【作者】 刘伟；

【导师】 李天红； 王忆；

【作者基本信息】 中国农业大学 ， 果树学， 2017， 博士

【摘要】 我国苹果的栽培面积和产量均居世界首位,但在北方栽培中经常会受到石灰性土壤的影响,出现缺铁黄化情况,导致苹果品质和产量下降,严重制约了苹果产业的发展。因此探究植物尤其是木本果树缺铁应答调控方式显得愈发重要。多年来,人们对植物抗缺铁机理进行了大量研究,从生理生化到功能基因克隆,直至近年来的信号转导方面的研究都在陆续展开。乙烯信号在缺铁响应中的调控作用也有一定研究,然而其通路中重要转录因子ERF家族如何参与缺铁响应则未见报道。本研究中我们以不同缺铁耐受性的苹果砧木为试材,通过缺铁胁迫及外源试剂处理,试图探讨转录因子ERF4和ERF72在缺铁引发的乙烯信号转导中的功能,并比较不同砧木间的差异。主要结果如下:1、缺铁早期小金海棠(Malus xiaojinensis ChengetJiang)和山定子(Malus baccata Borkh)根中都产生乙烯,但随着缺铁时间增加小金海棠乙烯生成量降低,而山定子根中乙烯积累则不断增加。2、外源乙烯试剂处理不同缺铁耐受性苹果砧木的响应方式不同。附加乙烯抑制剂AVG(0μMFe+ 10 μMAVG)可促使小金海棠幼叶黄化情况加重,叶绿素含量显著下降。附加乙烯利(40μMFe+100mg/LACP)并未使得植株表型及叶绿素含量有大幅变化。而外源乙烯的加入对小金海棠的铁吸收指标如根际三价铁还原酶活性,根际pH值,根毛区H+外泌以及根部铁转运蛋白基因IRT1、泌氢重要基因HA2的表达和根叶中活性铁含量都有显著影响,山定子的缺铁应答反应则受乙烯作用不明显。3、通过对小金海棠和山定子的缺铁试验,筛选出转录因子ERF4和ERF72作为重点研究对象。缺铁条件下小金海棠MxERF4和MxERF72前6 d的表达下降,而缺铁延长至9 d表达显著上调,其与正调控缺铁响应的MxEIN3和MxEIL1、MxFIT以及铁吸收相关基因MxIRT1,MxHA2的表达模式相反。利用酵母单杂交验证了 MxERF4的抑制转录激活是通过其氨基酸C末端的EAR-motif(LDLNL)抑制结构域实现的。将MxERF4和MxFIT同时注射烟草时可抑制MxFIT对MxIRT1和MxHA2启动子的激活能力;酵母双杂交和双分子荧光互补(BiFC)试验表明MxERF4与MxFIT和MxIR02可发生互作,进而影响这两个转录因子对MxIRT1和MxHA2的正调控作用。通过VIGS沉默小金海棠MxERF4后发现根中MxIRT1和MxHA2的表达上调,活性铁含量显著增加,H+外排增加,根际酸化。4、研究还发现MxERF72与MxERF4、MxFIT和MxIR02均存在蛋白互作。MxERF72既能直接结合MxHA2启动子,也能通过抑制MxFIT影响MxHA2基因的表达。分别沉默小金海棠和山定子中ERF4和ERF72基因后,缺铁处理下山定子根际pH值比小金海棠根际pH值显著低,干扰ERF4处理组对铁吸收的促进效果更为明显。研究还发现缺铁产生的乙烯能缓解小金海棠叶绿体的解体,MxERF4和MxERF72可影响叶绿素含量。5、另外通过观察同源基因的拟南芥突变体表型、检测生理指标和分子试验,证实了 ERF4和ERF72在缺铁响应中起重要作用,提出缺铁产生的乙烯诱发AtERF4和AtERF72表达,AtERF4通过结合AtIRT1和AtCLH1的启动子负调控缺铁响应,AtERF72负调控AtIRT1、AtHA2和AtCLH1。更多还原

