| 柑橘黄龙病菌侵染对甜橙叶片糖代谢的影响 |
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| 引用本文： | 吴越,苏华楠,黄爱军,周彦,李中安,刘金香,周常勇. 柑橘黄龙病菌侵染对甜橙叶片糖代谢的影响[J]. 中国农业科学, 2015, 48(1): 63-72. DOI: 10.3864/j.issn.0578-1752.2015.01.07 |
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| 作者姓名： | 吴越 苏华楠 黄爱军 周彦 李中安 刘金香 周常勇 |
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| 作者单位： | 1西南大学植物保护学院，重庆 4007152中国农业科学院柑桔研究所，重庆 400712 |
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| 基金项目： | 国家公益性行业(农业)科研专项 |
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| 摘 要： | 【目的】研究甜橙（Citrus sinensis）在柑橘黄龙病菌（Candidatus Liberibacter asiaticus）侵染下不同时期糖代谢的变化，以探讨其与柑橘叶片中淀粉积累间的关系，为进一步阐明柑橘黄龙病的发病机理提供理论依据。【方法】以甜橙为供试材料，采用腹接接种病原的方法，试验组植株用感染黄龙病菌的芽条进行嫁接，对照组植株用健康芽条进行接种，每个植株均嫁接3个芽条。在检测到感病的植株中选取生长状况良好且接近的3株作为后续试验材料，每月采集1次成熟叶片，至10月份结束。采集的叶片立即抽提DNA和RNA，并进行可溶性糖及淀粉含量的测定，其余叶片用液氮速冻后保存于-80℃用于测定糖代谢过程中关键酶活性的变化，同时利用实时荧光定量PCR技术比较二者关键酶基因表达量的差异。【结果】随着病原菌胁迫时间的延长，可溶性糖和淀粉含量总体呈升高趋势，均在6月达到峰值，分别为对照的1.59和3.73倍，在感病后期开始有所下降；通过对二者的比值分析发现，感病植株的比值随时间的延长不断下降。在黄龙病菌侵染的不同阶段，各种酶的作用程度有所不同。感病植株的蔗糖磷酸合成酶（SPS）活性在感病初期迅速上升至顶峰，高达58.44 μg·g-1·min-1，至后期已略微低于对照；酸性转化酶（AI）总体显示出较高活性，各个时期均有显著性差异（P＜0.05），感病中期活性最高为对照的2.91倍；中性转化酶（NI）活性在感病和对照中均维持较低水平，且变化趋势基本一致；可溶性淀粉合成酶（SSs）与束缚性淀粉合成酶（GBSS）协调作用，共同参与淀粉合成的调控；淀粉酶活性在感病不同时期均有所下降，在感病中期低至0.38 U·g-1。定量PCR分析表明，蔗糖磷酸合成酶基因SPS1表达量显著升高，与其酶活性具有显著相关性（P＜0.05）。蔗糖分解相关基因中，蔗糖合成酶基因SuSy在感病不同时期的表达差异不大，变化幅度小；细胞壁酸性转化酶基因CSCWI在不同时期均上调表达，最高上调约7.4倍，且表达量一直维持在较高水平；与CSCWI相比，液泡酸性转化酶基因CUAI1的表达水平较低。淀粉合成相关酶ADP-葡萄糖焦磷酸化酶基因最高上调3倍左右；淀粉分解相关基因BAM3、MEX1和DPE2在不同时期有所下调，达到显著性差异水平（P＜0.05）。【结论】柑橘黄龙病感染甜橙后，对植株叶片中光合产物的形成与运转产生影响，扰乱宿主糖代谢平衡，与宿主淀粉积累及后期症状的产生有密切关系。
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| 关 键 词： | 甜橙 柑橘黄龙病 柑橘黄龙病菌 糖代谢 淀粉积累 |
| 收稿时间： | 2014-06-20 |
Effect of Candidatus Liberibacter asiaticus Infection on Carbohydrate Metabolism in Citrus sinensis |
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| WU Yue,SU Hua-nan,HUANG Ai-jun,ZHOU Yan,LI Zhong-an,LIU Jin-xiang,ZHOU Chang-yong. Effect of Candidatus Liberibacter asiaticus Infection on Carbohydrate Metabolism in Citrus sinensis[J]. Scientia Agricultura Sinica, 2015, 48(1): 63-72. DOI: 10.3864/j.issn.0578-1752.2015.01.07 |
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| Authors: | WU Yue SU Hua-nan HUANG Ai-jun ZHOU Yan LI Zhong-an LIU Jin-xiang ZHOU Chang-yong |
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| Affiliation: | 1College of Plant Protection, Southwest University, Chongqing 4007152Citrus Research Institute, Chinese Academy of Agricultural Sciences, Chongqing 400712 |
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| Abstract: | 【Objective】The objective of this experiment is to study the effect of Huanglongbing (HLB) infection on carbohydrate metabolism in Citrus sinensis, elucidate the relationship between HLB infection and starch accumulation, and provide a theoretical basis for HLB pathogenesis. 【Method】C. sinensis were used as the experimental materials, plants were inoculated by grafting three bark pieces onto the rootstock portion of each plant. For infected plants, bark pieces were derived from trees infected with Candidatus Liberibacter asiaticus, which were confirmed positive for Ca. L. asiaticus by PCR. For non-infected plants, bark pieces were obtained from healthy trees. Three plants in good growth state were chosen for successive test, mature leaves were collected every month until October. DNA and RNA were extracted, the content of soluble sugar and starch were measured immediately, the other leaf tissue was snap-frozen with liquid nitrogen and stored at -80℃ for testing carbohydrate metabolism related key enzyme activities. At the same time, the expression levels were compared using real-time quantitative PCR analysis. 【Result】The content of soluble sugar and starch tended to increase with time extended, and their peak value were respectively reached 1.59 and 3.73 times of control in June, then slightly decreased later, the ratio of soluble sugar to starch dropped down. Various key enzymes imposed different hands on carbohydrate metabolism at different stages of infection. Sucrose phosphate synthase activity rapidly rose to the top 58.44 μg?g-1?min-1 and had become lower than control in late infection; acid invertase had been consistently high and the difference was significant (P＜0.05), the highest activity was 2.91 times of control at middle stage; the activity of neutral invertase was kept at a lower level and had the same change trend in experimental group and control group; soluble starch synthase activity was closely related to granule-bond starch synthase, coordinating the synthesis of acting on the starch, while amylase activity had dropped to some extend at each stage, even as low as 0.38 U?g-1 at middle stage. Real-time quantitative PCR indicated that there was a significant relationship between the expression level of sucrose phosphate synthase and its enzyme activity (P＜0.05). Among the sucrose decomposition related genes, sucrose synthase gene was not changed obviously and had lower change range while cell wall-bound invertase gene CSCWI was up-regulated with the highest value of about 7.4 times and expressed abundantly during the whole process. Starch decomposition-related genes BAM3, MEX1 and DPE2 had a relatively reduced expression and the difference was significant (P＜0.05) in different periods.【Conclusion】HLB infection has influence on the formation and transportation of photosynthate in C. sinensis and results in the disruption of carbohydrate metabolism balance, it is in deep connection with starch accumulation and the late stage symptoms. |
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| Keywords: | Citrus sinensis citrus Huanglongbing Candidatus Liberibacter asiaticus carbohydrate metabolism starch accumulation |
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