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作物学报 ›› 2023, Vol. 49 ›› Issue (4): 955-965.doi: 10.3724/SP.J.1006.2023.24089

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

木薯MYB转录因子基因MeMYB60表达特征分析及其互作蛋白筛选

徐子寅1(), 于晓玲2,3, 邹良平2,3, 赵平娟2,3, 李文彬2,3, 耿梦婷1,*(), 阮孟斌2,3,*()   

  1. 1海南大学热带作物学院, 海南海口 570228
    2中国热带农业科学院热带生物技术研究所/农业部热带作物生物学与遗传资源利用重点实验室, 海南海口 571101
    3海南热带农业资源研究院/海南省热带农业生物资源保护与利用重点实验室, 海南海口 571101
  • 收稿日期:2022-04-10 接受日期:2022-07-21 出版日期:2023-04-12 网络出版日期:2022-08-22
  • 通讯作者: *阮孟斌, E-mail: ruanmengbin@itbb.org.cn;耿梦婷, E-mail: mengtinggeng8908@163.com
  • 作者简介:E-mail: 670307392@qq.com
  • 基金资助:
    国家重点研发计划项目(2018YFD1000501);海南省重大科技计划项目(ZDKJ2021012);中央级公益科研院所基本科研业务费专项(1630052022036)

Expression pattern analysis and interaction protein screening of cassava MYB transcription factor MeMYB60

XU Zi-Yin1(), YU Xiao-Ling2,3, ZOU Liang-Ping2,3, ZHAO Ping-Juan2,3, LI Wen-Bin2,3, GENG Meng-Ting1,*(), RUAN Meng-Bin2,3,*()   

  1. 1College of Tropical Crops, Hainan University, Haikou 570228, Hainan, China
    2Key Laboratory of Biology and Genetic Resources of Tropical Crops, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, Hainan, China
    3Key Laboratory for Biology and Genetic Resources of Tropical Crops of Hainan Province, Hainan Institute for Tropical Agricultural Resources, Haikou 571101, Hainan, China
  • Received:2022-04-10 Accepted:2022-07-21 Published:2023-04-12 Published online:2022-08-22
  • Contact: *ruanmengbin@itbb.org.cn;E-mail: mengtinggeng8908@163.com
  • Supported by:
    National Key Research and Development Program of China(2018YFD1000501);Major Science and Technology Plan of Hainan Province(ZDKJ2021012);Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences(1630052022036)

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摘要：

Myeloblastosis (MYB)类转录因子在植物对非生物胁迫的响应过程中起重要调控作用。本研究在分析栽培木薯MYB家族成员表达模式的基础上, 筛选并克隆到了一个R2R3-MYB转录因子基因MeMYB60。基因表达特性分析表明, 该基因在叶片特异表达, 受干旱、低温负调控, 同时对ABA处理也有响应。启动子活性分析发现, MeMYB60可以在保卫细胞表达, 预示着该转录因子基因的表达可能与木薯气孔开闭调节有关。MeMYB60编码蛋白主要定位于细胞核中, 具有转录激活活性, 其转录激活结构域在蛋白C端第194~343氨基酸残基范围内。以MeMYB60蛋白N端第1~194氨基酸残基片段为诱饵, 从干旱胁迫后木薯叶片的cDNA文库中筛选到18种可能与MeMYB60互作的蛋白, 酵母双杂确定了MeCatlase1和MeCatalase2分别与MeMYB60存在互作关系。本研究为深入研究转录因子MeMYB60在木薯响应非生物胁迫过程中的功能并解析其调控网络奠定了基础。

关键词: 木薯, 非生物胁迫, MYB转录因子, 表达特征, 互作蛋白筛选

Abstract:

Myeloblastosis (MYB) transcription factors widely involve in a variety of physiological and biochemical processes in plants, and play important regulatory roles in response to abiotic stress in plant. Based on the expression pattern of MYB members in cassava cultivars, an R2R3-MYB transcription factor, namely MeMYB60 was screened and cloned. Gene expression characteristics showed that MeMYB60 was specifically expressed in leaves of cassava, and negatively regulated by drought stress and low temperature. Moreover, this gene was also responded to ABA treatment in leaves of cassava. Promoter activity analysis showed that MeMYB60 could be expressed in guard cells, indicating that the expression of this transcription factor gene may be related to stomatal movement regulation in cassava. MeMYB60 protein was predominately located in the nucleus and had transcriptional activation activity. Its transcriptional activation domain was in the range of 194th-343rd amino acid residues at the C-terminal of the protein. The cDNA library of drought stressed cassava leaves was screened by using the 1st-194th amino acid residues at the N-terminal of MeMYB60 protein as bait. Subsequently, 18 proteins had been that may interact with MeMYB60. Yeast-two-hybrid analysis determined that MeCatlase1 and MeCataase2 are potential interactors of MeMYB60, respectively. These results lay a foundation for further functional study of MeMYB60 in cassava in response to abiotic stress and are helpful for the regulatory network investigation of MeMYB60.

