白桦转BpGH3.5基因叶片早衰突变株的光合特性及生长分析
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摘要
【目的】植物叶片早衰将影响作物产量和品质,研究叶片早衰机制对于培育耐早衰优良品种具有重要意义。【方法】以转BpGH3.5基因的白桦叶片早衰突变株(G4)、非转基因白桦(WT)及叶片正常的转基因白桦(G21)等为材料,测定其叶绿素含量、净光合速率(P_n)、气孔导度(G_s)、胞间CO_2浓度(C_i)、蒸腾速率(T_r)等光合指标,测定苗高的时序变化规律。【结果】叶片早衰突变影响了叶片叶绿素的合成及积累,突变株的叶绿素相对含量(SPAD)均值为36.08,相对两个对照株系分别下降7.34%、7.48%。叶片早衰突变影响了白桦光合呼吸作用。突变株的净光合速率、气孔导度、蒸腾速率分别为WT野生株系的67.54%、64.44%、64.93%,胞间CO_2浓度达到234.33μmol/mol,显著高于G21对照株系(P<0.05)。突变株的当年高生长显著低于WT、G21两个对照株系,当年高生长分别是WT、G21两个对照株系的68.9%、85.0%。利用Logistic方程对3个参试株系当年苗高生长量的变化过程进行了拟合,其系数均高于0.98,同时,通过Logistic方程计算的生长参数揭示了早衰突变株高生长较两个对照株系低的原因是速生期苗高平均生长量(GR)、苗高日生长量的平均值(GD)等生长参数较低。【结论】转BpGH3.5基因的白桦发生了叶片早衰现象,使叶绿素提前降解,影响了光合呼吸作用,进而影响了苗高生长。
【Objective】 Knowing that precocious leaf senescence affects plant growth and wood quality, we aimed to understand the effect of precocious leaf senescence on photosynthesis and growth to reveal the mechanism underlying this phenomenon, and to create cultivars resistant to it. 【Method】We used BpGH3.5 transgenic Betula platyphylla plants lines showing precocious leaf senescence(G4), non-transgenic B. platyphylla plants(WT), and transgenic plants with normal leaves(G21) to perform molecular analyses. Genetic transformation results were analyzed by quantitative gene expression. Moreover, experiments were performed to determine the chlorophyll content, the net photosynthetic rate(P_n), the stomatal conductance(G_s), the intercellular carbon dioxide concentration(C_i), and the transpiration rate(T_r) for each plant. In addition, the seedling height was measured in a time series. Finally, a logistic model was used to perform fitting analysis and to judge the differences between growth parameters. 【Result】 The BpGH3.5 mutation affected both the synthesis and accumulation of chlorophyll in leaves. The average SPAD value of the mutant line was 36.08, which was 7.34% and 7.48% lower than that of the WT and the G21 plants, respectively. The B. platyphylla BpGH3.5 transgenic line G4 showed significant alterations in photosynthetic respiration values: net photosynthetic rate, stomatal conductance and transpiration rate in mutant lines were 67.54%, 64.44% and 64.93% compared with those from the WT line, respectively. The intercellular carbon dioxide concentration(234.33 μmol/mol) was significantly higher in G4 lines than in that G21 lines(P < 0.05). Tree height current increment of BpGH3.5 transgenic line G4 was significantly lower than that from WT and G21 lines(representing the 68.9% and 85.0% of WT and G21, respectively). A Logistic model was used to fit tree height current increment of the three tested lines. All fitting coefficients were higher than 0.98. Moreover, calculated growth revealed that the height of the G4 mutant was lower than that of the two control lines, G21 and WT. This was due to a slow growth during the fast-growing period(GR) and a low daily growth rate in plant height(GD). 【Conclusion】 A mutation leading to precocious leaf senescence led to early degradation of chlorophyll in early leaf shedding. As a result, it affected the photosynthetic and respiration pathways, leading to a reduction in Betula platyphylla plant height.
