秦艽种子质量评价及引发机制和药材采收干燥的研究
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摘要
本文以秦艽(Gentiana macrophylla Pall.)为研究对象,通过秦艽种子生物学的研究,初步建立了秦艽种子质量检验方法和质量分级标准；通过引发剂种类及引发条件的研究,建立了一种提高种子萌发能力的引发方法；通过对引发过程物质代谢、能量代谢、信号转导等生理和分子指标的研究,探讨了种子引发提高种子活力的机制；以秦艽药用部位为对象,研究了不同年限、不同采收期以及不同采收部位有效成分含量的差异；并初步探讨了不同干燥方法对药材各项理化指标的影响。主要研究结果如下：
1.通过研究秦艽种子发芽试验、生活力测定等指标分析,初步建立了秦艽种子质量检验方法；利用制定的检验方法,对24份不同产地的秦艽种子进行质量分析,初步建立了秦艽种子的质量分级标准。
2.优化建立了一种秦艽种子的引发方法：引发的种子初始发芽时间提前1-2d,终止发芽时间提前6d以上,种子发芽率增加了15.6%,第6d的发芽势增加了49.5倍,可达75%以上,发芽指数增加了90.6%。
3.引发处理影响了秦艽种子内部储藏物质动态,其中葡萄糖、蔗糖含量降低了12.8%、9.6%,可溶性糖、可溶性蛋白、粗蛋白含量升高了1.9%、49.7%、2.6%；脂肪酸中豆蔻酸、木焦油酸甲酯的含量增加了4.9%、9.0%,油酸、亚油酸、亚麻酸、山嵛酸的含量降低了8.1%、8.4%、6.7%、6.7%。
4.引发处理改变了秦艽种子内能量、代谢相关酶及内源激素水平,降低了引发种子电导率,提高了种子内部酸性磷酸酶、异柠檬酸裂解酶活性的活性；种子内部ABA含量降低了64.9%,GA含量升高21.6倍；ATP含量增加了2.17倍；线粒体内COX、MDH活性分别增加了68.1%、72.3%,同时,增加了抗氧化系统中的CAT、SOD、APX、GSH、GSH/GSSG、GR、ASC的酶活性。
5.通过蛋白组学研究,找到了差异在1.5倍以上的13个蛋白差异点,其中有6个已经确定名称的蛋白分别为硫氧还蛋白H、翻译起始因子、谷胱甘肽过氧化物酶、丙酮酸脱氢酶E1组件单元、UDP葡萄糖焦磷酸化酶、热休克蛋白70,与种子内糖、蛋白、淀粉调控、生物生长发育、衰老及环境抗性相关。
6.建立了有关秦艽种子的Unigene库,该库中unigene有81492条,其中35722条于NR、Swissport、COG、CO、KEEG数据库中得到基因功能注释信息,为研究秦艽提供丰富的分子基础。通过引发种子的转录组数据分析,得到了H2O、FeSO4引发与对照相比较存在的差异表达基因,分别为914个和2050个,这些差异基因分别涉及20、23个COG功能分类,40、49个GO分类,66、90个代谢途径。通过基因功能注释,进一步从中筛选出因FeSO4引发导致的,与物质代谢基因调控有关的酶36个,与能量代谢基因调控有关的酶3个,与抗性基因调控有关的酶5个,与激素代谢基因有关的酶7个。
7.秦艽单株植株根、茎、叶、花的干物积累和有效成分含量(马钱酸、獐牙菜苦苷、龙胆苦苷、獐牙菜苷)会随着生育期和生长年限的变化而变化；三年生植株干物质量、有效成分含量要高于两年生。两年生、三年生花中,龙胆苦苷含量分别为7.47%、7.54%；獐牙菜苦苷分别是同一生长年限枯萎期根中含量的1.98、2.25倍,獐牙菜苷为7.15、4.28倍,马钱酸含量占87.0%、42.4%。。
8.通过药材干燥方法比较分析,真空冷冻干燥和40℃恒温烘干方法,秦艽药材可溶性糖、粗蛋白含量较高；容积密度较低、孔隙度较高；色泽更接近于鲜样的颜色；最大限度的减少了有效成分龙胆苦苷含量的损失,提高了马钱酸、獐牙菜苦苷、獐牙菜苷在干燥样品中的含有量。
This study has preliminarily proposed a method to test and classify the quality of Gentiana macrophylla L. seeds by researching the seed biology; We also proposed a sound system to improve the ability of seed germination by selecting the best initiator and the optimal condition for priming. At the same time, we tried to uncover the physiological and molecular mechanism of seed priming by study the changes of material metabolism, energy metabolism, and signal transduction in priming seed. In addition, we took Gentiana macrophylla L. as medical herb in this study, analyzing the differences of the effective ingredients'concentration between different years, harvest time and different sections of the plant; we also tested the effects of5different dry methods on several physiological indexes. Here are the main results as below:
1. Setting up the preliminary standards applied to the G. macrophylla seed quality test by analysing the germination, viability and other indexex. Using these methods, we analyzed the seeds from multiple locations in the past2years, and set up the priciples for classifying the seed quality.
