【作者】 李静；

【导师】 荣廷昭； 卢艳丽； 吴锋锴；

【作者基本信息】 四川农业大学 ， 作物遗传育种， 2021， 硕士

【摘要】 玉米（Zea mays L.）是重要粮食、饲料和工业原料作物之一,其产量和品质受缺磷胁迫的制约。磷（Phosphorus,Pi）是作物吸收利用的三大元素之一,在生物体能量代谢、结构支撑中起着重要作用。由于土壤中的磷有效利用率极低,现已成为限制植物生长发育的主要因子之一。研究发现,植物对生长素和磷（Pi）饥饿的反应紧密相连。低磷条件下生长素受体TIR1基因的表达显著上调,导致AUX/IAA蛋白降解,从而释放ARF7/19,促进拟南芥侧根的生长发育。然而,生长素与磷饥饿（-Pi）反应相关的潜在机制尚不清楚。生长素响应因子（auxin response factor,ARF）是一类受生长素调节的转录因子,在生长素信号传导途径和根系建成中起着重要的作用。研究ZmARF生物学功能及其对缺磷胁迫的响应机制,有助于为生长素与磷饥饿（-Pi）反应之间的联系提供新的证据,并为玉米有效利用Pi提供新的策略。课题组前期通过对缺磷胁迫下ZmARF家族基因初步定量,筛选到若干个关键候选基因ZmARFs响应玉米根系的缺磷胁迫。本研究将重要候选基因之一ZmARF4作为研究对象,克隆了玉米低磷极端自交系中ZmARF4基因全长序列进行比对,探究了其启动子活性差异,同时分析缺磷胁迫下ZmARF4基因的表达模式,并结合拟南芥和玉米的遗传转化实验初步探讨了ZmARF4的生物学功能及其对缺磷胁迫的响应,再利用酵母双杂交文库筛选ZmARF4的互作蛋白,为探究ZmARF4可能参与的生物学调控通路提供基础。具体研究结果如下:1、玉米耐低磷自交系178和低磷敏感自交系9782中ZmARF4的启动子活性存在显著差异,且均响应缺磷胁迫。克隆自交系178和自交系9782中ZmARF4基因全长序列比对发现,序列差异主要在启动子区域。p ZmARF4_178/9782分别与GUS报告基因融合,利用烟草叶片瞬时表达系统和GUS组织化学染色,结果表明耐低磷自交系178的ZmARF4启动子活性显著高于低磷敏感材料9782。组织特异性表达检测显示,pZmARF4₁₇₈-GUS在拟南芥叶脉、叶柄、初生根（成熟区的中柱）中表达。western blot检测发现,与未处理（0 h）相比,随着缺磷胁迫处理时间增加,转基因拟南芥的GUS蛋白积累量逐渐增多。2、ZmARF4在玉米耐低磷自交系178和低磷敏感自交系9782根系中响应缺磷胁迫,表达模式差异显著。正常供磷条件下,ZmARF4在自交系178和9782根系中的表达量均显著高于叶片,且在耐低磷自交系178根系与叶片中的相对表达量均显著高于低磷敏感自交系9782,在根系中的表达丰度差异高达4倍。缺磷胁迫处理后,叶片中ZmARF4的相对表达量维持稳态水平。在耐低磷自交系178根系中,ZmARF4总体呈先上升后下降的表达趋势,而在低磷敏感自交系9782根系中显著上调表达。亚细胞定位结果显示,ZmARF4蛋白在玉米中主要定位在细胞核,细胞质有少量表达。3、ZmARF4具有促进植物根系生长发育的生物学功能,促进了玉米株高和产量的提高。拟南芥arf7 arf19双突突变体丧失了侧根的发生,ZmARF4过表达能部分恢复突变体侧根的发生和伸长。在玉米KN5585中过表达ZmARF4增强了玉米根系的生长发育,株高和穗重都显著高于野生型。4、ZmARF4在低磷条件下促进磷的吸收,并增强了对盐和渗透胁迫的耐受性。在拟南芥Col-0中过表达ZmARF4显著抑制主根的伸长。而在低磷胁迫条件下,拟南芥正常生长受到抑制,表现出侧根增多,根长变短的表型,但过表达ZmARF4的拟南芥株系叶片更绿,根毛更密集,其根直径显著大于Col-0。正常供磷条件下,拟南芥Col-0中的磷含量与ZmARF4过表达株系差异不显著。低磷处理后,过表达株系中磷含量的下调幅度显著低于野生型,磷转运基因AtPht1:4上调幅度极显著高于野生型。同时还发现,盐、渗透胁迫下过表达株系的总根长、根表面积和根尖数均显著高于野生型。5、ZmARF4具有转录激活活性,酵母文库筛选检测到与ZmChc5、ZmILL4互作。酵母双杂交文库筛选与ZmARF4互作结果表明,与ZmARF4互作的部分候选基因功能注释主要与植物的生长发育、防御反应相关。酵母回转验证ZmARF4能与几丁质酶ZmChc5以及IAA氨基酸水解酶ZmILL4互作。克隆ZmChc5和ZmILL4的全长CDS序列,分别构建Ca MV35S::ZmChc5:RFP和Ca MV35S::ZmILL4:RFP的融合表达载体。亚细胞定位显示,ZmChc5与ZmILL4在玉米中主要定位于细胞质。更多还原

