He-Ne激光对增强UV-B辐射损伤菘蓝幼苗的防护、修复效应及机理研究
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摘要
本研究以菘蓝(Isatis indigotica Fort)为实验材料，运用生物化学、分子生物学、热分析(DTA、TG和DTG)以及热力学等方法研究了He-Ne激光辐照(5.23mW·mm~(-2))菘蓝种子的生物学效应、He-Ne激光辐照对增强UV-B(10.08KJ.d~(-1))辐射损伤菘蓝幼苗的防护、修复效应及激光对生物学的作用机制。该研究从生理水平、生化水平、分子水平、个体生长发育以及板蓝根与大青叶的品质与产量(主要药用化学成分和生物量)上系统分析，全面揭示激光对菘蓝的生物学效应以及对增强紫外线的防护、修复作用。结果表明：
1、He-Ne激光预处理菘蓝种子不但显著地提高了萌发种子的焓、能熵流、化学熵产生、能熵流率、化学熵产生率，而且促进了幼苗的生长发育，改善了大青叶品质。其作用效果集中表现为：与对照(种子未经激光预处理)相比，激光预处理提高了菘蓝幼苗的光合色素含量、水分利用率、净光合速率、光合产物含量、蛋白质含量、丙酮酸含量、转氨酶活性、淀粉酶活性以及大青叶的产量与品质。这一结果将为我国传统中药材—大青叶品质的改善、产量的提高提供了有效手段，同时也为激光在中药材栽培上的应用提供了理论依据。
2、He-Ne激光预处理菘蓝种子对增强UV-B辐射损伤具有良好的防护效果。与对照(菘蓝种子既未经He-Ne激光预处理也未经UV-B辐射)相比，UV-B辐射显著的降低了抗氧化酶SOD、POD和CAT活性及抗氧化物质AsA、紫外吸收物质和脯氨酸浓度，提高了MDA浓，加强了膜脂氧化：而种子经He-Ne激光预处理后再暴露在UV-B辐射下，其幼苗MDA浓度显著的降低，抗氧化酶SOD、POD和CAT活性显著地增强，抗氧化物质AsA、紫外吸收物质和脯氨酸浓度提高。这说明激光预处理可以帮助酶促与非酶促抗氧化系统有效的清除由UV-B辐射损伤引起的自由基，增强幼苗抵抗UV-B辐射能力，保护细胞免受增
强UV-B伤害。
3、与对照(箱蓝幼苗既未经日e一Ne激光处理也未经UV-B辐射)相比，U协B
辐射私蓝幼苗显著地抑制了生理生化代谢，降低了板蓝根与大青叶的产量品质;
激光辐照落蓝幼苗显著地促进生理生化代谢，提高了板蓝根与大青叶的产量品
质;落蓝幼苗经增强UV-B辐射损伤后再经He一Ne激光辐照，其幼苗在生理生
化代谢以及板蓝根与大青叶的产量品质均有不同程度的改善，说明日e一Ne激光
辐照对增强UV-B辐射损伤落蓝幼苗具有良好的修复效应。集中表现在以下几个
方面:
(，)、UV-B辐射落蓝幼苗显著地提高了MOA浓度，降低了AsA、紫外吸
收物质和脯氨酸浓度以及500、POO和CAT活性;日e一Ne激光辐照经增强
U认B损伤后的落蓝幼苗，明显的降低了MOA浓度，提高AsA、紫外吸收物质
和脯氨酸浓度，增强500、POO和CAT活性。这说明激光可以帮助酶促与非
酶促抗氧化系统有效的清除由UV-B辐射损伤引起的自由基，增强幼苗抵抗UV-B
辐射能力，保护细胞免受增强UV-日伤害。
(2)、根据澳化乙锭只能插入双链ONA中并且在激发光下发射荧光的原理，
采用荧光光谱法测定了不同处理组落蓝幼苗双链ONA浓度，检测日e一Ne激光帮
助幼苗对增强OV-B损伤ONA的暗修复效果。与单纯U认B处理组相比，经增
强UV-B损伤幼苗再经日e一Ne激光辐照，其ONA双链浓度得到明显地提高。这
说明He一Ne激光具有帮助植物对UV-B损伤ONA进行修复的功能。
(3)、激光提高了落蓝幼苗光合作用色素含量、水分利用率、净光合速率及
气孔导度水平;U协B辐射降低了落蓝幼苗光合作用色素含量、水分利用率、净
光合速率及气孔导度水平;幼苗经增强UV-B辐射后再经日e一Ne激光处理，光
合作用色素含量、水分利用率、净光合速率及气孔导度与UV-B处理组相比有不
同程度的提高。这说明激光在生理上可以帮助藕蓝对UV-B辐射损伤引起的损伤
进行修复，增强幼苗抵抗UV-B辐射能力。
(4)、激光处理幼苗后，蛋白质含量、游离氨基酸含量、蛋白酶活性、GP丁
与GO丁活性均有不同程度的提高，而UV-B辐射降低蛋白质含量、游离氨基酸
含量、蛋白酶活性、GP丁与GO丁活性，幼苗经增强UV-B辐射后再经日e一Ne
激光处理，其蛋白质含量、游离氨基酸含量、蛋白酶活性、GPT与GO丁活性均
得到不同程度的恢复，基本接近对照水平。这说明激光在生化上可以帮助落蓝对
UV-B辐射损伤引起的损伤在生化水平进行修复，增强幼苗抵抗UV-B辐射能力。
(5)、本研究首次发现，He一Ne激光对增强紫外线B辐射裕蓝地上部分和
地下的影响效果明显的不同。对大青叶而言，靛蓝、靛玉红含量和焙值与生物量
成正相关性。与对照相比，激光处理促进了地上部分生长，提高了生物量、靛蓝、
靛玉红含量和焙值;UV-日辐射抑制了地上部分生长，降低了生物量、靛蓝、靛
玉红含量和焙值;而UV-B辐射后再用激光辐照，激光明显地减轻了UV-B辐射
