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bZIP44基因在植物缺铁响应中的作用研究

The Role of bZIP44 Gene in Plant Response to Iron Deficiency

【作者】 王媛媛

【导师】 曹树青;

【作者基本信息】 合肥工业大学 , 植物学, 2019, 硕士

【摘要】 铁是人类、动物和植物生长所必需的微量元素,铁在土壤中的溶解度较低,尤其在碱性土壤中的含量更低,但是全球约三分之一的土壤都是碱性土壤,所以植物缺铁问题是全球性的。植物是人类和动物主要的膳食来源,植物缺铁导致人类和动物无法摄取到足够的铁元素,正常的生理活动无法完成,容易导致抵抗力的下降,增加缺铁性贫血的发病率。本论文主要研究拟南芥bZIP44转录因子在植物缺铁胁迫响应中的作用,以期为分子育种改善作物矿质营养提供科学依据。主要研究结果如下:(1)对bZIP44基因的组织特异性表达进行分析,发现bZIP44基因在拟南芥花中的表达量最高。在缺铁胁迫下,bZIP44基因被诱导表达,推测bZIP44基因可能参与植物缺铁胁迫应答反应的调节。(2)鉴定了bzip44纯合突变体植株。对其表型分析发现,在正常情况下,bzip44突变体和野生型生长状况无明显差异。然而,在缺铁胁迫下,与野生型相比,bzip44突变体表现更加敏感,表明bZIP44基因可能正向调节植物缺铁胁迫应答。(3)对野生型和bzip44突变体根和茎中的铁含量及其根茎比例进行分析,结果表明,在正常生长条件下,野生型和bzip44-1突变体根中的铁含量无显著差异,而野生型茎中的铁含量明显高于bzip44-1突变体;在缺铁胁迫下,野生型和bzip44-1突变体根中铁含量均降低,但bzip44-1突变体根中铁含量较野生型降低更多;然而,在茎中,野生型铁含量较正常条件升高,但bzip44-1突变体茎中的铁含量较正常条件无显著性差异;在缺铁条件下,野生型和bzip44-1突变体铁含量根茎比值均升高,但野生型植株升高幅度更大,表明bZIP44基因可能通过调节铁转运相关基因表达来调控铁离子从根部向茎部运转,进而调节植物对缺铁胁迫响应。(4)对缺铁胁迫相关的基因表达分析,发现在缺铁胁迫下,bzip44突变体中,编码烟酰胺合成酶的NAS2、NAS4基因和转录因子MYB10基因转录水平显著低于野生型,而两者的FIT1、FRO2和bHLH38基因的转录水平无显著差异,表明bZIP44基因可能通过调节NAS2、NAS4和MYB10基因的表达,来调控植物缺铁耐受性。(5)进一步通过烟草瞬时表达实验,证明bZIP44基因可激活NAS2及MYB10基因的转录,表明bZIP44基因可能直接调节NAS2和MYB10基因的表达来调控植物缺铁响应。(6)为进一步研究bZIP44在植物缺铁胁迫响应中的作用,构建了bZIP44基因过表达载体:克隆拟南芥bZIP44基因,运用基因重组技术,获得bZIP44基因过表达阳性植株,发现在相同生长时期,bZIP44基因过表达植株长势弱于野生型。(7)在缺铁培养基上,bZIP44基因过表达植株表现出耐受表型,叶片叶绿素含量显著高于野生型,表明bZIP44基因在调控植物缺铁耐受中起重要作用。综上所述,bZIP44基因参与植物缺铁胁迫应答,其调控机制可能是可能直接调节NAS2和MYB10的表达基因的表达,进而提高植物在缺铁胁迫下的耐受能力。

【Abstract】 Iron is an essential micronutrients for the human,animal and plant.It is noteworthy that aproximately one-third soil is alkalescent,which contains lower iron concentration.Plants are the main dietary sources of human and animal.Therefore,iron deficiency in plants has tremendously impacted on human health so that normal physiological activities cannot be completed,which easily leads to the decline of resistance and the increase of the incidence of iron-deficiency anemia.Therefore,it is a golabl threat urged to be solved for us.This paper mainly studies the role of the bZIP44 transcription factor in response to the iron deficiency stress in Arabidopsis thaliana,and will provide scientific basis for molecular genetic breeding to improve crop mineral nutrion content.The main results are as follows:(1)Pattern of bZIP44 tissue expression was analyzed.It was found that the transcript of bZIP44 was the highest in flowers of Arabidopsis thaliana.Expression of bZIP44 gene was induced by iron deficiency,suggesting that bZIP44 gene might be involved in the regulation of plant response to iron deficiency.(2)The bzip44 mutant homozygous plants were identified.The phenotype analysis showed that there was no significant difference between the bzip44 mutant and the wild type under normal condition.However,under iron deficiency,the bzip44 mutant was more sensitive than the wild type.This implyed that the bZIP44 gene positively regulates the iron deficiency stress response in plants.(3)Iron contents in roots and shoots were analyzed in wild-type and bzip44 mutant plants.It was found that under the normal condition,in roots,there was no significant difference in the iron content bewteen the wild type and bzip44-1 mutant,while in shoots,the iron content was significantly higher in the wild type than in bzip44-1 mutant.Under iron deficiency,the root iron contents in root and shoot were decreased in both the wild type and the bzip44-1 mutant,but the iron content in root of the bzip44-1 mutant was significantly lower than that of the wild type;however,in shoot,the wild-type iron content was higher than that in normal growth conditions,but the iron content in the bzip44-1 mutant shoot is similar to that under the normal condition.Under the iron deficiency stress,the ratios of root and shoot of iron content in the wild type and the bzip44-1 mutant were increased,but it was higher in the wild type than the bzip44-1 mutant,suggseting that bZIP44 gene might regulate the iron ion tansport from the root to shoot by regulating the expression of iron transport-related genes,thereby modulating the plant response to the iron deficiency stress.(4)Based on the analysis of gene expression related to iron deficiency,it was found that under iron deficiency,in bzip44 mutant,the transcription level of NAS2 and NAS4 genes encoding nicotinamide synthase and the MYB10 gene encoding transcription factor were significantly lower than those of the wild type,and there was no significant difference in the transcription levels of the FIT1,FRO2 and bHLH38 genes between the wild type and the bzip44-1 mutant,suggesting that the bZIP44 gene may regulate plant iron deficiency tolerance by regulating the expression of the NAS2,NAS4 and MYB10 genes.(5)Through the tobacco transient expression assays,it was found that bZIP44 activates the transcription of the NAS2 and MYB10 gene,suggesting that bZIP44 gene regulates the response to iron deficiency by regulating the expression of NAS2 and MYB10.(6)To further determine the role of bZIP44 in regulating plant iron deficiency response,a 35S:bZIP44 overexpression vector was constructed: the Arabidopsis thaliana bZIP44 gene was cloned,and the bZIP44 gene overexpression plants were obtained by gene recombination technology,and it was found that in the normal growth period,the bZIP44-overexpressing plants grew weaker than the wild type.(7)On the iron-deficient medium,the bZIP44-overexpressing plants showed a tolerant phenotype,and its the chlorophyll content is higher than that in the wild-type,indicating that bZIP44 gene played an important role in the regulation of iron deficiency tolerance in plants.In summary,bZIP44 gene regulates the response to iron deficiency stress in plants by activating NAS2 and MYB10 gene expression,and thereby improves the tolerance of plants to iron deficiency.

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