【Objective】 Knowing that precocious leaf senescence affects plant growth and wood quality, we aimed to understand the effect of precocious leaf senescence on photosynthesis and growth to reveal the mechanism underlying this phenomenon, and to create cultivars resistant to it. 【Method】We used BpGH3.5 transgenic Betula platyphylla plants lines showing precocious leaf senescence(G4), non-transgenic B. platyphylla plants(WT), and transgenic plants with normal leaves(G21) to perform molecular analyses. Genetic transformation results were analyzed by quantitative gene expression. Moreover, experiments were performed to determine the chlorophyll content, the net photosynthetic rate(P_n), the stomatal conductance(G_s), the intercellular carbon dioxide concentration(C_i), and the transpiration rate(T_r) for each plant. In addition, the seedling height was measured in a time series. Finally, a logistic model was used to perform fitting analysis and to judge the differences between growth parameters. 【Result】 The BpGH3.5 mutation affected both the synthesis and accumulation of chlorophyll in leaves. The average SPAD value of the mutant line was 36.08, which was 7.34% and 7.48% lower than that of the WT and the G21 plants, respectively. The B. platyphylla BpGH3.5 transgenic line G4 showed significant alterations in photosynthetic respiration values: net photosynthetic rate, stomatal conductance and transpiration rate in mutant lines were 67.54%, 64.44% and 64.93% compared with those from the WT line, respectively. The intercellular carbon dioxide concentration(234.33 μmol/mol) was significantly higher in G4 lines than in that G21 lines(P < 0.05). Tree height current increment of BpGH3.5 transgenic line G4 was significantly lower than that from WT and G21 lines(representing the 68.9% and 85.0% of WT and G21, respectively). A Logistic model was used to fit tree height current increment of the three tested lines. All fitting coefficients were higher than 0.98. Moreover, calculated growth revealed that the height of the G4 mutant was lower than that of the two control lines, G21 and WT. This was due to a slow growth during the fast-growing period(GR) and a low daily growth rate in plant height(GD). 【Conclusion】 A mutation leading to precocious leaf senescence led to early degradation of chlorophyll in early leaf shedding. As a result, it affected the photosynthetic and respiration pathways, leading to a reduction in Betula platyphylla plant height.
引文
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[19] XU W D,HAN R,XU S J,et al.Expression of BpIAA10 from Betula platyphylla (birch) is differentially regulated by different hormones and light intensities[J].Plant Cell,Tissue and Organ Culture (PCTOC),2018,132(2):371-381.DOI:10.1007/s11240-017-1336-y.
[20] LIN L,YAO Q C,XU H W,et al.Characteristics of the staminate flower and pollen from autotetraploid Betula platyphylla[J].Dendrobiology,2012,69:3-11.DOI:10.12657/denbio.069.001.
[21] LIU C Y,XU H W,JIANG J,et al.Analysis of the promoter features of BpCUC2 in Betula platyphylla×Betula pendula[J].Plant Cell,Tissue and Organ Culture (PCTOC),2018,132(1):191-199.DOI:10.1007/s11240-017-1324-2.
[22] YANG G,CHEN S,JIANG J.Transcriptome analysis reveals the role of BpGH3.5 in root elongation of Betula platyphylla× B.pendula[J].Plant Cell,Tissue and Organ Culture (PCTOC),2015,121(3):605-617.DOI:10.1007/s11240-015-0731-5.
[23] YANG G,CHEN S,WANG S,et al.BpGH3.5,an early auxin-response gene,regulates root elongation in Betula platyphylla× Betula pendula[J].Plant Cell,Tissue and Organ Culture (PCTOC),2015,120(1):239-250.DOI:10.1007/s11240-014-0599-9.
[24] 龚盼,黎坤瑜,黄福灯,等.水稻叶片早衰突变体ospls3的生理特征和基因定位[J].作物学报,2016,42(5):667-674.DOI:10.3724/SP.J.1006.2016.00667.GONG P,LI K Y,HUANG F D,et al.Physiological characteristics and gene mapping of a precocious leaf senes-cence mutant ospls3 in rice[J].Acta Agronomica Sinica,2016,42(5):667-674.
[25] 王复标.水稻早衰突变体(psf)叶片衰老形成与衰老速率调控的生理机制研究[D].杭州:浙江大学,2017.WANG F B.Physiological mechanism of leaf senescence formation and its metabolic regulation in premature senescence rice(psf) mutant leaves[D].Hangzhou:Zhejiang University,2017.
