葡萄铁蛋白基因Ferritin的鉴定、克隆及其在果实发育不同时期对氨基酸铁复合肥喷施的响应

Identification and Cloning of Ferritin Family Genes in Grape and Response to Compound Amino Acid-Iron Spraying During Different Fruit Developmental Stages

【目的】铁蛋白Ferritin在植物生长发育过程中发挥重要作用,其在果树中的生物学功能尚未见报道。克隆葡萄Ferritin家族基因并揭示其在果实发育不同时期的表达模式及其对叶面喷施氨基酸-铁(Fe)复合肥处理的响应差异,可为研究果树铁素营养与代谢的分子机制提供理论依据。【方法】通过同源克隆法从‘马瑟兰’中克隆Ferritin家族基因,利用生物信息学手段分析葡萄Ferritin及其编码蛋白的详细特征;设置叶面喷施氨基酸-Fe复合肥处理,利用荧光实时定量PCR技术分析Ferritin在葡萄果实发育不同时期的表达特征及其对叶面喷施处理的差异响应。【结果】在葡萄基因组中检索并克隆获得4个Ferritin家族基因,命名为VvFer1—VvFer4,分布于第6、8和13号三条染色体上,均含有7个长度不一的内含子,且主要定位于叶绿体和细胞核。本文所选16种植物Ferritin蛋白序列的一致性高达61.48%,系统发育树分析表明同一属的Ferritin同源蛋白如十字花科的拟南芥和芜菁,茄科的烟草和马铃薯,豆科的大豆、落花生和鹰嘴豆,大戟科的橡胶树、木薯和蓖麻,蔷薇科的苹果、桃和草莓,遗传进化关系较近,葡萄VvFer3和茄科同源蛋白遗传关系最近。葡萄VvFer基因在五年生‘马瑟兰’果实发育不同时期、不同组织中的表达水平差异较大,其中,VvFer3在不同组织中的表达量均最高,最大值出现在硬核期至成熟期的果实中,其次是VvFer2和VvFer4。‘马瑟兰’葡萄发育不同时期果实中的Fe含量略有差异,从幼果期开始逐渐增加,并在转色期果实中达到最高值,从转色期到成熟期略有下降,但仍高于幼果期和硬核期果实中的Fe含量,叶面喷施处理显著提高了成熟期果实中的Fe含量、乌头酸酶ACO(aconitase)、硝酸还原酶NIR(nitrite reductase)和琥珀酸脱氢酶SDH(succinate dehydrogenase)的酶活性。VvFer2—4在转录水平易受叶面喷施铁肥的诱导而显著增强,且与葡萄组织/器官分布和果实发育的不同时期密切相关,VvFer2在葡萄发育整个周期仅在果实中对叶面喷施铁肥处理比较敏感,VvFer3在所有组织中仅从幼果期至转色期对叶面喷施铁肥有响应,VvFer4在葡萄发育整个周期的韧皮部和叶片中持续受叶面喷施铁肥的诱导,而在果实中仅从幼果期至转色期对叶面喷施铁肥有响应;VvFer1在本文所选组织中的表达量相对较低,但很均匀,且在转录水平对叶面喷施铁肥没有响应。【结论】从葡萄中克隆并鉴定了4个铁蛋白基因Ferritin,其在葡萄发育不同时期、不同组织中的表达水平差异较大,并在转录水平易受叶面喷施铁肥的诱导;VvFer3在葡萄所有组织中的整体表达量最高(特别是果实中最为突出),且在幼果期至转色期易受铁肥叶面喷施的调控。

【Objective】Ferritin plays an important role in plant growth and development,and its biological function in fruit trees are essentially unknown.Cloning of grape Ferritin family genes and revealing their expression patterns at different fruit developmental stages and their response differences to foliar spraying of amino acid iron(Fe)compound fertilizers could provide a theoretical basis for studying the molecular mechanisms of Fe nutrition and metabolism in fruit trees.【Method】The Ferritin family genes were screened and identified in grape genome by homologous cloning.The detailed characteristics of Ferritin genes and their encoded proteins were analyzed by using bioinformatical methods.The tissue-specific expression patterns of Ferritin family genes during distinct fruit developmental stages and differential response to foliar spraying of amino acid-iron compound fertilizer were determined by carrying out real-time fluorescent quantitative PCR.【Result】In total,4 Ferritin family genes were retrieved and cloned from grape,entitled with VvFer1-VvFer4,which were distributed on No.6,8 and 13 chromosomes,containing 7 introns with different lengths.VvFer proteins were mainly located in chloroplast and nucleus.The amino acid sequence identity of Ferritin homologs from 16 plant species was as high as 61.48%.Phylogenetic tree analysis indicated that Ferritin homologs belonging to the same genus,such as Arabidopsis and turnip of Cruciferae,tobacco and potato of Solanaceae,soybeans,peanuts and chickpeas of Leguminosae,rubber trees,cassava and castor of Euphorbiaceae,and apples,peaches and strawberries of Rosaceae,possessed a closer genetic distance during evolution.Grape VvFer3 was closely clustered with Solanaceae homologs.The expression levels of VvFer genes were different among distinct tissues or organs of 5-year-old Mathelan grape trees during different fruit developmental stages.In particular,the expression level of VvFer3 was the most abundant,and the maximum expression was observed in fruits from hard core stage to mature stage,followed by VvFer2 and VvFer4.The content of Fe Marselan fruits was slightly different among distinct grape developmental stages,which was gradually increased from young fruit stage,and reached the highest value at veraison stage,and then slightly decreased until mature stage,but still higher than that of young fruit stage and hard core stage.Foliar spraying treatment significantly enhanced Fe content of fruits at mature stage,accompanied by ACO(aconitase),NIR(nitrate reductase)and SDH(succinate dehydrogenase).Genes of VvFer2-4 were significantly up-regulated by foliar spraying of amino acid-iron compound fertilizer,which was closely related to distinct grape tissues/organs and different fruit developmental stages.In details,the expression of VvFer2 in fruits was sensitive to foliar spraying treatment during the whole period of grape development.The expressions of VvFer3 in all tested tissues were sensitive to foliar spraying treatment from young fruit stage to veraison stage.The expressions of VvFer4 in phloem and leaves were continuously induced by foliar spraying treatment during the whole period of grape development,whereas in fruits from young fruit stage to veraison stage.The expression of VvFer1 was relatively low,but very uniform,and there was no response to foliar spraying treatment at the transcription level.【Conclusion】Four Ferritin family genes were cloned and identified in grape,whose expression were significantly different among distinct tissues during different fruit developmental stages and were prone to be up-regulated under foliar spraying treatment of amino acid-iron compound fertilizer.The overall expression level of VvFer3 gene was the highest in all tested tissues(especially in fruits)during the whole fruit development stage,and was up-regulated in fruits under foliar spraying treatment from young fruit stage to the verason stage.