摘要：

百合鳞茎膨大对百合栽培与育种具有重要意义,PLETHORA(PLT)转录因子在植物器官膨大中起关键调控作用。以穿梭百合(Tresor)为材料,探究LaPLT5基因在其鳞茎膨大过程中的作用机制。通过分析穿梭百合小鳞茎S1~S5时期的转录组数据,筛选出可能参与鳞茎膨大的LaPLT5基因,利用克隆技术从鳞茎cDNA中克隆该基因,结合生物信息学手段预测其编码蛋白质的结构特征,采用qRT-PCR技术分析LaPLT5在鳞茎膨大过程中的表达模式,通过病毒诱导的基因沉默(VIGS)及瞬时过表达技术验证该基因在鳞茎膨大中的功能。结果表明,LaPLT5基因全长1 500 bp,编码499个氨基酸,其中39个氨基酸残基带有正电荷,38个氨基酸残基带有负电荷,蛋白质相对分子质量为53 754.25,不稳定系数为40.77,蛋白亲水性平均值为0.56,为不稳定的疏水性蛋白。qRT-PCR检测显示,LaPLT5在穿梭百合鳞茎的中层鳞片中表达量最高,小鳞茎中表达量次之,且在小鳞茎膨大的S2~S4时期表达量持续升高。VIGS沉默LaPLT5后,小鳞茎的直径、周径和质量均极显著高于对照组,而瞬时过表达LaPLT5的结果则与之相反,说明LaPLT5负调控穿梭百合小鳞茎的膨大。基于LaPLT5沉默后百合小鳞茎细胞数目增多但面积变小的表型,qRT-PCR分析显示,细胞壁松弛相关基因LaPME34、LaXTH1和LaXTH6表达极显著下调;而细胞分裂的正向调控基因LaEBP1和LaANT1表达极显著上调,负调控基因LaCKX4表达极显著下调,表明LaPLT5可能通过协调细胞壁重塑和细胞分裂相关基因的表达,共同调控小鳞茎细胞的膨大与分裂。综上,LaPLT5参与穿梭百合小鳞茎的膨大过程,且对小鳞茎膨大起负调控作用。

关键词: 百合, LaPLT5, 功能分析, 鳞茎, 膨大

Abstract:

Bulblet enlargement is of significant importance for lily cultivation and breeding.The PLETHORA(PLT)transcription factors play a key regulatory role in plant organ expansion.This study used Lilium Tresor as experimental material to investigate the mechanism of the LaPLT5 gene during its bulblet enlargement process.By analyzing transcriptome data from small bulblets at stages S1 to S5 of Lilium Tresor,the LaPLT5 gene,which is potentially involved in bulblet enlargement,was identified.The gene was cloned from bulblet cDNA using molecular cloning techniques,and the structural characteristics of its encoded protein were predicted through bioinformatics analysis.The expression pattern of LaPLT5 during bulblet enlargement was examined using qRT-PCR,and its function was validated via virus-induced gene silencing(VIGS)and transient overexpression assays.The results showed that the LaPLT5 gene had a full length of 1 500 bp,encoding 499 amino acids,including 39 positively charged residues and 38 negatively charged residues.The protein had a relative molecular weight of 53 754.25,an instability index of 40.77,and an average hydrophilicity value of 0.56,indicating that it is an unstable hydrophobic protein.qRT-PCR analysis revealed that LaPLT5 expression was highest in the middle scale layers of Lilium Tresor bulblets,followed by that in small bulblets,and its expression level continuously increased from stages S2 to S4 during the enlargement of small bulblets.After VIGS-mediated silencing of LaPLT5,the diameter,circumference,and weight of small bulblets were highly significantly greater than those in the control group,whereas transient overexpression of LaPLT5 yielded opposite results.This indicated that LaPLT5 negatively regulated the enlargement of small bulblets in Lilium Tresor.Based on the phenotype of increased cell number but reduced cell area in small bulblets after LaPLT5 silencing,qRT-PCR analysis showed that the expression of cell wall relaxation-related genes,LaPME34,LaXTH1 and LaXTH6,was highly significantly downregulated.Meanwhile,the positive cell wall regulatory genes LaEBP1 and LaANT1 were highly significantly upregulated, whereas the negative regulatory gene LaCKX4 was highly significantly downregulated.These findings suggested that LaPLT5 likely regulated the expansion and division of small bulblet cells by coordinating the expression of genes involved in cell wall remodeling and cell division.In summary,LaPLT5 participates in the enlargement of small bulblets in Lilium Tresor and functions as a negative regulator of this process.

Key words: Lily, LaPLT5, Function analysis, Bulblet, Dilated

中图分类号: 

• S682.2