[26] 王平荣,张帆涛,高家旭,等.高等植物叶绿素生物合成的研究进展[J].西北植物学报,2009,29(3):629-636.DOI:10.3321/j.issn:1000-4025.2009.03.032.WANG P R,ZHANG F T,GAO J X,et al.An overview of chlorophyll biosynthesis in higher plants[J].Acta Botanica Boreali-Occidentalia Sinica,2009,29(3):629-636.
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[2] 张文旭.紫花苜蓿荚的光合性能及产物转运作用机理研究[D].北京:中国农业大学,2014.ZAHNG W X.Study on pod photosynthesis and photosynthate transporting and acting mechanism in alfalfa[D].Beijing:China Agricultural University,2014.
[3] 孟军,陈温福,徐正进,等.水稻剑叶净光合速率与叶绿素含量的研究初报[J].沈阳农业大学学报,2001,32(4):247-249.DOI:10.3969/j.issn.1000-1700.2001.04.002.MENG J,CHEN W F,XU Z J,et al.Study on photosynthetic rate and chlorophyll content[J].Journal of Shenyang Agricultural University,2001,32(4):247-249.
[4] 毛剑飞,李凯伟,杨再强,等.弱光胁迫及强光恢复对设施红地球葡萄叶片光合及衰老特性的影响[J].江苏农业科学,2018,46(6):105-111.DOI:10.15889/j.issn.1002-1302.2018.06.027.MAO J F,LI K W,YANG Z Q,et al.Effects of weak light stress and strong light recovery on photosynthetic and senescence characteristics of grape leaves in greenhouse[J].Jiangsu Agricultural Sciences,2018,46(6):105-111.
[5] 王复标.水稻早衰突变体(psf)叶片衰老形成与衰老速率调控的生理机制研究[D].杭州:浙江大学,2017.WANG F B.Physiological mechanism of leaf senescence formation and its metabolic regulation in premature senescence rice(psf) mutant leaves[D].Hangzhou:Zhejiang University,2017.
[6] 周建平.植物衰老相关基因的克隆与分析[D].成都:电子科技大学,2007.ZHOU J P.Cloning and analysis of the senescence-associated genes from plants[D].Chengdu:University of Electric Science and Technology of China,2007.
[7] LI Z H,PENG J Y,WEN X,et al.Gene network analysis and functional studies of senescence-associated genes reveal novel regulators of Arabidopsis leaf SenescenceF[J].Journal of Integrative Plant Biology,2012,54(8):526-539.
[8] 刘连涛,李存东,孙红春,等.棉花叶片衰老生理研究进展[J].中国农学通报,2006,22(7):316-321.DOI:10.3969/j.issn.1000-6850.2006.07.082.LIU L T,LI C D,SUN H C,et al.Advances of research on cotton leaf senescence physiology[J].Chinese Agricultural Science Bulletin,2006,22(7):316-321.
[9] HUNG K T,KAO C H.Hydrogen peroxide is necessary for abscisic acid-induced senescence of rice leaves[J].Journal of Plant Physiology,2004,161(12):1347-1357.DOI:10.1016/j.jplph.2004.05.011.
[10] 张林,罗天祥.植物叶寿命及其相关叶性状的生态学研究进展[J].植物生态学报,2004,28(6):844-852.DOI:10.17521/cjpe.2004.0110.ZHANG L,LUO T X.Advances in ecological studies on leaf lifespan and associated leaf traits[J].Acta Phytoecologica Sinica,2004,28(6):844-852.
[11] REICH P B,UHL C,WALTERS M B,et al.Leaf lifespan as a determinant of leaf structure and function among 23 amazonian tree species[J].Oecologia,1991,86(1):16-24.DOI:10.1007/bf00317383.
[12] 段俊,梁承邺,黄毓文.杂交水稻开花结实期间叶片衰老[J].植物生理学报,1997,23(2):139-144.DOI:10.3321/j.issn:1671-3877.1997.02.006.DUAN J,LIANG C Y,HUANG Y W.Studies on leaf senescence of hybrid rice at flowering and grain formation stage[J].Acta Photophysiologica Sinica,1997,23(2):139-144.
[13] NAVABPOUR S.Expression of senescence-enhanced genes in response to oxidative stress[J].Journal of Experimental Botany,2003,54(391):2285-2292.DOI:10.1093/jxb/erg267.
