普通白菜CYP79B2同源基因的克隆与表达

doi:10.13560/j.cnki.biotech.bull.1985.2022-0341

摘要/Abstract

摘要：

CYP79B是拟南芥参与生长素合成的关键基因，克隆白菜CYP79B2的同源基因BrcCYP79B2-2，并分析该基因的组织表达情况，为研究普通白菜开花调控的基因功能验证提供理论依据。采用RT-PCR方法克隆普通白菜CYP79B2的同源基因BrcCYP79B2-2，并对其进行生物信息学分析，利用RT-qPCR方法研究BrcCYP79B2-2在普通白菜不同组织中的表达情况。结果表明，BrcCYP79B2-2的CDS全长为1 554 bp，编码517个氨基酸。经氨基酸序列多重比对发现，BrcCYP79B2-2氨基酸序列与大白菜的同源性为99.42%，BrcCYP79B2-2在白菜不同变种中高度保守。RT-qPCR分析表明，该基因在不同组织中均有表达，在根中表达量最高，花蕾中较低，而在茎中的表达量最低。经低温处理后，BrcCYP79B2-2在茎尖的表达量降低。另外，BrcCYP79B2-2在不同时期的表达量也不同，在营养生长期表达量较高，但是在花芽即将分化时表达量降低。BrcCYP79B2-2对低温处理有响应，且在不同组织中的表达差异显著，推测该基因的表达可能影响花芽分化。

关键词: 普通白菜, 生长素, BrcCYP79B2-2, 基因克隆, 表达分析

Abstract:

CYP79B is a key gene involved in auxin synthesis in Arabidopsis thaliana. Cloning the homologous gene BrcCYP79B2-2 of CYP79B2 and analyzing the tissue expression of this gene may provide a theoretical basis for the functional verification of genes regulating flowering in pak choi（Brassica rapa ssp. chinensis）. Here，RT-PCR was used to clone the BrcCYP79B2-2 of pakchoi’s CYP79B2 and the gene was analyzed by bioinformatics. RT-qPCR was employed to study the expression of BrcCYP79B2-2 in different tissues of pakchoi. The results showed that the CDS of BrcCYP79B2-2 was 1 554 bp in length，encoding 517 amino acids. The amino acid sequence of BrcCYP79B2-2 protein was of 99.42% homology with that of Brassica pekinensis，suggesting that BrcCYP79B2-2 was highly conserved in different varieties of B. pekinensis. RT-qPCR analysis revealed that the gene was expressed in different tissues，with the highest expression in the roots，lower in the buds，and lowest in stems. The expression of BrcCYP79B2-2 in the stem tip after low-temperature treatment decreased. Besides，the expressions of BrcCYP79B2-2 varied among different growth stages，higher in the vegetative growth stage but lower in the critical stage of flower bud differentiation. The expression of BrcCYP79B2-2 responded to low temperature and significantly varied in different tissues. It is speculated that the gene may affect flower bud differentiation.

Key words: Brassica rapa ssp. chinensis, auxin, BrcCYP79B2-2, gene cloning, expression analysis

胡琪, 侯玉翔, 李璿, 李梅兰. 普通白菜CYP79B2同源基因的克隆与表达[J]. 生物技术通报, 2022, 38(12): 168-174.

HU Qi, HOU Yu-xiang, LI Rui, LI Mei-lan. Cloning and Expression of CYP79B2 Homologous Genes in Brassica rapa ssp. chinensis[J]. Biotechnology Bulletin, 2022, 38(12): 168-174.

图/表 5

图1 BrcCYP79B2-2的扩增电泳图

Fig. 1 Amplified electrophoresis of BrcCYP79B2-2

图2 不同物种CYP79B2-2氨基酸序列的多重比对

Fig. 2 Multiple alignment of CYP79B2-2 amino acid sequences from different species

图3 不同物种CYP79B2-2同源蛋白的系统进化树

Fig. 3 Phylogenetic tree of CYP79B2-2 homologous prote-ins from different species

图4 BrcCYP79B2-2蛋白质三级结构预测

Fig. 4 Protein tertiary structure prediction of BrcCYP-79B2-2

图5 BrcCYP79B2-2在不同组织的表达和低温处理后不同时期茎尖部位的表达 V0：低温处理后0 d（DAV0）；V10：营养生长期（DAV10）；V15：花芽分化临界期（S0）；V16：花芽分化期（S1）

Fig. 5 Expressions of BrcCYP79B2-2 in different tissues and in stem tip in different stages after low te-mperature treatment V0：0 d after vernalization（DAV0）；V10：10 d after vernalization（DAV10）；V15：critical stage of flower bud differentiation（S0）；V16：flower bud differentiation stage（S1）

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