【Abstract】 China’s apple cultivation area and production rank first in the world.In the north of our country,the influence of calcareous soil is often encountered,which leads to the decrease of the quality and yield of apple,and it seriously restricted the development of apple industry.Therefore,it is more and more important to explore the regulation of iron in plants,especially in the fruit trees.Over the years,many researches have carried on the mechanism of plant resistance to iron deficiency,including physiological and biochemical,functional gene cloning,and the research of signal transduction has been carried out.It is also known that ethylene signaling is involved in the regulation of iron deficiency response in plants.However,there is no report on how the ERF family is involved in the response to iron deficiency.In this study,we used the apple rootstocks with different iron tolerance under iron deficiency and exogenous reagent treatment,to investigate the function of transcription factor ERF4 and ERF72 in the ethylene signal transduction induced by iron deficiency and in comparing the differences between different rootstocks.The main results are as follow:Ethylene was produced in roots of Malus xiaojinensis Cheng et Jiang and Malus baccata Borkh under early iron deficiency,but with the increase of iron deficiency time,the ethylene production in M.xiaojinensis root decreased,and the ethylene accumulation of M.baccata root cells increasedThe response patterns in different iron deficiency tolerant apple rootstocks have been showed differently under different exogenous ethylene treatments.The additional ethylene inhibitor AVG(0 μM Fe + 10 μM AVG)can promote M.xiaojinensis chlorosis symptoms to be increased,and the chlorophyll content to be decreased significantly.Additional ethephon(40 μM Fe + 100 mg/L ACP)did not make the phenotype and chlorophyll content of plants to be significantly changed.At the same time,exogenous ethylene made other iron absorption parameters such as root ferric reductase(FCR)activity,rhizosphere pH value,root hair zone H+ velocity,the iron transporter gene IRT1 and hydrogen excretion gene HA2 expression and active Fe content in roots and leaves of M.xiaojinensis to be significantly influenced.But the iron deficiency response of M.baccata was not obvious by ethylene action.Transcription factor ERF4 and ERF72 were selected as the key research object through the iron deficiency test of M.xiaojinensis and M.baccata.The expression of MxERF4 and MxERF72 decreased during the first 6 d under iron deficiency in M.xiaojinensis,and MxERF4 and MxERF72 expression significantly increased in response to Fe deficiency,compared with Fe sufficiency on the ninth day.The expression patterns of genes(MxEIN3,MxEIL1,and MxFIT)which play an important role in positive regulation of iron deficiency and iron uptake related genes(MxIRTl and MxHA2)were opposite to that of MxERF4.The inhibition of transcriptional activation of MxERF4 was realized by the amino acid C terminal EAR-motif(LDLNL)inhibitory domain by yeast one-hybrid.The promoters of MxIRTl and MxHA2 was inhibited in the tobacco leaves by coinfiltrated with agrobacterium containing MxERF4 and MxFIT.Yeast two-hybrid and bimolecular fluorescence complementation(BiFC)assays showed that MxERF4 could interact with MxFIT and MxIR02.Furthermore,the positive regulation effect of these two transcription factors on MxIRT1 and MxHA2 were inhibited.This study displayed that MxIRTl and MxHA2 expression,active iron content and H+ efflux in M.xiaojinensis root were increased significantly after silencing MxERF4 by VIGS.This study also found that MxERF72 could interact with MxERF4,MxFIT and MxIR02.MxERF72 could not only directly bind to MxHA2 promoter,but alsoit inhibited the expression of MxHA2 by MxFIT.ERF4 and ERF72 genes in M.xiaojinensis and M.baccata were silenced by VIGS,rhizosphere pH value in M.baccata was significantly lower than in M.xiaojinensis.The effect of iron absorption in interference ERF4 treatment group was more significant than that of ERF72 treatment group.This study also found that iron deficiency produces ethylene and it can alleviate the disintegrate of M.xiaojinensis chloroplast,while MxERF4 and MxERF72 could effect chlorophyll content.The phenotypic,physiological and molecular studies of Arabidopsis mutants revealed that ERF4 and ERF72 play an important role in the response to iron deficiency.We proposed that ethylene induced by iron deficiency lead to the increase of the expression of AtERF4 and AtERF72.AtERF4 negatively regulated iron deficiency response by combining AtIRT1 and AtCLHl promoter,and AtERF72 negatively regulated AtIRTl,AtHA2 and AtCLH1 expression.更多还原