铁对番茄幼苗生长的影响及其柠檬酸转运基因SlFRD3的克隆与分析

发布时间：2018-08-14 17:37

【摘要】：铁是参与植物呼吸作用和光合作用等一系列生命活动的必需微量金属元素,对植物发挥正常生理代谢功能至关重要。由于其在土壤中多以可溶性极低的氧化物形式存在,并不能使植物直接吸收利用,因此植物缺铁是农业中普遍存在的问题。缺铁胁迫不仅阻碍植物生长,影响籽粒中的铁含量的积累,干扰植物对土壤条件的耐受性和适应性,而且它还会通过食物链直接影响到人类自身的铁营养水平。了解铁在植物内的代谢平衡规律是解决植物缺铁问题的基础,解决植物缺铁问题又是改善动物和人类铁营养的关键。番茄是世界各地广泛栽培的一种重要的蔬菜作物。因此,番茄铁分子代谢机制的研究已成为当今植物界的研究热点。本课题研究了不同铁含量的营养液对番茄幼苗生理形态指标的影响,结果表明,在不同的铁浓度供给下,番茄幼苗形态和生理指标差异明显,缺铁处理(OmMEDTA-Fe)会显著抑制番茄幼苗的生长,并且随着胁迫时间增加,抑制生长的效应愈加明显;而高铁(200mMEDTA-Fe)处理的番茄幼苗与正常处理(100mMEDTA-Fe)和缺铁处理相比,在根活、叶绿素含量和丙二醛含量方面表现更佳,整体表现出促进番茄幼苗的生长。本研究通过查询NCBI,根据SlFRD3基因的编码区设计特异性引物,克隆获得鉴定了该基因。本研究利用荧光定量PCR研究了SlFRD 在番茄根、茎、叶、花和果实中中的表达情况,结果显示该基因在番茄根中表达量最高,其次为叶、茎、成熟果(花后38-40d)、花、幼果(花后8-10d)、膨大果(花后28-30d)、青果(花后18-20d);在不同铁浓度下SlFRD3在不同部位随处理时间的表达差异。结果显示:在处理96h内的番茄根中,随着处理时间增加,三种浓度铁处理的番茄根中SlFRD3基因表达量都出现了升高,这可能和根系的生长有关;同时和正常铁处理相比,缺铁胁迫不影响SlFRD3基因的表达,而高铁胁迫则表现出促进SlFRD3基因的表达;在叶中,正常处理随处理时间的增加,表达量差异不显著;缺铁处理在8h内会使SlFRD3基因表达升高,然后随着处理时间增长,表达量有所降低,高铁处理则在32h内缓慢升高,32h到72h又缓慢降低,96h又出现升高。高铁胁迫比缺铁胁迫诱导SlFRD3基因表达量显著增加。本研究对SlFRD3蛋白进行了生物信息学分析,表明其分子式为C2613H4170N652 0703S21,分子量为56641D,等电点为9.19,是一种可溶性蛋白;无信号肽位点,稳定系数为29.38,是稳定蛋白;平均亲水系数为0.652,存在12个跨膜结构域,是跨膜蛋白。含有两个 MatE(PF01554)(158-259,316-475)和一个 Polysacc synt C(PF14667)(212-341)结构域,属于典型的MATE家族。SlFRD3蛋白质的二级结构预测结果为48.76%的α-螺旋,21.9%。的延长链,8.95%的β3-转角,20.38%的随机卷曲。氨基酸序列比对和进化树分析发现番茄SlFRD3与潘娜丽番茄、马铃薯、辣椒、烟草、美花烟草和茸毛烟草的相关蛋白等亲缘关系较近,但与水稻和拟南芥等生物学模式作物亲缘关系较远,氨基酸序列比对不在一个分支,表明番茄SlFRD3蛋白和现在已经研究较多的拟南芥AtFRD3蛋白、水稻OsFRDL1蛋白等在行使柠檬酸木质部加载转运功能的过程可能有些不同。本研究克隆了SlFRD 基因一段长为1831bp的一段启动子片段,构建了PBl121-SlFRD3P融合表达载体。启动子预测分析软件Plantcare分析了顺式作用原件,预测结果表明,有很多重要的顺式作用元件位于SlFRD3基因的这一启动子区。在此序列中除了存在能使转录精确起始的TATA-box,和控制着起始的频率CAAT-box等核心启动子元件外,还存在着2种与胁迫相关的作用元件,分别参与热应激和防御胁迫;6种与激素相关的作用元件,它们是分别是参与茉莉酸、乙烯、赤霉素和水杨酸的相关反应;还有多个与组织特异性相关的元件,如参与和调节胚乳表达的作用元件;以及一些最大激发剂介导的激活元件等,为后续进一步转化鉴定SlFRD3基因的亚细胞定位以及SlFRD3基因启动子调控的研究奠定了基础。
[Abstract]:Iron is an essential trace metal element involved in a series of life activities, such as respiration and photosynthesis, and it is very important for plants to play a normal physiological and metabolic function. Iron deficiency stress not only hinders plant growth, affects iron accumulation in grains, interferes with plant tolerance and adaptability to soil conditions, but also directly affects the iron nutrition level of human beings through the food chain. Understanding the iron metabolism balance in plants is the basis to solve the problem of iron deficiency in plants and solve the problem of planting. The problem of iron deficiency is also the key to improve iron nutrition of animals and humans.Tomato is an important vegetable crop widely cultivated all over the world.Therefore, the study of iron metabolism mechanism of tomato has become a hot topic in the plant field.The effects of different iron content nutrient solution on physiological and morphological indexes of tomato seedlings were studied. The results showed that the morphological and physiological indices of tomato seedlings were significantly different under different iron concentrations. Iron deficiency treatment (OmMEDTA-Fe) could significantly inhibit the growth of tomato seedlings, and the inhibition effect became more obvious with the increase of stress time; while iron deficiency treatment (200 mMEDTA-Fe) could significantly inhibit the growth of tomato seedlings compared with normal treatment (100 mMEDTA-Fe) and iron deficiency treatment (100 mMEDTA-Fe). In this study, the gene was cloned