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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (12): 3108-3119.doi: 10.3724/SP.J.1006.2022.14241

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

Identification and relative expression levels of PEPC gene family members in cassava

LI Xiang-Chen1(), SHEN Xu3, ZHOU Xin-Cheng2, CHEN Xin2, WANG Hai-Yan2(), WANG Wen-Quan3()   

  1. 1College of Agricultural, Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang, China
    2Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture and Rural Affairs / Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences / Key Laboratory for Biology and Genetic Resources of Tropical Crops of Hainan Province, Hainan Institute for Tropical Agricultural Resources, Haikou 571101, Hainan, China
    3College of Tropical Crops, Hainan University, Haikou 570228, Hainan, China
  • Received:2021-12-20 Accepted:2022-03-25 Online:2022-12-12 Published:2022-04-19
  • Contact: WANG Hai-Yan,WANG Wen-Quan E-mail:l13j45q3k2a@126.com;wanghaiyan@itbb.org.cn;wangwenquan@itbb.org.cn
  • Supported by:
    Natural Science Foundation of Hainan Province(320RC708);Project of the National Key Research and Development Program of China(2018YFD1000500);Major Science and Technology Plan of Hainan Province(ZDKJ2021012)

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Abstract:

Phosphoenolpyruvate carboxylase (PEPC) is the key enzyme of photosynthesis in C4 plants. Cassava is a C3-C4 plant, and the relative expression levels of MePEPC genes in cultivated species are significantly higher than wild type. So far, systematic studies on MePEPC have not been done. To investigate the basic information of MePEPC family in cassava, five MePEPCs members were identified in the whole genome of cassava by bioinformatics method. The MePEPC family of cassava were comprehensively analyzed by bioinformatics software, including basic physical and chemical properties analysis, prediction of subcellular localization, evolutionary tree analysis, chromosome localization, gene and protein structure, and promoter cis-element prediction. The results showed that five MePEPC family members were identified and distributed on five chromosomes, respectively. Among them, MePEPC2 was distributed on chromosome 3 with underwent variable splicing, early termination of sequence, and loss of function. The phylogenetic tree revealed that MePEPC family could be divided into two subfamilies (plant type and bacterial type), and the distribution of exons of the same group were similar. The promoter region of MePEPC members contained different numbers of light corresponding elements, hormone corresponding elements, and stress response elements, indicating that different MePEPC member may participate in different growth and development regulation processes. The relative expression levels of MePEPC1, MePEPC4, and MePEPC5 were relatively higher, and the different expression patterns were in different light time, different development stages, drought and ABA stress. The relative expression levels of MePEPC2 and MePEPC3 were lower and almost invisible. This study provides basic data for in-depth study of the function of MePEPC family in cassava and candidate genes for cassava high light efficiency breeding.

Key words: cassava, phosphoenolpyruvate carboxylase, relative expression profile, drought, ABA, circadian rhythm

Table 1

Primers used in the study"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 用途
Purpose
MePEPC1F GTCGACATGGCTGCTAGGAATCTGGA 扩增全长Full length amplification
MePEPC1R GGTACCACCAGTGTTTTGCATGCCA
MePEPC2F GTCGACATGCAACCTAAGAATGTTGAGAAG 扩增全长Full length amplification
MePEPC2R GGTACCACCAGTATTCTGCATTCCAGC
MePEPC3F GTCGACATGACGGATACAACAGATGATATAGC 扩增全长Full length amplification
MePEPC3R GGTACCACCTGTGTTCCTCATCCCAG
MePEPC4F GTCGACATGGAGAAACTTGAAAGGATTCG 扩增全长Full length amplification
MePEPC4R ACTAGTACCCGTGTTTCTCATCCCA
MePEPC5F GTCGACATGCAACCTCGGAATCTTGAG 扩增全长Full length amplification
MePEPC5R GGTACCACCAGTGTTCTGCATTCCAG
Actin-F TCTTCTCAACTGAGGAGCTGCT RT-qPCR
Actin-R CCTTCGTCTGGACCTTGCTG
qMePEPC1F ATACGAGCAGAAGTGAAGGTG RT-qPCR
qMePEPC1R CTTATTCTTCTCGTGTGGTTCAC
qMePEPC2F ATATCGTCGAAGGCAAAAGTTG RT-qPCR
qMePEPC2R TGCCAAGCAATTACGAATCCTT
qMePEPC3F GTCCACCTCTCCACTGCT RT-qPCR
qMePEPC3R TGTTGTATCCGTCATTGTACCC
qMePEPC4F GTGGCAGGGATAGAGGATACG RT-qPCR
qMePEPC4R CTGGAGAAACTTCACCCTGTAAT
qMePEPC5F GGAGATCCCAGAGGAGGG RT-qPCR
qMePEPC5R GTCCTGTCGGCGTTCTTC

