秋水仙碱对银杏和毛新杨雄株生殖细胞的生理影响
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摘要
【目的】多倍化是树木遗传改良的重要途径,秋水仙碱诱导生殖细胞是树木多倍体诱变的重要方式,但还存在某些树种多倍体诱导困难、多倍化诱导质量难以判别等问题。因此,探究多倍化诱导机制,找到一种能够对秋水仙碱人工诱导树木生殖细胞多倍化效果进行初步判别的科学方法很有必要。【方法】本文以银杏和毛新杨雄性生殖细胞为研究对象,采用茚三酮显色法、CBA法、四氮唑蓝法、愈创木酚法、硫代巴比妥酸法,分别测定了受秋水仙碱诱导后其各自细胞内游离脯氨酸(PRO)含量、可溶性蛋白(Protein)的含量,超氧化物歧化酶(SOD)、过氧化物酶(POD)的活性、丙二醛(MDA)含量等变化趋势,并对比分析了银杏和毛新杨雄性生殖细胞在秋水仙碱加倍诱导状态下各自生理生化指标差异动态变化节点。【结果】秋水仙碱处理后,银杏生殖细胞内脯氨酸含量在12～36 h先增高后降低,可溶性蛋白含量1～36 h内降低,SOD活性有所降低,POD活性在3～36 h内大幅度升高,MDA含量6 h开始升高,12～48 h处在顶峰状态。毛新杨生殖细胞脯氨酸含量0.5～1 h内升高后期变化趋势不明显,可溶性蛋白含量24～48 h内略降低,SOD活性在12～48 h内明显升高,POD活性与空白组变化差异不明显,MDA含量与空白组、对照组相似。【结论】秋水仙碱诱导对银杏与毛新杨生殖细胞基因表达机制影响不同,对银杏生殖细胞的胁迫强于毛新杨。毛新杨受水分与秋水仙碱胁迫伤害较小,生理指标变化差异不大,但保护酶变化银杏相反,12～48 h内SOD活性上升明显,POD活性变化不明显。银杏生殖细胞POD活性上升,SOD活性有所降低,在此之前脯氨酸与MDA含量有明显上升趋势,可溶性蛋白含量下降。所以秋水仙碱胁迫损伤积累后的基因表达调控可能是其生殖细胞多倍化困难的主要原因,对基因表达调控的影响主要在秋水仙碱胁迫后期。研究结果将为探究秋水仙碱诱导过程基因表达调控机制奠定基础,并为初步判别不同树木种质生殖细胞多倍化诱导难易提供了理论和实例参考。
[Objective] Polyploidization is an important approach to the genetic improvement of trees,colchicine inducing reproductive cells is an important method of tree polyploid induced, but there are problems such as some tree polyploid induction is difficult, and polyploidization induced quality is difficult to distinguish. Therefore, it is necessary to explore the mechanism of polyploidy induction and to find a scientific method for the preliminary discrimination of the polyploidy effect of colchicine artificially induced forest germ cells. [Method] Taking ginkgo, Populus tomentosa × P. bolleana male reproductive cells as the research object, by ninhydrin colorimetry, CBA method,tetrazolium blue method,guaiacol method and thiobarbituric acid method, their intracellular free proline(PRO) content, soluble protein(Protein) content,superoxide dismutase(SOD), peroxidase(POD) activity, malondialdehyde(MDA) content changes were determined by colchicine inducing, and then the dynamic change nodes of the physiological and biochemical indexes of the male germ cells of Ginkgo biloba and Populus tomentosa × P. bolleana under the double induction of colchicine were compared and analyzed. [Result] After colcine treatment, in ginkgo germ cells, proline content increased first at 12-36 hour and then decreased, soluble protein content decreased at1-36 hour, SOD activity decreased at 12-48 hour, POD activity increased significantly at 3-36 hour, MDA content began to increase at 6 hour and peaked at 12-48 hour. In the Populus tomentosa × P. bolleana, germ cell proline content increased within 0.5-1 hour and the change trend was not obvious at the later stage, the soluble protein content decreased within 24-48 hour, the SOD activity increased significantly within 12-48 hour, the POD activity was not significantly different from the blank group, and the MDA content was similar to the blank group and the control group. [Conclusion] Colchicine induction had different effects on gene expression mechanism of ginkgo and Populus tomentosa × P. bolleana generative cells, and the stress on ginkgo germ cells was stronger than that on poplar. However, the change of protective enzyme of the poplar was opposite to that of ginkgo, and the SOD activity increased significantly during 12-48 hour period, while the POD activity did not change significantly. Therefore, the regulation of gene expression after damage accumulation under colchicine stress may be the main reason for the difficulty of tree germ cell polyploidy, and the influence on gene expression regulation is mainly in the late stage of colchicine stress.The results of this study will lay a foundation for exploring the regulation mechanism of gene expression during colchicine induction and provide theoretical and practical references for the preliminary identification of the difficulty of inducing polyploidy in germplasm of different trees.
