陆地棉漆酶基因家族成员表达模式分析

Relative expression patterns of laccase gene family members in upland Gossypium hirsutum L.

漆酶是铜蓝氧化酶蛋白家族的一员,在植物木质素合成和提高植株抵御胁迫能力等方面发挥着重要作用。本研究从陆地棉基因组中鉴定到104个漆酶基因(GhLAC)家族成员,进行了系统进化树和组织表达图谱的构建,并随机选取了20个基因进行荧光定量PCR分析,验证了表达热图的结果。为进一步探索漆酶在棉花中担当的角色,本研究采用启动子-GUS融合载体转化拟南芥,通过转基因拟南芥不同发育过程不同组织GUS染色结果,分析了陆地棉漆酶基因家族6个成员(GhLAC12A、GhLAC14A、GhLAC20A、GhLAC25D、GhLAC59D、GhLAC63D)的精细表达模式。为探究漆酶在逆境中发挥的作用,对该6个漆酶基因进行了切割和刺洞2种创伤胁迫诱导表达分析,并利用2个棉花品系‘84021’(高温耐受型)和‘H05’(高温敏感型)在常温和高温胁迫条件下不同时期的花药进行相应基因的荧光定量PCR分析。研究结果表明,随机挑选的20个基因在根、茎、叶、花瓣、花药和柱头6个组织中差异表达,大多数基因的表达与转录组结果一致。6个漆酶基因的启动子能够不同程度地驱动GUS基因在种子萌发时期、二叶期、四叶期表达;创伤处理结果显示GhLAC12A、GhLAC14A的启动子受创伤诱导后驱动GUS蛋白在叶片中表达的能力显著提高,暗示着这2个基因可能参与创伤胁迫响应。6个GhLACs基因在棉花耐高温品系‘84021’的四分体时期和花药开裂期高温胁迫后都有表达量显著下调的趋势,推测GhLACs基因可能负调控陆地棉花药高温耐受性。本研究结果为进一步探索漆酶家族基因功能提供了参考。

Laccase,a member of the blue copper oxidase protein family,plays an important role in plant lignin synthesis and improving plant resistance to stress.In this study,104 members of the Laccase gene(GhLAC)family were identified from the upland cotton genome.Phylogenetic tree and tissue expression map were constructed.Twenty genes were randomly selected for qRT-PCR analysis to verify the results of expression heat map.To further explore the role of laccase in cotton,promoter-GUS fusion vectors were transformed into Arabidopsis thaliana.The detailed expression patterns of six members of the Laccase gene family(GhLAC12A,GhLAC14A,GhLAC20A,GhLAC25D,GhLAC59D,and GhLAC63D)were studied by GUS staining in different tissues during different developmental period of transgenic Arabidopsis thaliana.To explore the role of laccase in stress,the expression of the six laccase genes was analyzed by cutting and piercing,and the corresponding genes were analyzed by qRT-PCR using the anther of two cotton strains‘84021’(high temperature tolerant)and‘H05’(high temperature sensitive)at different stages under normal and high temperature conditions.The results showed that 20 randomly selected genes were differentially expressed in six tissues of root,stem,leaf,petal,anther,and stigma,and the relative expression levels of most genes were consistent with the transcriptome data.The promoter of six laccase genes could drive GUS gene expression in different levels at germination,two-leaf,and four-leaf stages.The trauma treatment indicated that the promoter of GhLAC12A and GhLAC14A significantly improved the ability to drive GUS protein expression in leaves after trauma induction,suggesting that the two genes might be involved in traumatic stress response.In addition,the relative expression levels of the six GhLACs genes were significantly down-regulated after high temperature stress at the tetrad stage and anther dehiscence stage of cotton strain‘84021’,suggesting GhLACs gene might negatively regulate the high temperature tolerance of cotton anthers.The results of this study provide the reference for further exploring the function of laccase family genes.