【摘要】 【目的】克隆‘早钟6号’枇杷(Eriobotrya japonica‘Zaozhong 6’)的一个质膜水孔蛋白(plasma membrane intrinsic protein,PIP)基因,分析其序列特征,研究接种丛枝菌根(Arbuscular mycorrhizal,AM)真菌对枇杷水分利用效率(water use efficiency,WUE)及对质膜水孔蛋白基因表达模式的影响。【方法】以‘早钟6号’枇杷实生苗为材料,通过盆栽试验,设置2个处理(AM和NM),5个重复,利用光合测定系统对叶片水平上水分利用效率进行测定;采用RT—PCR和RACE技术克隆该基因的cDNA全长,结合生物信息学软件对其序列特征进行分析,利用实时荧光定量PCR(Real time—PCR)分析接种AM真菌后该基因在枇杷叶片和根中的表达模式。【结果】在有效的光合同化时间(7:00—17:00)内,接种AM真菌显著的提高了枇杷的水分利用效率。克隆得到枇杷质膜水孔蛋白基因EjPIP1(GenBank登录号:JX041 627),cDNA全长为1 142 bp,编码区含861 bp,共编码286个氨基酸。氨基酸序列分析表明,EjPIP1具有水孔蛋白家族高度保守的2个NPA(Asn—Pro—Ala,天冬氨酸-脯氨酸-丙氨酸)基序。同源性分析显示,枇杷EjPIP1氨基酸序列与已报道的苹果(Malus domestica)、桃(Prunus persica)和智利草莓(Fragaria chiloensis)等高等植物的氨基酸序列同源性很高,分别为97%、92%和90%。Rea1 time-PCR分析结果证实,EjPIP1在枇杷叶片和根中均有表达,并且叶片中的相对表达量略高于根中。正常供水条件下,接种AM真菌后,该基因在根中表达上调,在叶片中表达下调。【结论】接种AM真菌提高了‘早钟6号’枇杷叶片水平上水分利用效率。从枇杷叶片中克隆得到了一个质膜水孔蛋白类的基因EjPIP1,实时荧光定量PCR分析表明该基因在枇杷叶片和根中表达受AM真菌的影响,该基因的表达模式有利于提高AM植株的水分利用效率。更多还原

【Abstract】 【Objective】The aim of this study was to clone a plasma membrane aquaporin gene EjPIP1 from Eriobotrya japonica variety ’Zaozhong 6’,and to analyze its sequence characteristics,then to study the effects of AM fungi on the PIP expression pattern and water use efficiency(WUE).【Method】A pot experiment with seedlings of loquat cultivar(’Zaozhong 6’),two treatments(AM and NM) and five replicates were conducted.The leaf water use efficiency was measured by using LI-6400photosynthesis determination system.The full-length cDNA of EjPIPl was obtained by the technology of reverse transcription PCR(RT-PCR) and RACE.The cDNA sequence and the deduced amino acid sequence were analyzed by bioinformatics method.The expression patterns of EjPIPl in leaves and roots by AM fungi inoculation were determined by real-time RT-PCR.[Result]In the effective photosynthetic time(7:00—17:00),the water use efficiency of loquat leaves were significantly increased by AM fungi inoculation.Loquat plasma membrane aquaporin gene EjPIPl(GenBank Accessin No.JX041627) was cloned.The full-length cDNA of EjPIPl gene consists of 1 142 bp and contains a 861 bp open reading frame(ORF) encoding 286 amino acid proteins.Two highly conserved NPA(Asn-Pro-Ala,asparagine-proline-alanine) motifs of aquaporin were identified in EjPIPl.The homologue analysis revealed that the amino acids sequence of loquat PIP1 was highly homologous with other species,especially apple(Malus domestica),peach(Prunus persica) and strawberry(Fragaria chiloensis),which was up to 97%,92%and 90%,respectively.Real-time PCR analysis revealed that EjPIPl could be expressed in different loquat tissues including root and leaf,and the relative expression in leaf was higher than that in root.Furthermore,under well-watered condition,the expression of EjPIPl in loquat leaf was significantly down-regulated by AM fungi inoculation,while the expression of EjPIPl in root was up-regulated.[Conclusion]The water use efficiency of loquat leaves was significantly increased by AM fungi inoculation.A plasma membrane intrinsic proteins(PIPs) gene,designated as EjPIPl,was cloned from leaf of loquat(E.japonica ’Zaozhong 6’).And the expression of this corresponding gene in loquat leaves and roots were influenced by AM fungi,which helped to improve water use efficiency of loquat seedlings after being inoculated with AM fungi.更多还原