[Objective] Polyploidization is an important approach to the genetic improvement of trees,colchicine inducing reproductive cells is an important method of tree polyploid induced, but there are problems such as some tree polyploid induction is difficult, and polyploidization induced quality is difficult to distinguish. Therefore, it is necessary to explore the mechanism of polyploidy induction and to find a scientific method for the preliminary discrimination of the polyploidy effect of colchicine artificially induced forest germ cells. [Method] Taking ginkgo, Populus tomentosa × P. bolleana male reproductive cells as the research object, by ninhydrin colorimetry, CBA method,tetrazolium blue method,guaiacol method and thiobarbituric acid method, their intracellular free proline(PRO) content, soluble protein(Protein) content,superoxide dismutase(SOD), peroxidase(POD) activity, malondialdehyde(MDA) content changes were determined by colchicine inducing, and then the dynamic change nodes of the physiological and biochemical indexes of the male germ cells of Ginkgo biloba and Populus tomentosa × P. bolleana under the double induction of colchicine were compared and analyzed. [Result] After colcine treatment, in ginkgo germ cells, proline content increased first at 12-36 hour and then decreased, soluble protein content decreased at1-36 hour, SOD activity decreased at 12-48 hour, POD activity increased significantly at 3-36 hour, MDA content began to increase at 6 hour and peaked at 12-48 hour. In the Populus tomentosa × P. bolleana, germ cell proline content increased within 0.5-1 hour and the change trend was not obvious at the later stage, the soluble protein content decreased within 24-48 hour, the SOD activity increased significantly within 12-48 hour, the POD activity was not significantly different from the blank group, and the MDA content was similar to the blank group and the control group. [Conclusion] Colchicine induction had different effects on gene expression mechanism of ginkgo and Populus tomentosa × P. bolleana generative cells, and the stress on ginkgo germ cells was stronger than that on poplar. However, the change of protective enzyme of the poplar was opposite to that of ginkgo, and the SOD activity increased significantly during 12-48 hour period, while the POD activity did not change significantly. Therefore, the regulation of gene expression after damage accumulation under colchicine stress may be the main reason for the difficulty of tree germ cell polyploidy, and the influence on gene expression regulation is mainly in the late stage of colchicine stress.The results of this study will lay a foundation for exploring the regulation mechanism of gene expression during colchicine induction and provide theoretical and practical references for the preliminary identification of the difficulty of inducing polyploidy in germplasm of different trees.
引文
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[3]裴新澍.人工诱导多倍体的原理和技术[J].生物学通报,1958,22(3):36-39.Pei X S.Principle and technique of artificially inducing polyploids[J].Bulletin of Biology,1958,22(3):36-39.
[4]李兆龙,卢耀明.含银杏叶提取物的保健饮料[J].软饮料工业,1996(2):29-32.Li Z L,Lu Y M.Health drinks containing Ginkgo biloba extract[J].Soft Drink Industry,1996(2):29-32.