伤害，使大青叶的产量、靛蓝含量、靛玉红含量和焙值得到了不同程度的恢复。
就板蓝根的产量而言，激光、UV-B辐射、二者复合处理均有利于提高板蓝根产
量。比较而言，激光效果最好，二者复合作用效果次之，UV-B处理产量稍高于
对照。但从板蓝根的品质来考虑，虽然激光可以改善其品质，但是效果不如二者
复合处理，更不如单纯UV-B辐射。因此，在生产实践上，适量补充UV-B
The Isatis indigotica seeds and the seedlings damaged by enhanced UV-B irradiation (10.08 kJ.m-2.d-1) were exposed to He-Ne laser (5.23 m W mm-2, radiated for 5 min) radiation In order to determine whether or not He-Ne laser caused the changes of thermodynamic parameters of seeds and had a long-term physiochemical effects and whether or not He-Ne laser has the protects and repairs capacity of the seedlings from enhanced UV-B damage. The results from the experiments indicated chiefly that:
1. The seeds embryos of Isatis Indogotica were exposed to He-Ne laser irradiation. Subsequently, the thermodynamic parameters were calculated according to thermograms of seeds germination tested with calorimeter at 25C, And the changes in physiological characters ( net photosynthetic rate, stomatal conductance, water utilization efficiency and concentration of chlorophyll) , biochemical characters (soluble sacchrides, soluble protein, pyruvic acid, and the activities of three kinds of enzymes) and the growth (biomass and leaves area) were measured when the seedlings of He-Ne laser pretreated seeds developed at stage of 24-25 days in comparison with those of untreated seeds. We found that the thermodynamic parameters in H,( S)e,( S)c,
and during germination at 250C changed significantly with He-Ne laser pretreatment seeds , and that the physiological characters, e.g. stomatal
conductance, water utilization efficiency, net photosynthetic rate and chlorophyll concentration, the biochemical characters, e.g. the concentration of soluble sacchrides, soluble protein and the activities of a-amylase, GPT and GOT, and the biomass and leaf areas were statistically significant improved. These changes suggested that the pretreatment with He-Ne laser not only had a short-term biology effect (which enhanced inner energy of seeds) but also a long-term biology effect (which contributed to the acceleration of the growth and development of seedlings).