Key words: cassava, the abiotic stress, MYB transcription factor, the relative expression pattern, the interaction protein screening

表1

PCR引物名称及序列"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 用途
Function
MeMYB60_SalI_EcoRI_5° TCGAATTCGAATTCATGGGAAGACCTCCTTGCTGTG 基因克隆Gene cloning
MeMYB60-full_BamHI_3° TAGGATCCGAATATTGGAGACAGTTCCATG 基因克隆Gene cloning
MeMYB60_Promoter_PstI_5° TACTGCAGGGAAGACGAACTCATTGATATC 启动子克隆Isolation of promoter
MeMYB60_Promoter_SalI_3° TCGAATTCACAGCAAGGAGGTCTTCCCAT 启动子克隆Isolation of promoter
Me_ACT1_QP1 GGAATGGTGAAGGCTGGGTTTGC 荧光定量PCR qRT-PCR
Me_ACT1_QP2 AGTTGCTGACAATACCATGCTCA 荧光定量PCR qRT-PCR
MeMYB60_QP1 CCATTGCAGCACATGACTCGTCATC 荧光定量PCR qRT-PCR
MeMYB60_QP2 CATCTGGGTTCTCCAAGTTATTGTC 荧光定量PCR qRT-PCR
MeMYB60-308_BamHI-3° GAGGATCCCTGCTTCCTGATCTCTGCTGC 转录激活分析Transcriptional activation assay
MeMYB60-271_BamHI-3' GAGGATCCTTTGTCCCATGCAACAGTGTT 转录激活分析Transcriptional activation assay
MeMYB60-234_BamHI-3' GAGGATCCGTAACACTGCAGAGAAGTTGT 转录激活分析Transcriptional activation assay
MeMYB60-194_BamHI-3' GAGGATCCAGGCTTTGGAGATGATCTCAT 转录激活分析Transcriptional activation assay
MeCAT1-SalI-5' TCGTCGACATGGACCCAAGCAAGTTCATTCC 互作验证Protein interaction assay
MeCAT1-BamHI-3' TAGGATCCGAAATTTGGCCTCACGTTGAGAC 互作验证Protein interaction assay
MeCAT2-SalI-5' TCGTCGACATGAGCAACAATCACCCAAGTTCTC 互作验证Protein interaction assay
MeCAT2-BamHI-3' TCGGATCCAAAACTTGGCCTCACATTGAGAC 互作验证Protein interaction assay

图1

木薯叶片RNA及MeMYB60基因PCR扩增产物电泳图"

图2

MeMYB60 (XP_021622445.1)蛋白与其他植物MYB60蛋白的系统进化树及序列一致性"

图3

木薯MeMYB60与其他植物的同源蛋白氨基酸序列比对 黑色区块: 氨基酸一致性为100%。"

图4

木薯MeMYB60表达的组织特异性分析(A)及其在叶片中对不同胁迫的响应分析(B) 内参基因为MeActin1。**表示不同处理样品与对照样品(Control)相比差异极显著(P < 0.01)。误差线为每组处理的标准误差(n = 3)。"

图5

MeMYB60-eGFP融合蛋白的亚细胞定位"

图6

MeMYB60:eGFP融合基因在MeMYB60启动子驱动下的表达分析"

图7

MeMYB60蛋白序列及转录激活结构域分析"

表2

MeMYB60酵母cDNA文库筛选结果"

登录号
Accession No.		 注释
Annotation		 克隆数
Number of clones
Manes.13G077148.1 细胞色素b6-f 复合体铁硫亚基Cytochrome b6-f complex iron-sulfur subunit 2
Manes.04G085600.1 组织蛋白酶F Cathepsin F 1
Manes.14G167100.1 捕光复合物ii叶绿素a/b结合蛋白6
Light-harvesting complex ii chlorophyll a/b binding protein 6		 1
Manes.01G009451.4 锚重复蛋白35 Ankyrin repeat protein skip 35 1
Manes.12G158200.5 线粒体核糖体蛋白147 Ribosomal protein l47, mitochondrial-related 1
Manes.08G108200.1 S-腺苷甲硫氨酸合酶4 S-adenosylmethionine synthase 4 1
Manes.09G074600.1 ATP依赖RNA解旋酶21 ATP-dependent RNA helicase DDX21 1
Manes.10G060650.1 DNAJ同源亚家族A成员2 DNAJ homolog subfamily A member 2 3
Manes.09G105200.1 吡哆醛5’-磷酸合酶PDXs亚基 Pyridoxal 5’-phosphate synthase PDXs subunit 2
Manes.07G116700.1 磷酸三糖异构酶 Triose-phosphate isomerase 1
Manes.08G168900.1 N-末端乙酰转移酶 N-terminal acetyltransferase 1
Manes.06G110100.1 BRCA1基因1旁蛋白 Next to BRCA1 gene 1 protein 1
Manes.01G011500.2 核酮糖二磷酸羧化酶 Ribulose-bisphosphate carboxylase 1
Manes.01G103400.7 FRNE类蛋白 FRNE protein-like 1
Manes.05G127800.3 叶绿体PSBp结构蛋白3 Chloroplastic PSBp domain-containing protein 3 2
Manes.05G130500.1 过氧化氢酶1 Catalase 1 1
Manes.02G113300.1 过氧化氢酶2 Catalase 2 1
Manes.10G066200.1 未知蛋白 Uncharacterized protein 2

图8

酵母双杂验证MeMYB60与木薯过氧化氢酶的蛋白互作关系"

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