普通白菜CYP同源基因克隆与表达分析

doi:10.7668/hbnxb.20193182

摘要/Abstract

摘要：

CYP79B2是拟南芥中调控生长素合成的有关基因,通过克隆普通白菜CYP79B2的同源基因BrcCYP79B2-1,分析其在不同组织及时期的表达情况,研究其对普通白菜春化开花的调控作用机理,旨在为后续普通白菜生长素编码基因的功能验证奠定理论基础。通过qRT-PCR方法从普通白菜中克隆到CYP79B2的同源基因,命名为BrcCYP79B2-1,利用生物信息学方法对其蛋白的结构、理化性质和亲缘关系进行分析,并利用qRT-PCR方法分析其在普通白菜中不同组织和生育期的表达量。结果表明,BrcCYP79B2-1基因编码序列全长为1 623 bp,编码540个氨基酸;对其蛋白质的理化分析结果表明,蛋白质分子质量为60.849 73 ku,理论等电点为8.71。与其他物种的氨基酸序列进行比对发现,BrcCYP79B2-1在十字花科植物中高度保守,且与芜菁同源性最高,达99.52%;采用qRT-PCR分析普通白菜不同器官的表达量发现,BrcCYP79B2-1在根中的表达量最高,在叶中次之,而在花蕾中的表达量最低;分析幼苗在低温处理0,10,15,16 d的表达发现,BrcCYP79B2-1在低温处理第10天,即营养生长期的表达量达到峰值,而在花芽分化期的表达量有所降低,说明该基因的表达与低温春化有关,可以影响花芽分化。

关键词: 普通白菜, BrcCYP79B2-1, 生物信息学, 基因克隆, 表达分析, 开花

Abstract:

CYP79B2 is a related gene that regulates auxin synthesis in Arabidopsis.By cloning the homologous gene BrcCYP79B2-1 of CYP79B2 in B.rapa ssp. cninensis,analyzing its expression in different tissues and periods,and studying its regulatory mechanism on vernal flowering in B.rapa ssp. cninensis,to lay a theoretical foundation for the subsequent functional verification of the auxin-encoding gene in B.rapa ssp. cninensis.The homologous gene of CYP79B2 was cloned from B.rapa ssp. cninensis by qRT-PCR and named as BrcCYP79B2-1.The structure,physicochemical properties and relationship of its protein were analyzed by bioinformatics method,and its protein was analyzed by qRT-PCR method.Expression levels in different tissues and growth stages in B.rapa ssp. cninensis.And its expression in different tissues and growth stages of B.rapa ssp. cninensis was analyzed by qRT-PCR method.The results showed that the full-length coding sequence of BrcCYP79B2-1 gene was 1 623 bp,encoding 540 amino acids.The physicochemical analysis of the protein showed that the molecular mass of the protein was 60.849 73 ku,and the theoretical isoelectric point was 8.71.Compared with the amino acid sequences of other species,it was found that BrcCYP79B2-1 was highly conserved in cruciferous plants,and had the highest homology with turnip,up to 99.52%;the expression levels of different organs of B.rapa ssp. cninensis were analyzed by qRT-PCR, and it was found that the expression level of BrcCYP79B2-1 was the highest in roots, followed by leaves, and the lowest in flower buds.The expression of BrcCYP79B2-1 in seedlings after 0,10,15 and 16 days of low temperature treatment showed that the expresion of BrcCYP79B2-1 reached its peak on the 10th day of low temperature treatment, that is, the vegetative growth period, while the expression of BrcCYP79B2-1 decreased during flower bud differentiation, which indicated that the expression of BrcCYP79B2-1 was related to low temperature vernalization and could affect flower bud differentiation.

Key words: B.rapa ssp. chinensis Makino, BrcCYP79B2-1, Bioinformatics, Gene cloning, Expression analysis, Flowering

侯瑞泽, 侯玉翔, 许小勇, 李璿, 李梅兰. 普通白菜CYP同源基因克隆与表达分析[J]. 华北农学报, 2022, 37(5): 45-51. doi: 10.7668/hbnxb.20193182.

HOU Ruize, HOU Yuxiang, XU Xiaoyong, LI Xuan, LI Meilan. Cloning and Expression Analysis of CYP Homologous Genes in Brassica rapa ssp. chinensis[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(5): 45-51. doi: 10.7668/hbnxb.20193182.

http://www.hbnxb.net/CN/Y2022/V37/I5/45

图/表 8

图1 BrcCYP79B2-1琼脂糖凝胶电泳

Fig.1 Agarose gel electrophoresis of the PCR products of BrcCYP79B2-1 M.DNA Marker;1—3.BrcCYP79B2-1。

图2 BrcCYP79B2-1基因及氨基酸序列

Fig.2 Gene and amino acid sequence of BrcCYP79B2-1

图3 BrcCYP79B2-1的氨基酸序列多重比对结果

Fig.3 Multiple alignment results of the amino acid sequence of BrcCYP79B2-1

图4 BrcCYP79B2-1同源蛋白的系统进化树

Fig.4 Phylogenetic tree of BrcCYP79B2-1 homologous protein

图5 BrcCYP79B2-1蛋白质二级结构预测

Fig.5 Protein secondary structure of BrcCYP79B2-1

图6 BrcCYP79B2-1蛋白质三级结构预测

Fig.6 Protein tertiary structure prediction of BrcCYP79B2-1

图7 BrcCYP79B2-1在不同器官的表达分析不同小写字母代表相对表达量在0.05水平上差异显著。

Fig.7 Expression analysis BrcCYP79B2-1 in different organs Different lowercase letters represent significant differences in relative expression at the 0.05 level.

图8 BrcCYP79B2-1在不同发育时期茎尖部位的表达

Fig.8 Expression of BrcCYP79B2-1 in shoot apex at different developmental

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