摘要
铁是植物生长发育必需的一种微量元素,但土壤中植物可直接吸收利用的铁非常有限。缺铁使作物生长受限,进而影响人类膳食健康。植物体能通过调节一系列基因表达的变化来响应缺铁,但目前对该调控系统的研究仍不完善。CYP82C4(At4g31940)是拟南芥中一个强烈响应缺铁的基因,本研究将该基因启动子连接荧光素酶报告基因LUC2并转化拟南芥,进一步通过T-DNA的随机插入得到一个响应缺铁信号的突变体库。通过筛选该突变体库,我们得到一个强烈响应缺铁信号的突变体L22-8。和野生型相比,正常情况下L22-8地上部和地下部内源CYP82C4的表达量均显著增高,缺铁处理时其地上部表达量仍高于野生型,而地下部则不明显。定量结果显示FIT,b HLH38和b HLH39等植物铁代谢关键调控因子的表达发生了显著变化,但植株总铁、磷、锌的含量较野生型并没有显著区别,表明该T-DNA的插入虽影响了植株对缺铁胁迫的响应,但并不直接作用于植株对铁的吸收、转运上。反向PCR分析发现L22-8的T-DNA插入位点位于At3g51950和At3g51960之间,且这两个基因的转录表达在正常生长条件下均略低于Col-0。基因互补实验发现仅有At3g51960能部分互补L22-8的荧光信号,表明At3g51960基因的表达影响了CYP82C4对缺铁胁迫的响应。本研究进一步扩展了植物吸收利用铁的分子调控网络,为分子育种工作提供了指导。
Iron is an essential nutrient for plant growth, but also a limiting factor for crop production due to its low solubility and availability in soil. The expression of a variety of genes would be induced under iron deficiency, while to many of these genes, the mechanisms remains unclear. CYP82C4（At4g31940） is one of the iron starvation induced gene and regulated by FIT in Arabidopsis. In this study, the promotor of CYP82C4 was linked to Luciferase to screen the T-DNA insertion mutant library for genes in response to iron deficiency in Arabidopsis. After screening, a line L22-8 was identified to remarkably intensify the fluorescence of CYP82C4 in both iron sufficient and deficient conditions. Real time qPCR analysis showed that the expression levels of endogenous CYP82C4 was increased in root and shoot under normal growth condition, and in shoot under iron deficiency condition, but not the shoot in shortage of iron. The expression of several Fe-marker genes, such as FIT, bHLH38 and bHLH39, et al., were also significantly changed. These results indicated that the T-DNA insertion of L22-8 affected the molecular response of Arabidopsis to iron deficiency stress and CYP82C4 may play an important role in the formation of heterodimer of FIT withbHLH38 and bHLH39. The T-DNA insertion site of the L22-8 strain was detected to be located between At3g51950 and At3g51960 by reverse PCR, and the transcriptional expression of these two genes was slightly lower in mutant than Col-0 under normal conditions. Complementary analysis confirmed that At3g51960 could partially recover the L22-8 fluorescence signal, suggesting its role in regulating the response of CYP82C4 to iron deficiency stress. In addition, the contents of total Fe, P and Zn in shoot of L22-8 had no differences compared with Col-0, indicating that there maybe exsit some other interactive genes of CYP82C4 in Arabidopsis. Our results further extend the molecular regulation network of iron uptake and utilization in plant, and provide guidance for molecular breeding.
作者
王立赛
闫明科
王晗
沈仁芳
兰平
WANG Lisai;YAN Mingke;WANG Han;SHEN Renfang;LAN Ping(State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China;University of Chinese Academy of Sciences, Beijing 100049, China)
出处
《土壤》
CAS
CSCD
北大核心
2018年第3期476-484,共9页
Soils
基金
国家重点研发计划项目(2016YFD0200308)
国家重点基础研究发展计划(973计划)项目(2015CB150501)资助
关键词
土壤
缺铁胁迫
突变体鉴定
分子调控
Soil
Iron-deficiency stress
Mutant characterization
Molecular regulation