2. Establishing a priming method for G. macrophylla seed. The germination rate of the treated seeds was one or two days ahead the untreated seeds. The proportion of the treated seed germination is up to94.8%, increased15.6%, and compared with the untreated seed. On the6th day, the germination potential has been increased49.5fold, reaching up to75%; the germination index also has been increased90.6%.
3. The priming has changed the metabolisms of seed internal storage, inducing the seed germination. Compared with untreated seeds, the electric conductivity of the FeSO4treated seeds has been decreased; the content of glucose and sucrose have been reduced byl2.8%and9.6%respectively, and the content of soluble sugar, soluble proteins and raw proteins have been increased1.9%,49.7%and2.6%.The myristic acid and methyl lignocerate contents increased by4.9%and9.0%, the oleic acid, linoleic acid, docosanoic acid and linolenic acid content decreased by8.1%,8.4%,6.7%and6.7%, respectively;
4. The priming treatment also changed the energy materials, enzymes and hormones in the seeds. Compared with the untreated seeds, the activity of acid phosphatase and isocitrate lyase have been increased by18.6%and7.3%respectively; the internal ABA content has been reduced by64.9%; there were21.6folds increase in GA content; the ATP content reached up to2.87E-05μmol/g, increasing2.17folds; the activity of COX and MDH in mitochondrion have been increased by68.1%and72.3%respectively. Meanwhile, the activity of CAT, SOD, APX, GSH, GR, GSH/GSSG and ASC in the peroxisome has also been increased.
5. The priming caused the protein changes in the seeds. We found13differentially expressed proteins which relative intensity reached more than1.5times,6of which have been confirmed the names and functions. They were respectively Thioredoxin h, Translation initiation factor5A, Glutathione peroxidase, Pyruvate dehydrogenase E1component, UDP-glucose pyrophosphorylase, Heat shock protein70isoform3, which related to the regulation of reserve substance, growth and development of organisms, environmental resistance, and so on.
6. Unigene bank of G. macrophylla seed was established. Average length of81492unigenes was721kb in this bank. Gene functions of35722unigenes were annoted in data banks of NR, Swissportm, COG, CO and KEEG, offering better understandings of molecular biological study for G. macrophylla. H2O and FeSO4priming caused914and2050differential expression genes compared to control, respectively. These differential expression genes designed20and23COG functional classifications,40and49GO functional classifications,66and90metabolic pathways, respectively. According to Gene function annotation,36enzymes of substences metabolism genes regulation,3enzymes of energy metabolism genes regulation,5enzymes of resistant gene regulation, and7enzymes of hormone metabolism genes regulation were screened due to FeSO4priming.
7. The dry matter quality and the effective ingredients of plant roots, stems, leaves and flowers always change with the growth cycles and years. The triennial plants have much more dry matters and effective ingredients than the biennial ones. In biennial and triennial flowers, the contents of gentiopicroside were7.47%,7.54%, respectively; the content of swertiamarin were1.98,2.25times compared to withering period root at the same year; sweroside were7.15,4.28times respectively; longanic acid took up87.0%,42.4%. Further researches would be launched to explore the medicinal values of the flowers.