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[6]胡君艳.银杏小孢子发育进程及其染色体加倍方法研究[D].北京:北京林业大学,2008.Hu J Y.The development and chromosome doubling of the microspore in Ginkgo biloba L.[D].Beijing:Beijing Forestry University,2008.
[7]程金新,李云,王勇,等.秋水仙碱诱导银杏花粉染色体加倍的研究[J].北京林业大学学报,2006,28(6):15-21.Cheng J X,Li Y,Wang Y,et al.Pollen chromosome doubling of Ginkgo biloba induced by colchicine[J].Journal of Beijing Forestry University,2006,28(6):15-21.
[8]王孟宇.作物遗传育种[M].北京:北京农业大学,2009.Wang M Y.Crop genetic breeding.[M].Beijing:Beijing Agricultural University,2009.
[9]刘晓露,王冬旭,杨蔚然,等.秋水仙素诱导黑白杨多倍体研究[J].安徽农业科学,2010,38(20):10532-10534.Liu X L,Wang D X,Yang W R,et al.Polyploidy induction of aigeiros-luece hybrids of Populus by colchicine[J].Anhui Agricultural Sciences,2010,38(20):10532-10534.
[10]梁君瑛.水分胁迫对桑树苗生长及生理生化特性的影响[D].北京:北京林业大学,2008.Liang J Y.Effect of water stress on growth and physiologicalbiochemical characteristics of Meorus alba L.seedling[D].Beijing:Beijing Forestry University,2008.
[11]吴高殷,韦小丽,王晓,等.林木体细胞胚胎发生机理研究进展[J/OL].世界林业研究[2019-06-05].https://doi.org/10.13348/j.cnki.sjlyyj.2019.0046.y.Wu G Y,Wei X L,Wang X,et al.Research progress of mechanism of somatic embryogenesis in forest trees[J/OL].World Forestry Research[2019-06-05].https://doi.org/10.13348/j.cnki.sjlyyj.2019.0046.y.
[12]刘宏伟.秋水仙素对北柴胡的诱导研究[D].绵阳:西南科技大学,2017.Liu H W.Study on polyploid induction of Bupleurum chinense DC[D].Mianyang:Southwest University of Science and Technology,2017.
[13]李霖锋,刘宝.植物多倍化与多倍体基因组进化研究进展[J].中国科学:生命科学,2019,49(4):327-337.Li L F,Liu B.Recent advances of plant polyploidy and polyploid genome evolution[J].Science in China:Life Sciences,2019,49(4):327-337.
[14]康向阳,朱之悌,林惠斌.杨树花粉染色体加倍有效处理时期的研究[J].林业科学,1999,35(4):21-26.Kang X Y,Zhu Z T,Lin H B.Study on the effective treating period for pollen chromosome doubling of Populus tomentosa×P.bolleana[J].Forestry Science,1999,35(4):21-26.
[15]石进校,刘应迪,陈军,等.土壤涝渍胁迫对淫羊藿叶片膜脂过氧化和SOD活性的影响[J].生命科学研究,2002,6(4):29-31.Shi J X,Liu Y D,Chen J,et al.Activation of SOD and content of MDA in Epimedium grandiflorm Morr.leaves under waterlogging stress[J].Life Science Research,2002,6(4):29-31.
[16]Yang N N,Sun Y H,Wang Y R,et al.Proteomic analysis of the low mutation rate of diploid male gametes induced by colchicine in Ginkgo biloba L.[J/OL].PLoS One,2013a,8(10):e76088[2018-12-01].https://www.researchgate.net/publication/258117809_Proteomic_Analysis_of_the_Low_Mutation_Rate_of_Diploid_Male_Gametes_Induced_by_Colchicine_in_Ginkgo_biloba_L.
[17]郝建军,卢环,黄春花,等.不同绿豆品种主要理化特性的比较[D].长春:吉林农业科学,2013.Hao J J,Lu H,Huang C H,et al.Comparison of main physical and chemical properties of different mung bean varieties[D].Changchun:Jilin Agricultural Sciences,2013.
