结球甘蓝BoLH01和BoLH02基因的克隆与表达

doi:10.3724/SP.J.1006.2014.01371

摘要/Abstract

摘要：

bHLH类转录因子在植物叶片形态建成和发育过程中发挥重要的生理功能。本文通过结球甘蓝转录组数据分析，筛选出莲座期和结球期的茎尖间与叶片间均显著差异表达的2个bHLH类转录因子，命名为BoLH01和BoLH02基因，获得了2个基因的编码序列，其中BoLH01基因CDS全长为966 bp、编码321个氨基酸；BoLH02基因CDS全长870 bp、编码289个氨基酸；序列分析表明BoLH01和BoLH02分别与拟南芥AtbHLH18和AtbHLH19氨基酸同源相似性最高，达81%和74%，均具典型bHLH结构域特点和完全保守的13E-16R和9H-13E-17R以及Leu23对应的关键氨基酸位点；分子进化上BoLH01、BoLH02与AtTCP2/3/4/10/24亲缘关系较近，与AtTCP9/11/14/15较远；转录组和荧光定量PCR分析表明BoLH01和BoLH02在平展的莲座叶中表达量较低，在卷曲的球叶中高量表达；序列分析BoHB7、BoHB12和BoILL6的ATG上游均包含能被bHLH功能域识别的E-box序列，对BoHB7、BoHB12和BoILL6荧光定量PCR分析表明, 三者与BoLH01和BoLH02在不同时期叶片中表达量的变化趋势完全一致；说明BoLH01和BoLH02可能通过正向调控BoHB7、BoHB12、BoILL6基因的表达来参与甘蓝叶片卷曲的调控。

关键词: 甘蓝, 叶片卷曲, 基因克隆, 表达分析

Abstract:

Basic helix-loop-helix (bHLH) transcription factors play an important role in the leaf morphogenesis and leaf development. In this study, through transcriptome analyses, two bHLH transcription factor genes, differentially expressed in leaves and shoot tips between cabbage rosette stage and heading stage, were screened, named BoLH01 and BoLH02 genes. The cDNA sequences of BoLH01and BoLH02 were cloned from cabbage (Brasscia oleracea var. Capitata L). The CDS of BoLH01 was 966 bp, which encoded 321 amino acids, and the CDS of BoLH02 was 870 bp, which encoded 289 amino acids. Sequence analysis showed that the similarity of BoLH01 and BoLH02 with AtbHLH18 and AtbHLH19 was 81% and 74% respectively. They contained the typical bHLH domains, conservative 13E-16R, 9E-13R-17H and Leu23 corresponding key amino acid sites. Phylogenetic tree analysis displayed that BoLH01and BoLH02 were closest to AtTCP2/3/4/10/24, and had no close genetic relationship with AtTCP9/11/14/15. Moreover, the transcriptome and real-time fluorescence quantitative PCR analysis indicated that the relative expression levels of BoLH01 and BoLH02 were low in rosette leaves, and high in heading stage. Sequence analysis showed that the ATG upstream of BoHB7, BoHB12 and BoILL6 contained the E-box motif, and those motifs could been recognized by bHLH functional domains. The real-time fluorescence quantitative PCR analysis indicated that the variation in expression levels of BoHB7, BoHB12 and BoILL6 was similar with that of BoLH01 and BoLH02 in leaves (1 cm). These results meant that BoLH01 and BoLH02 maybe participate in the regulation of cabbage leaf curl by positively regulating BoHB7, BoHB12, BoILL6.

Key words: Brassica oleracea L., Leaf curl, Gene cloning, Expression analysis

张林成,高启国,蒲全明,任雪松,刘豫东,朱利泉,王小佳. 结球甘蓝BoLH01和BoLH02基因的克隆与表达[J]. 作物学报, 2014, 40(08): 1371-1379.

ZHANG Lin-Cheng,GAO Qi-Guo,PU Quan-Ming,REN Xue-Song,LIU Yu-Dong,ZHU Li-Quan,WANG Xiao-Jia. Cloning and Expression Analysis of BoLH01and BoLH02 Genes in Cabbage[J]. Acta Agron Sin, 2014, 40(08): 1371-1379.

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