2. The seeds pretreated with He-Ne laser was cultivated in petri dishes in a growth chamber with humidity (Model: 515HD.USA) until the seedlings were 2d old, and then were subjected to UV-B radiation for 6d under photosynthetically active radiation (220 umol m-2 s-1). Afterwards, great changes of UV-B injured seedlings, respectively, in the activities of enzymes (SOD, CAT and POD) and the concentration of malonndiadlehyde (MDA), proline and UV absorbing compounds were measured to test the protect capacity of He-Ne laser pretreatment. Results from this experiment indicated that: MDA concentration of the seedlings was decreased, the activities (SOD, CAT and POD), the concentration of proline and UV absorbing compounds were enhanced with laser pretreatment; and finally UV-B stress resistance were enhanced significantly with He-Ne laser pretreatment.
3 The seedlings damaged by enhanced UV-B irradiation were exposed to He-Ne laser radiation under photosynthetically active radiation, the great changes were observed from followed results:
3.1 Great changes of UV-B injured seedlings not only' in the activities of enzymes (SOD, CAT and POD) but also in the concentration of malonndiadlehyde (MDA), proline and UV absorbing compounds were measured to test the repair capacity of He-Ne laser on cleaning the free radical induced by UV-B radiaon. Results from this experiment indicated that MDA concentration of the seedlings was decreased, the activities (SOD, CAT and POD), the concentration of proline and UV absorbing compounds were
enhanced with laser treatment; and finally UV-B stress resistance were enhanced significantly with He-Ne laser.
3.2 Great changes in physiological characters (net photosynthetic rate, stomatal conductance, water utilization efficiency and concentration of chlorophyll); biochemical characters (soluble protein, the activities of proteiase, GOT, GPT and free amino acid) were measured to test the repair capacity of He-Ne laser. We found that the physiological characters, e.g. stomatal conductance, water utilization efficiency, net photo
1、He-Ne激光预处理菘蓝种子不但显著地提高了萌发种子的焓、能熵流、化学熵产生、能熵流率、化学熵产生率，而且促进了幼苗的生长发育，改善了大青叶品质。其作用效果集中表现为：与对照(种子未经激光预处理)相比，激光预处理提高了菘蓝幼苗的光合色素含量、水分利用率、净光合速率、光合产物含量、蛋白质含量、丙酮酸含量、转氨酶活性、淀粉酶活性以及大青叶的产量与品质。这一结果将为我国传统中药材—大青叶品质的改善、产量的提高提供了有效手段，同时也为激光在中药材栽培上的应用提供了理论依据。
2、He-Ne激光预处理菘蓝种子对增强UV-B辐射损伤具有良好的防护效果。与对照(菘蓝种子既未经He-Ne激光预处理也未经UV-B辐射)相比，UV-B辐射显著的降低了抗氧化酶SOD、POD和CAT活性及抗氧化物质AsA、紫外吸收物质和脯氨酸浓度，提高了MDA浓，加强了膜脂氧化：而种子经He-Ne激光预处理后再暴露在UV-B辐射下，其幼苗MDA浓度显著的降低，抗氧化酶SOD、POD和CAT活性显著地增强，抗氧化物质AsA、紫外吸收物质和脯氨酸浓度提高。这说明激光预处理可以帮助酶促与非酶促抗氧化系统有效的清除由UV-B辐射损伤引起的自由基，增强幼苗抵抗UV-B辐射能力，保护细胞免受增
强UV-B伤害。
3、与对照(箱蓝幼苗既未经日e一Ne激光处理也未经UV-B辐射)相比，U协B
辐射私蓝幼苗显著地抑制了生理生化代谢，降低了板蓝根与大青叶的产量品质;
