高羊茅FaFKF1基因克隆、亚细胞定位及节律表达分析

Cloning,subcellular location and rhythmic expression analysis of FaFKF1 gene in Festuca arundinacea

【目的】克隆高羊茅蓝光受体家族基因(FaFKF1),分析其序列特征及亚细胞定位,并检测其在不同光照处理下的节律表达特征,为探索其在成花中的调控机制及高羊茅光周期适应性育种提供理论依据。【方法】采用RACE方法克隆FaFKF1基因cDNA全长序列,对其进行生物信息学分析,并构建pCAMBIA1300-FaFKF1-GFP融合表达载体,通过农杆菌介导转染烟草表皮细胞,观察荧光信号以确定蛋白亚细胞定位情况。同时,利用实时荧光定量PCR检测FaFKF1基因不同光照处理和不同生长阶段的节律表达特征。【结果】克隆获得FaFKF1基因cDNA全长为2266 bp,开放阅读框(ORF)为1881 bp,编码626个氨基酸残基,蛋白分子量为68.89 kD,理论等电点(pI)为5.76,脂肪系数为80.99,不稳定指数为39.81,属于较稳定的亲水性蛋白,亚细胞定位于细胞核。FaFKF1蛋白的二级结构包含α-螺旋(24.92%),无规则卷曲(44.89%)、延伸链(23.16%)和β-转角(7.03%)。FaFKF1蛋白与大豆(NP_001235886.2)、大麦(KAE8795993.1)和二穗短柄草(XP_003577479.1)的氨基酸序列相似性较高,为79.50%~87.68%,且与二穗短柄草FKF1蛋白的亲缘关系最近,其次是大麦和小麦。长日照处理下,FaFKF1基因在苗期、分蘖期、孕穗期和抽穗期的叶片中均有表达,相对表达量变化趋势也较相似,均在ZT8时(即下午16:00)达最高峰,呈现出24 h的昼夜节律,但在孕穗期和抽穗期的相对表达量明显较高。在长日照和短日照基础上施加不同光照处理下,FaFKF1基因均能通过自身调节来适应环境的变化,虽然表达峰出现时间不一致,但整体上能维持24 h的昼夜节律。【结论】FaFKF1基因在细胞核发挥作用,且在不同光照下均呈现节律表达,受光周期的诱导调控。

【Objective】The blue light receptor family gene(FaFKF1)in Festuca arundinacea was cloned,its se-quence characteristics and subcellular localization were analyzed,and its rhythmic expression characteristics under diffe-rent light treatments were detected,so as to provide a theoretical basis for exploring its regulatory mechanism in adult flowers and the adaptive breeding of F.arundinacea optoperiood.【Method】The RACE technology was used to clone the full length cDNA of FaFKF1 gene.Its bioinformatics analysis was conducted.pCAMBIA1300-FaFKF1-GFP fusion ex-pression vector was constructed,infected tobacco epidermal cells by Agrobacterium mediated transfection to observe the fluorescent signal to determine protein subcellular localization.Meanwhile,the rhythmic expression characteristics of dif-ferent light treatments and different growth stages of the FaFKF1 gene were detected using real-time fluorescence quanti-tative PCR.【Result】The sequence analysis results showed that the full-length cDNA(2266 bp)was obtained with a 1881 bp open reading frame(ORF),which encoded a small molecular protein containing 626 amino acids,the relative molecular weight of FaFKF1 protein was about 68.89 kD and its theoretical isoelectric point(pI)was 5.76,and the fat coefficient 80.99,instability index 39.81,which was a stable hydrophilic protein.Subcellular localization results showed that FaFKF1 was located in the nucleus.In the secondary structure of FaFKF1 protein,24.92%of alpha helix,44.98%of random coil,23.61%of extended strand and 7.03%of beta turn were observed.FaFKF1 protein had the high amino acid sequence simi-larity with FKF1 of Glycine max(NP_001235886.2),Hordeum vulgare(KAE8795993.1),and Brachypodium dilatatum(XP_003577479.1),reaching more than 85%.And the genetic distance with B.dilatatum was the shortest,followed by H.vulgare and wheat.Under long-day treatment,FaFKF1 gene was expressed in leaves during seedling,tillering,booting and heading stages,and the relative expression trend was similar,all peaked at ZT8(16:00 PM),showing a circadian rhythm of 24 h,but the relative expression was high in booting and heading stages.Under different light treatments based on long and short light exposure,the FaFKF1 gene could adapt to the environmental changes through autoregulation.Al-though the peak expression occurrence time was inconsistent,the overall circadian rhythm was maintained for 24 h.【Con-clusion】FaFKF1 gene may play an important role in the nucleus,and hasa rhythmic expression subcellular location under different lighting conditions,which is induced and regulated by the photoperiod.