and identified by inquiring NCBI and designing specific primers according to the coding region of SlFRD3 gene. In this study, quantitative PCR was used to study the SlFRD in tomato roots, stems, leaves, flowers and fruits. The results showed that the expression of the gene in tomato root was the highest, followed by leaves, stems, mature fruits (38-40 days after anthesis), flowers, young fruits (8-10 days after anthesis), expanded fruits (28-30 days after anthesis), green fruits (18-20 days after anthesis); the expression of SlFRD3 in different parts of tomato was different under different iron concentrations with different treatment time. In roots, the expression of SlFRD3 gene increased with the increase of treatment time, which may be related to root growth. Compared with normal iron treatment, iron deficiency stress did not affect the expression of SlFRD3 gene, while iron stress promoted the expression of SlFRD3 gene in leaves. The expression of SlFRD3 gene increased within 8 hours after iron deficiency treatment, then decreased with the increase of iron deficiency treatment, increased slowly within 32 hours, decreased slowly from 32 hours to 72 hours, and increased again at 96 hours after iron deficiency treatment. The bioinformatics analysis of SlFRD3 protein showed that its molecular formula was C2613H4170N652 0703S21, its molecular weight was 566441D, its isoelectric point was 9.19, and it was a soluble protein; the signal-free peptide site, its stability coefficient was 29.38, it was a stable protein; the average hydrophilic coefficient was 0.652, there were 12 transmembrane domains, and it was a transmembrane protein. E (PF01554) (158-259,316-475) and a polysacc synt C (PF14667) (212-341) domain belong to the typical MATE family. The secondary structure of SlFRD3 protein was predicted to be 48.76% alpha-helix, 21.9%. The elongated chain, 8.95% beta-3-rotation, 20.38% random curl. Amino acid sequence alignment and evolutionary tree analysis revealed that tomato SlFRD3 and Panari were related to each other. The relative proteins of tomato, potato, pepper, tobacco, American flowering tobacco and pubescent tobacco are close, but they are far from the biological model crops such as rice and Arabidopsis. Amino acid sequence alignment is not a branch, suggesting that tomato SlFRD3 protein and Arabidopsis AtFRD3 protein, rice OsFRDL1 protein have been studied extensively. This study cloned a 1 831 BP promoter fragment of the SlFRD gene and constructed a PBl121-SlFRD3P fusion expression vector. Acting elements are located in the promoter region of the SlFRD3 gene. In addition to TATA-box, which can make transcription start accurately, and core promoter elements such as CAAT-box, which control the initiation frequency, there are also two stress-related elements involved in heat stress and stress resistance, and six hormone-related elements. They are involved in the reactions of jasmonic acid, ethylene, gibberellin and salicylic acid, as well as many tissue-specific elements, such as those involved in and regulating endosperm expression, and some of the largest activator-mediated activating elements, for further transformation to identify the subcellular localization of the SlFRD3 gene and the SlFRD3 group. It laid the foundation for the study of promoter regulation.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S641.2

【相似文献】