[18]蒋洪恩.枣(Ziziphus jujuba Mill.)多倍体诱导研究[D].保定:河北农业大学,2003.Jiang H E.Study on poly ploid induction of Chinese jujube[D].Baoding:Hebei Agricultural University,2003.
[19]张蕊.低温下外源水杨酸对水稻幼苗生理生化特性的影响研究[D].重庆:西南大学,2006.Zhang R.Effects of salicylic acid on physiological and biochemical character of rice seedlings during chilling stress[D].Chongqing:Southwest University,2006.
[20]王嘉燕.涝害对樱桃砧木木生理生化特性的影响[D].泰安:山东农业大学,2006.Wang J Y.Effects of flood injury on physiological and biochemical characteristics of cherry rootstocks[D].Taian:Shandong Agricultural University,2006.
[21]万里强,李向林,石永红.PEG胁迫下4个黑麦草品种生理生化指标相应与比较分析研究[J].草业学报,2010,19(1):83-88.Wan L Q,Li X L,Shi Y H.A study on the response and on the comparison of physiological and biochemical indexes of four Lolium perenne varieties under PEG stress[J].Journal of Grass Industry,2010,19(1):83-88.
[22]Khan A A,Tao K L,Knypl J S.Oamotic conditining of seeds:physiological and biochemical changes[J].Acta Horticulturae,1978,83(83):267-278.
[2]张爱清.人工诱导植物多倍体的方法初探[J].中学生物学,2016,32(4):5-6.Zhang A Q.A preliminary study on the method of artificially inducing polyploids in plants[J].Middle School Biology,2016,32(4):5-6.
[3]裴新澍.人工诱导多倍体的原理和技术[J].生物学通报,1958,22(3):36-39.Pei X S.Principle and technique of artificially inducing polyploids[J].Bulletin of Biology,1958,22(3):36-39.
[4]李兆龙,卢耀明.含银杏叶提取物的保健饮料[J].软饮料工业,1996(2):29-32.Li Z L,Lu Y M.Health drinks containing Ginkgo biloba extract[J].Soft Drink Industry,1996(2):29-32.
[5]孙宇涵.秋水仙素诱导银杏2n雄配子低得率的机理研究[D].北京:北京林业大学,2011.Sun Y H.Study on mechanism of lower mutaton rate of 2n male gamete induced by colchicine in Ginkgo biloba L.[D].Beijing:Beijing Forestry University,2011.
[6]胡君艳.银杏小孢子发育进程及其染色体加倍方法研究[D].北京:北京林业大学,2008.Hu J Y.The development and chromosome doubling of the microspore in Ginkgo biloba L.[D].Beijing:Beijing Forestry University,2008.
[7]程金新,李云,王勇,等.秋水仙碱诱导银杏花粉染色体加倍的研究[J].北京林业大学学报,2006,28(6):15-21.Cheng J X,Li Y,Wang Y,et al.Pollen chromosome doubling of Ginkgo biloba induced by colchicine[J].Journal of Beijing Forestry University,2006,28(6):15-21.
[8]王孟宇.作物遗传育种[M].北京:北京农业大学,2009.Wang M Y.Crop genetic breeding.[M].Beijing:Beijing Agricultural University,2009.
[9]刘晓露,王冬旭,杨蔚然,等.秋水仙素诱导黑白杨多倍体研究[J].安徽农业科学,2010,38(20):10532-10534.Liu X L,Wang D X,Yang W R,et al.Polyploidy induction of aigeiros-luece hybrids of Populus by colchicine[J].Anhui Agricultural Sciences,2010,38(20):10532-10534.
[10]梁君瑛.水分胁迫对桑树苗生长及生理生化特性的影响[D].北京:北京林业大学,2008.Liang J Y.Effect of water stress on growth and physiologicalbiochemical characteristics of Meorus alba L.seedling[D].Beijing:Beijing Forestry University,2008.