[14] 孙玉莹,毕京翠,赵志超,等.作物叶片衰老研究进展[J].作物杂志,2013(4):11-19.DOI:10.16035/j.issn.1001-7283.2013.04.006.SUN Y Y,BI J C,ZHAO Z C,et al.The advancement on leaf senescence in crops[J].Crops,2013(4):11-19.
[15] 刘道宏.植物叶片的衰老[J].植物生理学通讯,1983,19(2):14-19.DOI:10.13592/j.cnki.ppj.1983.02.004.
[16] 李绍臣,高福玲,姜廷波,等.基于RAPD标记的白桦遗传连锁群分析[J].林业科学,2008,44(5):155-159.DOI:10.3321/j.issn:1001-7488.2008.05.028.LI S C,GAO F L,JIANG T B,et al.Analysis of genetic linkage groups on birch using RAPD markers[J].Scientia Silvae Sinicae,2008,44(5):155-159.
[17] HUANG H J,WANG S,JIANG J,et al.Overexpression of BpAP1induces early flowering and produces dwarfism in Betula platyphylla×Betula pendula[J].Physiologia Plantarum,2014,151(4):495-506.DOI:10.1111/ppl.12123.
[18] ZHANG W B,WEI R,CHEN S,et al.Functional characterization of CCRin birch (Betula platyphylla×Betula pendula) through overexpression and suppression analysis[J].Physiologia Plantarum,2015,154(2):283-296.DOI:10.1111/ppl.12306.
[19] XU W D,HAN R,XU S J,et al.Expression of BpIAA10 from Betula platyphylla (birch) is differentially regulated by different hormones and light intensities[J].Plant Cell,Tissue and Organ Culture (PCTOC),2018,132(2):371-381.DOI:10.1007/s11240-017-1336-y.
[20] LIN L,YAO Q C,XU H W,et al.Characteristics of the staminate flower and pollen from autotetraploid Betula platyphylla[J].Dendrobiology,2012,69:3-11.DOI:10.12657/denbio.069.001.
[21] LIU C Y,XU H W,JIANG J,et al.Analysis of the promoter features of BpCUC2 in Betula platyphylla×Betula pendula[J].Plant Cell,Tissue and Organ Culture (PCTOC),2018,132(1):191-199.DOI:10.1007/s11240-017-1324-2.
[22] YANG G,CHEN S,JIANG J.Transcriptome analysis reveals the role of BpGH3.5 in root elongation of Betula platyphylla× B.pendula[J].Plant Cell,Tissue and Organ Culture (PCTOC),2015,121(3):605-617.DOI:10.1007/s11240-015-0731-5.
[23] YANG G,CHEN S,WANG S,et al.BpGH3.5,an early auxin-response gene,regulates root elongation in Betula platyphylla× Betula pendula[J].Plant Cell,Tissue and Organ Culture (PCTOC),2015,120(1):239-250.DOI:10.1007/s11240-014-0599-9.
[24] 龚盼,黎坤瑜,黄福灯,等.水稻叶片早衰突变体ospls3的生理特征和基因定位[J].作物学报,2016,42(5):667-674.DOI:10.3724/SP.J.1006.2016.00667.GONG P,LI K Y,HUANG F D,et al.Physiological characteristics and gene mapping of a precocious leaf senes-cence mutant ospls3 in rice[J].Acta Agronomica Sinica,2016,42(5):667-674.
[25] 王复标.水稻早衰突变体(psf)叶片衰老形成与衰老速率调控的生理机制研究[D].杭州:浙江大学,2017.WANG F B.Physiological mechanism of leaf senescence formation and its metabolic regulation in premature senescence rice(psf) mutant leaves[D].Hangzhou:Zhejiang University,2017.
[26] 王平荣,张帆涛,高家旭,等.高等植物叶绿素生物合成的研究进展[J].西北植物学报,2009,29(3):629-636.DOI:10.3321/j.issn:1000-4025.2009.03.032.WANG P R,ZHANG F T,GAO J X,et al.An overview of chlorophyll biosynthesis in higher plants[J].Acta Botanica Boreali-Occidentalia Sinica,2009,29(3):629-636.
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