球茎甘蓝<i>MYB62</i>转录因子的克隆与表达特征分析

doi:10.7668/hbnxb.20194583

摘要/Abstract

摘要：

为了明晰球茎甘蓝MYB62转录因子的序列特征及其在逆境胁迫后的表达特征,进一步探索球茎甘蓝MYB62转录因子的生物学功能,为后续MYB62转录因子功能鉴定提供理论依据,以球茎甘蓝为研究对象,采用同源克隆的方法获得球茎甘蓝MYB62转录因子基因,并对其进行生物信息学分析,通过实时荧光定量PCR的方法分析MYB62在球茎甘蓝不同组织间以及逆境胁迫后的表达特征。基因克隆结果表明,BocMYB62基因gDNA全长1 353 bp,CDS为837 bp,包括4个外显子和3个内含子,编码278个氨基酸。序列结构分析结果显示,BocMYB62为亲水性蛋白,具有2个SANT-MYB结构域,属于R2R3-MYB型MYB转录因子;空间结构预测显示其具有典型的α-螺旋结构;系统发育分析表明,BocMYB62与甘蓝型油菜MYB62亲缘关系最近。时空表达结果显示,BocMYB62在绿色球茎甘蓝中的表达量始终高于在紫色球茎甘蓝中,且存在明显的组织特异性,在干旱胁迫后BocMYB62表达量显著升高,胁迫12 h表达量最高,低温胁迫后BocMYB62表达量显著低于对照,在4 ℃低温胁迫时表达量最低,推断BocMYB62可能参与花青素生物合成调控,并可能参与逆境胁迫的调控作用。

关键词: 球茎甘蓝, MYB62, 序列分析, 保守基序, 亲缘关系, 荧光定量PCR

Abstract:

In order to clarify the sequence characteristics of the MYB62 transcription factor,expression changes after stress,and further explore the biological function of the MYB62 transcription factor,kohlrabis were used as plant materials.MYB62 transcription factors were obtained by homologous cloning method and bioinformatics was analyzed.Spatiotemporal expression analysis of MYB62 and its expression after stress were performed by Real-time PCR.Results of gene cloning showed that gDNA length of BocMYB62 gene was 1 353 bp,the length of CDS was 837 bp,which contained four exons and three introns and encoded 278 amino acids.The sequence structure analysis showed that BocMYB62 was a hydrophilic protein with two SANT-MYB domains,belonging to the R2R3-MYB type in the MYB transcription factor family.The spatial structure prediction revealed a typical α-helix structure.Phylogenetic analysis indicated that BocMYB62 was closely related to MYB62 in Brassica napus.The spatiotemporal expression results showed that BocMYB62 was consistently higher in green kohlrabi than that in purple with clear tissue specificity.The expression of BocMYB62 was significantly increased during drought stress with the highest expression under 12 h.The BocMYB62 expression was significantly lower than control and the lowest at 4 ℃ cold stress.These results inferred that BocMYB62 may be involved in the regulation of anthocyanin biosynthesis and the regulation of stress.It provides a theoretical basis for the subsequent functional identification of MYB62 transcription factors.

Key words: Kohlrabi, MYB62, Sequence analysis, Conversed motifs, Relationship, Real-time PCR

王鑫淼, 赵孟良, 邵登魁, 尕桑, 任延靖. 球茎甘蓝MYB62转录因子的克隆与表达特征分析[J]. 华北农学报, 2024, 39(2): 62-70. doi: 10.7668/hbnxb.20194583.

WANG Xinmiao, ZHAO Mengliang, SHAO Dengkui, GA Sang, REN Yanjing. Cloning and Expression Analysis of MYB62 Transcription Factors from Kohlrabi[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(2): 62-70. doi: 10.7668/hbnxb.20194583.

http://www.hbnxb.net/CN/Y2024/V39/I2/62

图/表 10

图1 BocMYB62 基因扩增 M.DL2000 DNA Marker;1.绿色球茎甘蓝;2.紫色球茎甘蓝。左侧为gDNA扩增条带,右侧为cDNA扩增条带。

Fig.1 Amplification of BocMYB62 gene M.DL2000 DNA Marker;1.Green kohlrabi;2.Purple kohlrabi.The gDNA amplification bands were shown on the left and the cDNA amplification bands were shown on the right.

图2 球茎甘蓝BocMYB62基因结构

Fig.2 BocMYB62 gene structure of Brassica oleracea L.var.caulorapa

图3 球茎甘蓝BocMYB62基因的核苷酸序列及其编码的氨基酸序列 ^*.终止密码子;—.内含子;大写字母.氨基酸序列。

Fig.3 Nucleotide sequence of BocMYB62 gene and its encoded amino acid sequences of Brassica oleracea L.var.caulorapa ^*.Termination codon;—.Intron;Uppercase letter.Amino acid sequence.

图4 BocMYB62蛋白的多序列对比野甘蓝BooMYB108-like.XP_013596475.1;甘蓝型油菜BnMYB62-like.XP_013659722.1;白菜BrMYB62.XP_009115302.1;野甘蓝BooMYB24.XP_013584285.1。

Fig.4 Multiple alignment analysis of BocMYB62 protein Wild cabbage BooMYB108-like.XP_013596475.1;Brassica napus BnMYB62-like.XP_013659722.1; Brassica rapa BrMYB62.XP_009115302.1;Wild cabbage BooMYB24.XP_013584285.1.

图5 球茎甘蓝BocMYB62蛋白的二级结构预测蓝色曲线.α-螺旋;绿色曲线.β-转角;紫色曲线.无规则卷曲;红色曲线.延伸链。

Fig.5 Prediction of secondary structure of BocMYB62 protein in Brassica oleracea L.var.caulorapa Blue curve.α-helix;Green curve.β-angle;Purple curve.Irregular coil;Red curve.Extended strand.

图6 BocMYB62蛋白结构预测分析

Fig.6 BocMYB62 protein structure prediction analysis

图7 球茎甘蓝BocMYB62蛋白保守基序分析

Fig.7 Conserved motif analysis of the BocMYB62 protein of Brassica oleracea L.var.caulorapa

图8 球茎甘蓝BocMYB62与其他物种中相关MYB转录因子的系统发育关系

Fig.8 Phylogenetic relationship of BocMYB62 in Brassica oleracea L.var.caulorapa and anthocyanin-related MYB transcription factors in other species.

图9 BocMYB62在绿色球茎甘蓝与紫色球茎甘蓝不同组织中的表达量不同小写字母表示在P<0.05水平上差异显著。图10同。

Fig.9 Expression levels of BocMYB62 in different tissues of green and purple kohlrabi Different lowercase letters indicate significant difference at the P<0.05 level.The same as Fig.10.

图10 BocMYB62在逆境胁迫中的表达量 A.绿色球茎甘蓝BocMYB62在中干旱胁迫后的表达量变化;B.紫色球茎甘蓝BocMYB62在中低温胁迫的表达量变化。

Fig.10 Expression level changes of BocMYB62 under stress A.Expression level changes of BocMYB62 under drought stress in green kohlrabi;B.Expression level of BocMYB62 under cold stress in purple kohlrabi.

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[41]	Wang M L, Hao J, Chen X H, Zhang X C. SlMYB102 expression enhances low-temperature stress resistance in tomato plants[J]. PeerJ, 2020,8:e10059.doi:10.7717/peerj.10059.

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球茎甘蓝MYB62转录因子的克隆与表达特征分析

Cloning and Expression Analysis of MYB62 Transcription Factors from Kohlrabi

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