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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (4): 955-965.doi: 10.3724/SP.J.1006.2023.24089

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2023-04-12 Published: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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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

Table 1

Primers used in this study"

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

Fig. 1

Total RNA from cassava mature leaf and product of cDNA PCR amplification of MeMYB60 genes"

Fig. 2

Phylogenetic tree and protein sequence identity of MeMYB60 and MYB60 protein from other plants"

Fig. 3

Alignment of MeMYB60 and MeMYB2 of cassava (Manihot esculenta) with homologous proteins in other plants Black blocks: the amino acid identity is 100%."

Fig. 4

Relative expression pattern of MeMYB60 in different tissues (A) and after treated by drought, ABA, and cold stresses in mature leaves (B) MeActin1 was used as a home keeping gene. ** indicates significant differences at P < 0.01 in samples for different treatments compared to the sample of control. Error bar represents the standard error of each sample (n = 3)."

Fig. 5

Subcellular localization of MeMYB60-eGFP fusion proteins"

Fig. 6

Relative expression profile of MeMYB60:eGFP fusion genes driven by MeMYb60 promoter"

Fig. 7

Protein sequence and transcriptional activation domain identification of MeMYB60 proteins"

Table 2

Screening of MeMYB60 yeast in cDNA library"

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

Fig. 8

Interaction validation of MeMYB60 and cassava catalases by yeast two hybrid system"

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[3] YANG Jian-Chang;ZHANG Jian-Hua;WANG Zhi-Qin;ZH0U Qing-Sen. Changes in Contents of Polyamines in the Flag Leaf and Their Relationship with Drought-resistance of Rice Cultivars under Water Deficiency Stress[J]. Acta Agron Sin, 2004, 30(11): 1069 -1075 .
[4] Yan Mei;Yang Guangsheng;Fu Tingdong;Yan Hongyan. Studies on the Ecotypical Male Sterile-fertile Line of Brassica napus L.Ⅲ. Sensitivity to Temperature of 8-8112AB and Its Inheritance[J]. Acta Agron Sin, 2003, 29(03): 330 -335 .
[5] Wang Yongsheng;Wang Jing;Duan Jingya;Wang Jinfa;Liu Liangshi. Isolation and Genetic Research of a Dwarf Tiilering Mutant Rice[J]. Acta Agron Sin, 2002, 28(02): 235 -239 .
[6] WANG Li-Yan;ZHAO Ke-Fu. Some Physiological Response of Zea mays under Salt-stress[J]. Acta Agron Sin, 2005, 31(02): 264 -268 .
[7] TIAN Meng-Liang;HUNAG Yu-Bi;TAN Gong-Xie;LIU Yong-Jian;RONG Ting-Zhao. Sequence Polymorphism of waxy Genes in Landraces of Waxy Maize from Southwest China[J]. Acta Agron Sin, 2008, 34(05): 729 -736 .
[8] HU Xi-Yuan;LI Jian-Ping;SONG Xi-Fang. Efficiency of Spatial Statistical Analysis in Superior Genotype Selection of Plant Breeding[J]. Acta Agron Sin, 2008, 34(03): 412 -417 .
[9] WANG Yan;QIU Li-Ming;XIE Wen-Juan;HUANG Wei;YE Feng;ZHANG Fu-Chun;MA Ji. Cold Tolerance of Transgenic Tobacco Carrying Gene Encoding Insect Antifreeze Protein[J]. Acta Agron Sin, 2008, 34(03): 397 -402 .
[10] ZHENG Xi;WU Jian-Guo;LOU Xiang-Yang;XU Hai-Ming;SHI Chun-Hai. Mapping and Analysis of QTLs on Maternal and Endosperm Genomes for Histidine and Arginine in Rice (Oryza sativa L.) across Environments[J]. Acta Agron Sin, 2008, 34(03): 369 -375 .
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