8. The dry methods would affect the medicine quality. There were more soluble sugars and proteins left in the plants under vacuum frozen condition or40℃heat treatment; the starch degradability was slower, gelatinization degree increased, the bulk density decreased and porosity increased; color were more close to fresh samples; the loss of gentiopicroside content reduced to the minimum size, the content of swertiamarin, sweroside, longanic acid were increased compared to fresh samples; at the same times, the process decreased the content of element.
1.通过研究秦艽种子发芽试验、生活力测定等指标分析,初步建立了秦艽种子质量检验方法；利用制定的检验方法,对24份不同产地的秦艽种子进行质量分析,初步建立了秦艽种子的质量分级标准。
2.优化建立了一种秦艽种子的引发方法：引发的种子初始发芽时间提前1-2d,终止发芽时间提前6d以上,种子发芽率增加了15.6%,第6d的发芽势增加了49.5倍,可达75%以上,发芽指数增加了90.6%。
3.引发处理影响了秦艽种子内部储藏物质动态,其中葡萄糖、蔗糖含量降低了12.8%、9.6%,可溶性糖、可溶性蛋白、粗蛋白含量升高了1.9%、49.7%、2.6%；脂肪酸中豆蔻酸、木焦油酸甲酯的含量增加了4.9%、9.0%,油酸、亚油酸、亚麻酸、山嵛酸的含量降低了8.1%、8.4%、6.7%、6.7%。
4.引发处理改变了秦艽种子内能量、代谢相关酶及内源激素水平,降低了引发种子电导率,提高了种子内部酸性磷酸酶、异柠檬酸裂解酶活性的活性；种子内部ABA含量降低了64.9%,GA含量升高21.6倍；ATP含量增加了2.17倍；线粒体内COX、MDH活性分别增加了68.1%、72.3%,同时,增加了抗氧化系统中的CAT、SOD、APX、GSH、GSH/GSSG、GR、ASC的酶活性。
5.通过蛋白组学研究,找到了差异在1.5倍以上的13个蛋白差异点,其中有6个已经确定名称的蛋白分别为硫氧还蛋白H、翻译起始因子、谷胱甘肽过氧化物酶、丙酮酸脱氢酶E1组件单元、UDP葡萄糖焦磷酸化酶、热休克蛋白70,与种子内糖、蛋白、淀粉调控、生物生长发育、衰老及环境抗性相关。
6.建立了有关秦艽种子的Unigene库,该库中unigene有81492条,其中35722条于NR、Swissport、COG、CO、KEEG数据库中得到基因功能注释信息,为研究秦艽提供丰富的分子基础。通过引发种子的转录组数据分析,得到了H2O、FeSO4引发与对照相比较存在的差异表达基因,分别为914个和2050个,这些差异基因分别涉及20、23个COG功能分类,40、49个GO分类,66、90个代谢途径。通过基因功能注释,进一步从中筛选出因FeSO4引发导致的,与物质代谢基因调控有关的酶36个,与能量代谢基因调控有关的酶3个,与抗性基因调控有关的酶5个,与激素代谢基因有关的酶7个。
7.秦艽单株植株根、茎、叶、花的干物积累和有效成分含量(马钱酸、獐牙菜苦苷、龙胆苦苷、獐牙菜苷)会随着生育期和生长年限的变化而变化；三年生植株干物质量、有效成分含量要高于两年生。两年生、三年生花中,龙胆苦苷含量分别为7.47%、7.54%；獐牙菜苦苷分别是同一生长年限枯萎期根中含量的1.98、2.25倍,獐牙菜苷为7.15、4.28倍,马钱酸含量占87.0%、42.4%。。
8.通过药材干燥方法比较分析,真空冷冻干燥和40℃恒温烘干方法,秦艽药材可溶性糖、粗蛋白含量较高；容积密度较低、孔隙度较高；色泽更接近于鲜样的颜色；最大限度的减少了有效成分龙胆苦苷含量的损失,提高了马钱酸、獐牙菜苦苷、獐牙菜苷在干燥样品中的含有量。
This study has preliminarily proposed a method to test and classify the quality of Gentiana macrophylla L. seeds by researching the seed biology; We also proposed a sound system to improve the ability of seed germination by selecting the best initiator and the optimal condition for priming. At the same time, we tried to uncover the physiological and molecular mechanism of seed priming by study the changes of material metabolism, energy metabolism, and signal transduction in priming seed. In addition, we took Gentiana macrophylla L. as medical herb in this study, analyzing the differences of the effective ingredients'concentration between different years, harvest time and different sections of the plant; we also tested the effects of5different dry methods on several physiological indexes. Here are the main results as below:
1. Setting up the preliminary standards applied to the G. macrophylla seed quality test by analysing the germination, viability and other indexex. Using these methods, we analyzed the seeds from multiple locations in the past2years, and set up the priciples for classifying the seed quality.