Table 2

Basic physical and chemical properties of PEPC proteins in cassava"

基因号
Gene ID		 基因名称
Gene name		 氨基酸
Amino acid		 分子量
Molecular weight		 等电点
pI		 亲水系数
Hydrophilic coefficient		 染色体
Chr.		 染色体起始
Chr. start		 染色体终止
Chr. end		 亚细胞定位
Subcellular
localization
Manes.02G091300.1 MePEPC1 965 110,369.29 5.93 -0.421 2 6,822,458 6,829,902 细胞质Cytoplasm
Manes.03G101900.1 MePEPC2 295 33,742.35 5.26 -0.552 3 17,850,224 17,852,291 细胞质Cytoplasm
Manes.06G074600.1 MePEPC3 1020 114,385.76 6.51 -0.377 6 18,870,252 18,894,812 细胞质Cytoplasm
Manes.14G095900.1 MePEPC4 964 110,296.13 5.75 -0.412 14 7,740,112 7,757,686 细胞质Cytoplasm
Manes.15G093700.1 MePEPC5 965 110,554.63 6.05 -0.389 15 6,906,159 6,916,528 细胞质Cytoplasm

Fig. 1

Chromosome distribution of PEPC family in cassava"

Table 3

Secondary structure of PEPC family proteins in cassava"

基因名称
Gene name		 α-螺旋
Alpha helix		 延伸链
Extended strand		 β-转角
Beta turn		 无规卷曲
Random coil		 二级结构原件
Secondary structural elements
MePEPC1 62.38 5.91 4.15 27.56
MePEPC2 62.71 4.75 3.73 28.81
MePEPC3 56.45 6.36 4.46 32.73
MePEPC4 55.88 6.67 4.02 33.43
MePEPC5 61.93 6.02 4.25 27.8

Fig. 2

Phylogenetic tree of PEPC protein family members in cassava, corn, millet, rice, Arabidopsis, and soybean"

Fig. 3

Structure and conserved motif of MePEPC genes A: gene structure of MePEPCs; B: the conserved motif of MePEPCs."

Table 4

Cis-element analysis of promoter region of PEPC gene family in cassava"

名称Name PEPC1 PEPC2 PEPC3 PEPC4 PEPC5
光响应元件Light response element 6 16 12 13 12
脱落酸响应元件ABRE 1 4 2 1 2
生长素响应元件AuxRR-core 1
生长素响应元件TGA-element 1 2 1
水杨酸响应元件TCA-element 1 1
赤霉素响应元件P-box 1 1 1
茉莉酸甲酯响应元件TGACG-motif 3 1 1
干旱响应元件MBS 1 1 1
低温响应元件LTR 2 1
细胞周期响应元件MSA-like 1
昼夜节律响应元件Circadian 1
厌氧响应元件GC-motif 1
缺氧响应元件ARE 1 1 2
创伤响应元件WUN-motif 1
分生组织响应元件CAT-box 1 1 1

Fig. 4

Subcellular localization of MePEPC1, MePEPC4, and MePEPC5"

Fig. 5

Relative expression levels of cassava PEPC family in different tissues Three biological replicates are set for each treatment, and the values of relative expression are the average value of three biological replicates ± STDEVs. Different lowercase letters indicate that there are significant differences between different genes in the same treatment at P ≤ 0.05."

Fig. 6

Relative expression levels of MePEPC genes under different light conditions The first row of abscissa is the time point, and the second row is the light intensity corresponding to the time point. Three biological replicates are set for each treatment, and the values of relative expression are the average values of the three biological replicates ± STDEVs. Different lowercase letters indicate that there are significant differences between different genes in the same treatment at P ≤ 0.05."

Fig. 7

Relative expression levels of MePEPC family of leaves and roots under drought stress in cassava A: leaf of SC124; B: leaf of Arg7; C: root of SC124; D: root of Arg7. Three biological replicates are set for each treatment, and the values of relative expression are the average value of three biological replicates ± STDEVs. Different lowercase letters indicate that there are significant differences between different genes in the same treatment at P ≤ 0.05."

Fig. 8

Relative expression levels of MePEPC1, MePEPC4, and MePEPC5 genes in leaves and roots under ABA stress A: leaf; B: root. Three biological replicates are set for each treatment, and the values of relative expression are the average values of the three biological replicates ± STDEVs. Different lowercase letters indicate that there are significant differences between different genes in the same treatment at P ≤ 0.05."

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