激光辐照落蓝幼苗显著地促进生理生化代谢，提高了板蓝根与大青叶的产量品
质;落蓝幼苗经增强UV-B辐射损伤后再经He一Ne激光辐照，其幼苗在生理生
化代谢以及板蓝根与大青叶的产量品质均有不同程度的改善，说明日e一Ne激光
辐照对增强UV-B辐射损伤落蓝幼苗具有良好的修复效应。集中表现在以下几个
方面:
(，)、UV-B辐射落蓝幼苗显著地提高了MOA浓度，降低了AsA、紫外吸
收物质和脯氨酸浓度以及500、POO和CAT活性;日e一Ne激光辐照经增强
U认B损伤后的落蓝幼苗，明显的降低了MOA浓度，提高AsA、紫外吸收物质
和脯氨酸浓度，增强500、POO和CAT活性。这说明激光可以帮助酶促与非
酶促抗氧化系统有效的清除由UV-B辐射损伤引起的自由基，增强幼苗抵抗UV-B
辐射能力，保护细胞免受增强UV-日伤害。
(2)、根据澳化乙锭只能插入双链ONA中并且在激发光下发射荧光的原理，
采用荧光光谱法测定了不同处理组落蓝幼苗双链ONA浓度，检测日e一Ne激光帮
助幼苗对增强OV-B损伤ONA的暗修复效果。与单纯U认B处理组相比，经增
强UV-B损伤幼苗再经日e一Ne激光辐照，其ONA双链浓度得到明显地提高。这
说明He一Ne激光具有帮助植物对UV-B损伤ONA进行修复的功能。
(3)、激光提高了落蓝幼苗光合作用色素含量、水分利用率、净光合速率及
气孔导度水平;U协B辐射降低了落蓝幼苗光合作用色素含量、水分利用率、净
光合速率及气孔导度水平;幼苗经增强UV-B辐射后再经日e一Ne激光处理，光
合作用色素含量、水分利用率、净光合速率及气孔导度与UV-B处理组相比有不
同程度的提高。这说明激光在生理上可以帮助藕蓝对UV-B辐射损伤引起的损伤
进行修复，增强幼苗抵抗UV-B辐射能力。
(4)、激光处理幼苗后，蛋白质含量、游离氨基酸含量、蛋白酶活性、GP丁
与GO丁活性均有不同程度的提高，而UV-B辐射降低蛋白质含量、游离氨基酸
含量、蛋白酶活性、GP丁与GO丁活性，幼苗经增强UV-B辐射后再经日e一Ne
激光处理，其蛋白质含量、游离氨基酸含量、蛋白酶活性、GPT与GO丁活性均
得到不同程度的恢复，基本接近对照水平。这说明激光在生化上可以帮助落蓝对
UV-B辐射损伤引起的损伤在生化水平进行修复，增强幼苗抵抗UV-B辐射能力。
(5)、本研究首次发现，He一Ne激光对增强紫外线B辐射裕蓝地上部分和
地下的影响效果明显的不同。对大青叶而言，靛蓝、靛玉红含量和焙值与生物量
成正相关性。与对照相比，激光处理促进了地上部分生长，提高了生物量、靛蓝、
靛玉红含量和焙值;UV-日辐射抑制了地上部分生长，降低了生物量、靛蓝、靛
玉红含量和焙值;而UV-B辐射后再用激光辐照，激光明显地减轻了UV-B辐射
伤害，使大青叶的产量、靛蓝含量、靛玉红含量和焙值得到了不同程度的恢复。
就板蓝根的产量而言，激光、UV-B辐射、二者复合处理均有利于提高板蓝根产
量。比较而言，激光效果最好，二者复合作用效果次之，UV-B处理产量稍高于
对照。但从板蓝根的品质来考虑，虽然激光可以改善其品质，但是效果不如二者
复合处理，更不如单纯UV-B辐射。因此，在生产实践上，适量补充UV-B
The Isatis indigotica seeds and the seedlings damaged by enhanced UV-B irradiation (10.08 kJ.m-2.d-1) were exposed to He-Ne laser (5.23 m W mm-2, radiated for 5 min) radiation In order to determine whether or not He-Ne laser caused the changes of thermodynamic parameters of seeds and had a long-term physiochemical effects and whether or not He-Ne laser has the protects and repairs capacity of the seedlings from enhanced UV-B damage. The results from the experiments indicated chiefly that:
1. The seeds embryos of Isatis Indogotica were exposed to He-Ne laser irradiation. Subsequently, the thermodynamic parameters were calculated according to thermograms of seeds germination tested with calorimeter at 25C, And the changes in physiological characters ( net photosynthetic rate, stomatal conductance, water utilization efficiency and concentration of chlorophyll) , biochemical characters (soluble sacchrides, soluble protein, pyruvic acid, and the activities of three kinds of enzymes) and the growth (biomass and leaves area) were measured when the seedlings of He-Ne laser pretreated seeds developed at stage of 24-25 days in comparison with those of untreated seeds. We found that the thermodynamic parameters in H,( S)e,( S)c,
and during germination at 250C changed significantly with He-Ne laser pretreatment seeds , and that the physiological characters, e.g. stomatal
