弱光下菊花‘清露’的激素水平及相关基因表达

Hormone Levels and Gene Expression Analysis of Chrysanthemum Cultivar ‘Puma Sunny' Under Low Light Intensity

【目的】测量菊花在弱光条件下,出现庇荫症状过程中乙烯生成速率、赤霉素和生长素含量的变化及相关基因表达变化,从激素和基因水平探究庇荫机理。【方法】以菊花‘清露’为试验材料,设置55%光照(对照)和15%光照(弱光处理)。利用气相色谱仪Agilent 6890N GC在0、2、4、6和8 d测量乙烯生成速率,利用高效液相色谱仪Agilent HPLC-110在0、2、4、6和8 d测量赤霉素(GA3)和生长素(IAA)含量,以GAPDH为内参基因用荧光定量PCR方法测量0、2、4、6和8 d时PHYA、COP1、PIF4、GAI和IAA1的相对表达量。设置0.5、1.0、2.0和4.0 mmol·L-14个GA3浓度,用适量乙醇溶解,使乙醇终浓度为1%,0 d、4 d时各处理1次,采用地上部喷施;设10、20、50和100μmol·L-14个多效唑(paclobutrazol,PAC)浓度,用适量乙醇溶解,使乙醇终浓度是1%,0 d和4 d时各处理1次,根据结果以2.0 mmol·L-1GA3,50μmol·L-1PAC对植物生长影响显著,分别作为处理浓度。0 d、7 d各喷1次,14 d时取样进行基因表达分析。【结果】15%光照条件下,第2天和第4天时乙烯生成速率显著增加,第4天最显著,比对照增加了2.04倍,第6天以后对照和处理的植株乙烯生成速率差异不显著,15%弱光处理条件下植株地上部乙烯生成速率比55%光照条件增加显著;15%光照条件下,第2、4和6天时叶片赤霉素含量与对照相比显著增加,第6天时出现峰值,达到4 700.56 ng·g-1,比对照高出2.57倍,随后又迅速下降,第8天降到与对照相同的水平;15%光照条件下生长素水平与对照组相比显著下降,随着处理时间的延长,对照组生长素含量呈增加趋势,弱光处理组生长素含量呈下降趋势,第8天时处理组生长素含量为223.95 ng·g-1FW,对照为489.77 ng·g-1FW,对照比处理的生长素含量高出1.12倍。弱光处理第2天时PHYA表达量迅速增加,2—6 d以略小的速度增加,6—8 d保持不变,对照组从第2天开始下降,4—8 d时处理组PHYA变化量都高于对照组;对照和处理的PIF4表达量变化在0—4天趋势相同,不同的是处理组在4—8 d高于对照,尤其是第6天比对照高出1.85倍。对照组COP1呈缓慢降低趋势,处理组先增加后下降,峰值出现在第2天,第8天降到对照组含量以下。整个处理过程中,处理组的GAI一直增加,对照组0—4 d小幅增加,后以较快的速度下降。处理组和对照组IAA1在0—2 d均小幅上升,对照组在2—4 d又以较快速度下降,2 d后处理组IAA1表达量变化不大。外源赤霉素使COP1、PIF4的表达量上升,使GAI和IAA1表达量下降;PAC处理抑制COP1、PIF4、GAI和IAA1的表达。【结论】PHYA、COP1、IAA1和PIF4都受光的诱导,弱光引起GA3含量升高和GAI转录水平上调,赤霉素和乙烯参与菊花庇荫调节过程。

[ Objective ] In the study, ethylene accumulation rate, GA levels, IAA content and correlative gene expression levels in leaves of chrysanthemum under low light condition during shade avoidance symptoms was assayed, to explore the shade tolerant mechanism. [Method] Chrysanthemum ＇Puma Sunny＇ was assayed under 55% irradiance （control group） and 15% irradiance （low light treatment）. GA and IAA contents on 0, 2, 4, 6 and 8 d were measured by high performance liquid chromatograph Agilent 6890N GC system, the relative expression level was measured on 0, 2, 4, 6 and 8 d using GAPDH as reference gene. The concentrations of GA3 were set at 0.5, 1.0, 2.0 and 4.0 mmol.L-l, with a proper the amount of ethanol dissolution, the final concentration ethanol was 1%. The sample was treated on 4 d and 8 d using the above ground spraying. The concentrations of PAC were set at 10, 20, 50 andl00 pmol.L-l, with a proper amount of ethanol dissolution, the final concentration of ethanol was 1%. The results showed that 2.0 mmol.L-1 GA3, and 50 μmol.L-1 PAC showed impacts on plant growth significantly. After treatment on 0 d and 7 d, the sample was collected on 14 d for analysis of gene expression. [Result] The ethylene level increased markedly on 2 and 4 d, especially on 4 d, and it was 2.04 times more than that of CK. After 6 d, the ethylene level between CK and treatment was not significant. The plant ethylene generation rate under 15% low light conditions was higher than that of 55% irradiance. The gibberellin content in leaves increased significantly on day 2, 4 and 6 compared with the control, the peak appeared on the 6 d, reached 4700.56 ng.g-1, 2.57 times higher than the control, then decreased quickly, and down to control level on the 8 d. With the prolonging of treatment time, the content of auxin in control group showed an increasing trend, weak light treatment group decreased. On the 8 day, the auxin content in treatment group was 223.95 ng.g1 FW, and that in the control group was 489.77 ng.g1 FW. The IAA content of the control groupment was 1.12 times higher than that of the treatment group. In the second day of weak light treatment, the expression level of PHYA increased rapidly, 2-6 d with a smaller velocity, 6-8 d kept unchanged, but the control group began to decline from the second day. The change of the expression of PLYA in the treatment group from 4 to 8 day was higher than that of control group. The expression level of PIF4 of the control and the treatment kept the same change trend, from 0 to 6 day the difference was that on 4-8 day the treatment group was higher than the control, especially on the sixth day, it was 1.85 times higher than the control. COP1 gene in control group showed a slow decreasing trend while treatment group increased first and then decreased, the peak appeared on the second day, and on 8 d down to the control group. In the whole process, GA1 in the treatment group tended to increase continuously, the control group that in increased slightly on 0-4 day and then declined rapidly. The expression oflAA1 in the treatment group and control group on the 0-2 d increased slightly, and that in the control group on the 2-4 d decreased rapidly. After 2 days, the expression level of IAA1 changed little, lrradiance at 15% induced the PHYA, COP1, IAA1 and PIF4 gene expression. Irradiance at 15% increased GAI transcript level. Exogenous GA inhibited the expression of COP1 and PIF4 expression and down regulated GAI and IAA 1 level. PAC inhibited the expression of COP1, PIF4, GAI and IAA1. [ Conclusion ]All these data indicated that the expression of PHE4, COP1, IAA 1 and PIF4 was induced by low light, the GAI transcript level increased under low light. GA and ethylene involved in shade avoidance process of chrysanthemum.