2. Establishing a priming method for G. macrophylla seed. The germination rate of the treated seeds was one or two days ahead the untreated seeds. The proportion of the treated seed germination is up to94.8%, increased15.6%, and compared with the untreated seed. On the6th day, the germination potential has been increased49.5fold, reaching up to75%; the germination index also has been increased90.6%.
3. The priming has changed the metabolisms of seed internal storage, inducing the seed germination. Compared with untreated seeds, the electric conductivity of the FeSO4treated seeds has been decreased; the content of glucose and sucrose have been reduced byl2.8%and9.6%respectively, and the content of soluble sugar, soluble proteins and raw proteins have been increased1.9%,49.7%and2.6%.The myristic acid and methyl lignocerate contents increased by4.9%and9.0%, the oleic acid, linoleic acid, docosanoic acid and linolenic acid content decreased by8.1%,8.4%,6.7%and6.7%, respectively;
4. The priming treatment also changed the energy materials, enzymes and hormones in the seeds. Compared with the untreated seeds, the activity of acid phosphatase and isocitrate lyase have been increased by18.6%and7.3%respectively; the internal ABA content has been reduced by64.9%; there were21.6folds increase in GA content; the ATP content reached up to2.87E-05μmol/g, increasing2.17folds; the activity of COX and MDH in mitochondrion have been increased by68.1%and72.3%respectively. Meanwhile, the activity of CAT, SOD, APX, GSH, GR, GSH/GSSG and ASC in the peroxisome has also been increased.
5. The priming caused the protein changes in the seeds. We found13differentially expressed proteins which relative intensity reached more than1.5times,6of which have been confirmed the names and functions. They were respectively Thioredoxin h, Translation initiation factor5A, Glutathione peroxidase, Pyruvate dehydrogenase E1component, UDP-glucose pyrophosphorylase, Heat shock protein70isoform3, which related to the regulation of reserve substance, growth and development of organisms, environmental resistance, and so on.
6. Unigene bank of G. macrophylla seed was established. Average length of81492unigenes was721kb in this bank. Gene functions of35722unigenes were annoted in data banks of NR, Swissportm, COG, CO and KEEG, offering better understandings of molecular biological study for G. macrophylla. H2O and FeSO4priming caused914and2050differential expression genes compared to control, respectively. These differential expression genes designed20and23COG functional classifications,40and49GO functional classifications,66and90metabolic pathways, respectively. According to Gene function annotation,36enzymes of substences metabolism genes regulation,3enzymes of energy metabolism genes regulation,5enzymes of resistant gene regulation, and7enzymes of hormone metabolism genes regulation were screened due to FeSO4priming.
7. The dry matter quality and the effective ingredients of plant roots, stems, leaves and flowers always change with the growth cycles and years. The triennial plants have much more dry matters and effective ingredients than the biennial ones. In biennial and triennial flowers, the contents of gentiopicroside were7.47%,7.54%, respectively; the content of swertiamarin were1.98,2.25times compared to withering period root at the same year; sweroside were7.15,4.28times respectively; longanic acid took up87.0%,42.4%. Further researches would be launched to explore the medicinal values of the flowers.
8. The dry methods would affect the medicine quality. There were more soluble sugars and proteins left in the plants under vacuum frozen condition or40℃heat treatment; the starch degradability was slower, gelatinization degree increased, the bulk density decreased and porosity increased; color were more close to fresh samples; the loss of gentiopicroside content reduced to the minimum size, the content of swertiamarin, sweroside, longanic acid were increased compared to fresh samples; at the same times, the process decreased the content of element.
引文
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