conductance, water utilization efficiency, net photosynthetic rate and chlorophyll concentration, the biochemical characters, e.g. the concentration of soluble sacchrides, soluble protein and the activities of a-amylase, GPT and GOT, and the biomass and leaf areas were statistically significant improved. These changes suggested that the pretreatment with He-Ne laser not only had a short-term biology effect (which enhanced inner energy of seeds) but also a long-term biology effect (which contributed to the acceleration of the growth and development of seedlings).
2. The seeds pretreated with He-Ne laser was cultivated in petri dishes in a growth chamber with humidity (Model: 515HD.USA) until the seedlings were 2d old, and then were subjected to UV-B radiation for 6d under photosynthetically active radiation (220 umol m-2 s-1). Afterwards, great changes of UV-B injured seedlings, respectively, in the activities of enzymes (SOD, CAT and POD) and the concentration of malonndiadlehyde (MDA), proline and UV absorbing compounds were measured to test the protect capacity of He-Ne laser pretreatment. Results from this experiment indicated that: MDA concentration of the seedlings was decreased, the activities (SOD, CAT and POD), the concentration of proline and UV absorbing compounds were enhanced with laser pretreatment; and finally UV-B stress resistance were enhanced significantly with He-Ne laser pretreatment.
3 The seedlings damaged by enhanced UV-B irradiation were exposed to He-Ne laser radiation under photosynthetically active radiation, the great changes were observed from followed results:
3.1 Great changes of UV-B injured seedlings not only' in the activities of enzymes (SOD, CAT and POD) but also in the concentration of malonndiadlehyde (MDA), proline and UV absorbing compounds were measured to test the repair capacity of He-Ne laser on cleaning the free radical induced by UV-B radiaon. Results from this experiment indicated that MDA concentration of the seedlings was decreased, the activities (SOD, CAT and POD), the concentration of proline and UV absorbing compounds were
enhanced with laser treatment; and finally UV-B stress resistance were enhanced significantly with He-Ne laser.
3.2 Great changes in physiological characters (net photosynthetic rate, stomatal conductance, water utilization efficiency and concentration of chlorophyll); biochemical characters (soluble protein, the activities of proteiase, GOT, GPT and free amino acid) were measured to test the repair capacity of He-Ne laser. We found that the physiological characters, e.g. stomatal conductance, water utilization efficiency, net photo
引文
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