogenous hormones in maize [J].Maize Science,2013(3):144-148.
[7] 陈昆松,陈青俊,张上隆.本地早柑桔果实贮藏中内源ABA、IAA变化与枯水的关系[J].园艺学报,1997,24(3):291-292.CHEN K S,CHEN Q J,ZHANG SH L.Relationship between endogenous ABA and IAA changes and dry water in local citrus fruits [J].Acta Horticulturae Sinica,1997,24(3):291-292.
[8] 哈基姆,张上隆,陈大明,等.CPPU、GA3处理对藤稔葡萄生物学性状及其生理基础效应的研究[J].中国农业科学,1998,31(1):92-94.HA J M,ZHANG SH L,CHEN D M,et al.Study on the biological characters and physiological basic effects of CPPU and GA3 treatment on minoru tengica [J].Scientia Agricultura Sinica,1998,31(1):92-94.
[9] 陈昆松,李方,张上隆,等.ABA和IAA对猕猴桃果实成熟进程的调控[J].园艺学报,1999,26(2):81-86.CHEN K S,LI F,ZHANG SH L,et al.ABA and IAA regulation of kiwi fruit ripening process [J].Acta Horticultura Sinica,1999,26(2):81-86.
[10] 郑春霞,王强.HPLC分离和测定香梨三种植物激素[J].新疆农业大学学报,2000,23(1):54-56.ZHENG CH X,WANG Q.HPLC was used to isolate and determine three plant hormones of pear [J].Journal of Xinjiang Agricultural University,2000,23(1):54-56.
[11] 张占畅,孙志红,郑小能,等.高效液相色谱法测定库尔勒香梨幼果中的4种内源激素[J].新疆农业科学,2017,54(5):886-892.ZHANG ZH CH,SUN ZH H,ZHENG X N,et al.Determination of 4 endogenous hormones in young fruit of korla pear by HPLC [J].Xinjiang Agricultural Science,2017,54(5):886-892.
[12] 吴敏,陈昆松,贾惠娟,等.桃果实采后软化过程中内源IAA、ABA和乙烯的变化[J].果树学报,2003,20(3):157-160.WU M,CHEN K S,JIA H J,et al.Changes of endogenous IAA,ABA and ethylene in peach fruits during postharvest softening [J].Journal of Fruit Trees,2003,20(3):157-160.
[13] 陈波浪,郑春霞,盛建东,等.HPLC分离和测定棉花中3种植物激素[J].新疆农业大学学报,2006,29(1):28-30.CHEN B L,ZHENG CH X,SHENG J D,et al.Three plant hormones in cotton were isolated and determined by HPLC [J].Journal of Xinjiang Agricultural University,2006,29(1):28-30.
[14] 郭敏敏,王清连,胡根海.利用高效液相色谱法分离和测定棉花组织培养过程中4种内源激素[J].生物技术通讯,2009,20(2):213-216.GUO M M,WANG Q L,HU G H.HPLC was used to isolate and determine 4 endogenous hormones in cotton tissue culture [J].Biotechnology Communications,2009,20(2):213-216.
[15] 谢君.高效液相色谱测定多种植物内源激素方法研究[J].四川农业大学学报,1997,15(3):297-299.XIE J.Determination of various plant endogenous hormones by HPLC [J].Journal of Sichuan Agricultural University,1997,15(3):297-299.
[16] 朱智甲,郝振芳,张晓惠.高效液相色谱紫外检测法测定小麦幼苗中的内源激素[J].光谱实验室,1999,16(3):281-283.ZHU ZH J,HAO ZH F,ZHANG X H.Determination of endogenous hormones in wheat seedlings by HPLC ultraviolet detection [J].Spectroscopic Laboratory,1999,16(3):281-283.
[17] 陈洁,张永中,张谦,等.低磷胁迫下不同基因型玉米内源激素的动态变化[J].玉米科学,2013,21(5):6-12.CHEN J,ZHANG Y ZH,ZHANG Q,et al.Dynamic changes of endogenous hormones in maize of different genotypes under phosphorus stress [J].Maize Science,2013,21(5):6-12.
[18] 杨途熙,魏安智,郑元,等.高效液相色谱法同时分离测定仁用杏花芽中8种植物激素[J].分析化学,2007,35(9):1359-1361.YANG T X,WEI A ZH,ZHENG Y,et al.Simultaneous separation and determination of 8 plant hormones in flower bud of apricot kernel by HPLC [J].Analytical Chemistry,2007,35(9):1359-1361.
[19] 曾庆钱,陈厚彬,鲁才浩,等.HPLC测定荔枝不同器官中内源激素流程的优化[J].果树学报,2006,23(1):145-148.ZENG Q Q,CHEN H B,LU C H,et al.Optimization of endogenous hormone flow in litchi organs by HPLC [J].Acta Fruticosa Sinica,2006,23(1):145-148.
[20] 于玉梅,刘春香,朱妍妍,等.高效液相色谱法在黄瓜果实内源激素测定上的应用及改进[J].山东农业科学,2008,32(7):97-99.YU Y M,LIU CH X,ZHU Y Y,et al.Application and improvement of HPLC in determination of endogenous hormones in cucumber fruits [J].Shandong Agricultural Science,2008,32(7):97-99.
[21] 符继红,孙晓红,王吉德,等.植物激素定量分析方法研究进展[J].科学通报,2010,55(33):3163-3176.FU J H,SUN X H,WANG J D,et al.Advances in quantitative analysis of plant hormones [J].Chinese Science Bulletin,2010,55(33):3163-3176.
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