[11]吴高殷,韦小丽,王晓,等.林木体细胞胚胎发生机理研究进展[J/OL].世界林业研究[2019-06-05].https://doi.org/10.13348/j.cnki.sjlyyj.2019.0046.y.Wu G Y,Wei X L,Wang X,et al.Research progress of mechanism of somatic embryogenesis in forest trees[J/OL].World Forestry Research[2019-06-05].https://doi.org/10.13348/j.cnki.sjlyyj.2019.0046.y.
[12]刘宏伟.秋水仙素对北柴胡的诱导研究[D].绵阳:西南科技大学,2017.Liu H W.Study on polyploid induction of Bupleurum chinense DC[D].Mianyang:Southwest University of Science and Technology,2017.
[13]李霖锋,刘宝.植物多倍化与多倍体基因组进化研究进展[J].中国科学:生命科学,2019,49(4):327-337.Li L F,Liu B.Recent advances of plant polyploidy and polyploid genome evolution[J].Science in China:Life Sciences,2019,49(4):327-337.
[14]康向阳,朱之悌,林惠斌.杨树花粉染色体加倍有效处理时期的研究[J].林业科学,1999,35(4):21-26.Kang X Y,Zhu Z T,Lin H B.Study on the effective treating period for pollen chromosome doubling of Populus tomentosa×P.bolleana[J].Forestry Science,1999,35(4):21-26.
[15]石进校,刘应迪,陈军,等.土壤涝渍胁迫对淫羊藿叶片膜脂过氧化和SOD活性的影响[J].生命科学研究,2002,6(4):29-31.Shi J X,Liu Y D,Chen J,et al.Activation of SOD and content of MDA in Epimedium grandiflorm Morr.leaves under waterlogging stress[J].Life Science Research,2002,6(4):29-31.
[16]Yang N N,Sun Y H,Wang Y R,et al.Proteomic analysis of the low mutation rate of diploid male gametes induced by colchicine in Ginkgo biloba L.[J/OL].PLoS One,2013a,8(10):e76088[2018-12-01].https://www.researchgate.net/publication/258117809_Proteomic_Analysis_of_the_Low_Mutation_Rate_of_Diploid_Male_Gametes_Induced_by_Colchicine_in_Ginkgo_biloba_L.
[17]郝建军,卢环,黄春花,等.不同绿豆品种主要理化特性的比较[D].长春:吉林农业科学,2013.Hao J J,Lu H,Huang C H,et al.Comparison of main physical and chemical properties of different mung bean varieties[D].Changchun:Jilin Agricultural Sciences,2013.
[18]蒋洪恩.枣(Ziziphus jujuba Mill.)多倍体诱导研究[D].保定:河北农业大学,2003.Jiang H E.Study on poly ploid induction of Chinese jujube[D].Baoding:Hebei Agricultural University,2003.
[19]张蕊.低温下外源水杨酸对水稻幼苗生理生化特性的影响研究[D].重庆:西南大学,2006.Zhang R.Effects of salicylic acid on physiological and biochemical character of rice seedlings during chilling stress[D].Chongqing:Southwest University,2006.
[20]王嘉燕.涝害对樱桃砧木木生理生化特性的影响[D].泰安:山东农业大学,2006.Wang J Y.Effects of flood injury on physiological and biochemical characteristics of cherry rootstocks[D].Taian:Shandong Agricultural University,2006.
[21]万里强,李向林,石永红.PEG胁迫下4个黑麦草品种生理生化指标相应与比较分析研究[J].草业学报,2010,19(1):83-88.Wan L Q,Li X L,Shi Y H.A study on the response and on the comparison of physiological and biochemical indexes of four Lolium perenne varieties under PEG stress[J].Journal of Grass Industry,2010,19(1):83-88.
[22]Khan A A,Tao K L,Knypl J S.Oamotic conditining of seeds:physiological and biochemical changes[J].Acta Horticulturae,1978,83(83):267-278.