KT/HAK/KUP家族基因在桃开花期的表达及对钾肥施用的响应

doi:10.3864/j.issn.0578-1752.2015.06.13

摘要/Abstract

摘要： 【目的】从转录水平探索KT/HAK/KUP家族基因在桃花发育不同时期的表达模式及对钾肥施用的响应特征，明确关键基因并验证其功能。旨在揭示钾素营养状况与桃花开放之间的直接关系，为果树施钾及高效园艺作物的遗传改良与育种提供理论依据。【方法】以‘霞晖6号’桃树为研究对象，通过钾肥施用分析其对桃花生长发育、钾素含量及花朵开放时期的影响；通过HNO₃-HClO₄法消化样品，利用ICP-AES设备测定桃花开放不同时期的钾离子含量；利用荧光实时定量qRT-PCR技术，分析KT/HAK/KUP家族基因在桃花开放不同时期的表达水平，确定主效基因；进一步通过qRT-PCR技术分析KT/HAK/KUP家族基因在桃花开放不同时期对钾肥施用的差异响应；利用异源细菌缺失功能互补法验证主效基因的功能，将KUP11的CDS序列克隆到pPAB404中形成重组表达载体pPAB404-KUP11，测序验证的重组载体转化到大肠杆菌功能缺失突变菌株TK2420中，分析重组载体pPAB404-KUP11恢复TK2420突变细菌吸收外界K⁺（KCl或K₂SO₄）的功能。【结果】钾肥施用使‘霞晖6号’桃花提前2 d开放，并显著促进了盛开期桃花的发育，花朵鲜重增加21.5%。此外，盛开期桃花K⁺含量也最高，其次是初开期和花蕾期，败落期则最低。因此，钾肥施用显著增强了桃花开放过程中花朵K⁺的富集水平，在花蕾期、初开期、盛开期和败落期分别提高了24.3%、27.4%、29.1%和26.2%。KUP1—13在桃花开放不同时期的表达水平存在差异，但在盛开期的表达水平均达到最高，且在转录水平对钾肥施用的响应不同。KUP1和KUP5基因对钾肥施用最为敏感，其表达水平从花蕾期至桃花盛开期是持续被诱导的；KUP11在桃花开放的整个过程中表达量最高，钾肥处理显著诱导了其在花蕾形成初期的表达并抑制了其在桃花败落期的表达量。表达载体pPAB404-KUP11能恢复TK2420细菌重新吸收K⁺，且对外界KCl和K₂SO₄均有吸收，说明KUP11的表达水平与K⁺的吸收呈正相关性。【结论】钾肥施用促进桃花发育，改善钾营养状况，催使桃花提前开放，并对KUP家族基因在开花不同时期转录水平的调控有差异；KUP11转运体具有吸收外界K⁺的功能，在桃花开放过程中起重要作用。

关键词: 桃, 开花, 钾, KT/HAK/KUP, 钾素平衡, 桃, 开花, 钾, KT/HAK/KUP, 钾素平衡

Abstract: 【Objective】 The objective of this study is to analyze the transcriptional expression of KT/HAK/KUP family genes and response characteristics to potassium fertilizer application during different flowering stages, and key K⁺ transporter genes and its function. A close relationship between K⁺ nutritional status and flower development was discussed, thus providing a theoretical basis for K fertilization in orchards and genetic improvement and breeding of efficient horticulture crops.【Method】The K fertilizer treatment experiment was conducted via applying 834 g KCl (K₂O content 60%) to each ‘Xiahui6’ peach tree at flower bud expanding stage. The effect of K fertilizer application on flower development, K⁺ nutritional status and blooming date of peach trees was analyzed. The K⁺ concentration of peach flowers at different developmental stages was determined by using ICP-AES apparatus with HNO₃-HClO₄digestion method. Quantitative real-time PCR was used to analyze the expression profiles of KT/HAK/KUP family genes during the whole flowering process, and the key K⁺ transporter genes were identified. The response of KT/HAK/KUP family genes to K fertilizer application at different developmental stages was also revealed. Functions of the key genes were validated by using heterologous complementation of bacterial function lossing mutant. Coding sequence of KUP11 was cloned into pPAB404 vector to obtain the recombinant expression vector pPAB404-KUP11. Sequencing verified recombinant vector was then transformed into functional mutant E. coli strain TK2420. Whether the recombinant vector pPAB404-KUP11 can restore the ability of TK2420 mutant strain to uptake external K⁺, supplied with either KCl or K₂SO₄, was determined.【Result】Application of K fertilizer treatment favorably induced ‘Xiahui6’ flowers to bloom 2-day earlier, and specifically contributed to flower development at full bloom stage, with an increase of 21.5% in fresh weight. The highest K⁺ accumulation occurred at full-bloom stage, which was followed by begin bloom, bud period and petal fall stages. Potasium application significantly enhanced the K⁺ nutritional status, with an increase of 24.3%, 27.4%, 29.1% and 26.3% of K⁺ concentration, respectively, during four flowering stages. Genes of KUP1-13 were differentially expressed during the four stages, and the highest expression level appeared especially in full-bloom stage. The KT/HAK/KUP family genes were differentially regulated by K, i.e., KUP1 and KUP5 were most sensitive to Kapplication, whose expression was consistently induced from bud swell stage to full-bloom stage. KUP11 was the most expressed gene throughout the whole flowering process, which was highly up-regulated at bud swell stage but greatly reduced at petal fall stage by K treatment. The recombinant expression vector pPAB404-KUP11 can restore the K⁺ uptake capacity in TK2420 bacterial mutant, and can utilize either external KCl or K₂SO₄, indicating that the expression level of KUP11 was positively correlated with the K⁺ uptake of bacterial cells.【Conclusion】Application of K fertilizer favorably promotes peach flower development, improves the K⁺ nutritional status, makes peach flower early to open, and differentially regulated KT/HAK/KUP family genes at different flowering stages. KUP11 transporter possesses the capacity to uptake external K⁺, which may play an important role in peach flowering.

Key words: peach, flowering, potassium, KT/HAK/KUP gene family, potassium homeostasis, peach, flowering, potassium, KT/HAK/KUP gene family, potassium homeostasis

宋志忠，郭绍雷，马瑞娟，俞明亮. KT/HAK/KUP家族基因在桃开花期的表达及对钾肥施用的响应[J]. 中国农业科学, 2015, 48(6): 1177-1185.

SONG Zhi-zhong, GUO Shao-lei, MA Rui-juan, YU Ming-liang. Analysis of Expression of KT/HAK/KUP Family Genes and Their Responses to Potassium Fertilizer Application During Peach Flowering[J]. Scientia Agricultura Sinica, 2015, 48(6): 1177-1185.

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