【Abstract】 Maize(Zea mays L.)is one of the important food,feed and industrial raw materials crops,and its yield and quality are restricted by P deficiency stress.Phosphorus(Pi)is one of the three elements used by crops,and plays an important role in energy metabolism and structural support of organisms.Phosphorus in soil has become one of the main factors limiting plant growth and development due to its low effective utilization rate.The study found that plant responses to auxin and phosphorus(Pi)starvation are closely linked.The expression of auxin receptor TIR1 gene was significantly upregulated under low P condition,resulting in the degradation of AUX /IAA protein,thereby releasing ARF7/19 and promoting the growth and development of lateral roots of Arabidopsis thaliana.However,the underlying mechanisms by which auxin is associated with phosphorus starvation(-Pi)responses remain unclear.Auxin response factor(ARF)is a kind of transcription factor regulated by auxin,which plays an important role in auxin signal transduction pathway and root formation.The study of the biological function of ZmARF and its response mechanism to P deficiency stress will help to provide new evidence for the link between auxin and P starvation(-Pi)response,and provide new strategies for effective utilization of Pi in maize.Through preliminary quantification of ZmARF family genes under P deficiency stress,several key candidate genes ZmARFs were screened in response to P deficiency stress in maize roots.In this study,ZmARF4,one of the important candidate genes,was used as the research object to clone the full-length sequence of ZmARF4 gene in maize low-phosphorus extreme inbred lines for comparison,to explore the difference in its promoter activity,and to analyze the expression pattern of ZmARF4 gene under phosphorus deficiency stress.In addition,the biological functions of ZmARF4 and its response to phosphorus deficiency stress were preliminarily discussed in combination with the genetic transformation experiments of Arabidopsis thaliana and maize.The yeast two-hybrid library was used to screen the interaction proteins of ZmARF4,providing a basis for exploring the possible biological regulatory pathways that ZmARF4 may be involved in.The results of the study are as follows:1.The promoter activity of ZmARF4 in maize inbred line 178 with low phosphorus tolerance and 9782 with low phosphorus sensitivity was significantly different,and both of them responded to P deficiency stress.The comparison of the full length sequence of ZmARF4 gene in the cloned inbred line 178 and inbred line 9782 showed that the difference was mainly in the promoter region.p ZmARF4178 and p ZmARF49782 were fused with GUS reporter gene,respectively.The results showed that the activity of ZmARF4 promoter in inbred line 178 with low phosphorus tolerance was significantly higher than that in inbred line9782 with low phosphorus sensitivity.Tissue specific expression assay showed that p ZmARF4178/9782-GUS was expressed in Arabidopsis veins,petioles,and primary roots(medial columns in mature regions).Western blot analysis showed that GUS protein accumulation in transgenic Arabidopsis thaliana gradually increased with the increase of phosphorus deficiency treatment time compared with untreated(0 h).2.In response to P deficiency stress,ZmARF4 expression patterns were significantly different between maize inbred line 178 with low P tolerance and 9782 with low P sensitivity.Under normal phosphorus supply conditions,the expression level of ZmARF4 in roots of inbred line 178 and 9782 was significantly higher than that in leaves,and the relative expression level in roots and leaves of inbred line 178 with low phosphorus tolerance was significantly higher than that of inbred line 9782 with low phosphorus sensitivity,and the difference of expression abundance in roots was as high as 9 times.After P deficiency stress,the relative expression level of ZmARF4 in leaves did not change much and maintained a stable level.The overall expression of ZmARF4 was down-regulated in the roots of low phosphorus tolerance inbred line 178,while the expression was significantly up-regulated in the roots of low phosphorus sensitive inbred line 9782.ZmARF4 protein was localized in the nucleus of tobacco mesophyll cells,while mainly localized in the nucleus of maize,with a small amount of expression in the cytoplasm.3.ZmARF4 had the biological function of promoting the growth and development of plant roots,and promoted the increase of maize plant height and yield.ZmARF4 overexpression can partially restore the occurrence and elongation of lateral roots of Arabidopsis thaliana Arabidopsis arf7 arf19 mutants without the occurrence of lateral roots.The overexpression of ZmARF4 in maize KN5585 enhanced the growth and development of maize root system,and the plant height and panicle weight were significantly higher than those of the wild type.4.ZmARF4 promoted P uptake and enhanced tolerance to salt and osmotic stress under low P conditions.Overexpression of ZmARF4 in Arabidopsis Col-0 significantly inhibited taproot elongation.Under low P stress,the normal growth of Arabidopsis thaliana was inhibited,with more lateral roots and shorter root length.However,the Arabidopsis thaliana lines overexpressing ZmARF4 had greener leaves and denser root hairs,and their root diameter was significantly larger than Col-0.Under normal phosphorus supply condition,there was no significant difference in phosphorus content between Arabidopsis Col-0 and ZmARF4 overexpression lines.After low P treatment,the down-regulation of P content in overexpressed lines was significantly lower than that in wild type,and the up-regulation of P transporter At Pht1:4 was significantly higher than that in wild type.It was also found that the total root length,root surface area and root tip number of the overexpressed strains were significantly higher than those of the wild type under salt and osmotic stress.5.ZmARF4 has transcriptional activation activity,and interactions with ZmChc5 and ZmILL4 were detected by yeast library screening.The results of yeast two-hybrid library screening and ZmARF4 interaction showed that most of the functional annotations of candidate genes interacting with ZmARF4 were mainly related to plant growth,development and defense response.Yeast rotation confirmed that ZmARF4 could interact with chitinase ZmChc5 and IAA amino acid hydrolase ZmILL4.The full-length CDS sequences of ZmARF4,ZmChc5 and ZmILL4 were cloned to construct the fusion expression vectors of Ca MV35S::ZmARF4:e GFP,Ca MV35S::ZmChc5:RFP and Ca MV35S:: ZmILL4:RFP,respectively.The protoplast transformation results of ZmChc5 and ZmILL4 showed that the protoplast was mainly localized in the cytoplasm.更多还原