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作物学报

作物学报 ›› 2022, Vol. 48 ›› Issue (12): 3108-3119.doi: 10.3724/SP.J.1006.2022.14241

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

木薯PEPC基因家族成员鉴定及表达分析

李相辰1(), 沈旭3, 周新成2, 陈新2, 王海燕2(), 王文泉3()   

  1. 1黑龙江八一农垦大学农学院, 黑龙江大庆 163319
    2中国热带农业科学院热带生物技术研究所 / 农业农村部热带作物生物学与遗传资源利用重点实验室 / 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
    3海南大学热带作物学院, 海南海口 570228
  • 收稿日期:2021-12-20 接受日期:2022-03-25 出版日期:2022-12-12 网络出版日期:2022-04-19
  • 通讯作者: 王海燕,王文泉
  • 作者简介:E-mail: l13j45q3k2a@126.com
  • 基金资助:
    海南省自然科学基金项目(320RC708);国家重点研发计划项目(2018YFD1000500);海南省重大科技计划项目(ZDKJ2021012)

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 Published:2022-12-12 Published online:2022-04-19
  • Contact: WANG Hai-Yan,WANG Wen-Quan
  • 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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摘要：

磷酸烯醇式丙酮酸羧化酶(PEPC)是C4植物光合作用的关键酶, 而木薯是C3-C4型植物, MePEPC在栽培种的表达量显著高于野生型, 迄今尚未有人对MePEPC进行过系统研究。为了解木薯中MePEPC家族成员的基本信息, 本文运用生物信息学方法在木薯全基因组中确认了5个MePEPC基因家族成员, 并对MePEPC基因家族进行基本理化性质分析、亚细胞定位预测、进化树分析、染色体定位、二级结构预测、基因结构域及启动子顺式作用元件预测。结果表明, 鉴定到5个MePEPC基因家族成员, 分布在5条染色体上, 其中分布在3号染色体的MePEPC2发生可变剪切, 序列提前终止, 发生功能缺失。系统进化树表明, MePEPC可分为2个亚家族(植物型和细菌型), 同组成员基因结构和功能域相似。MePEPC家族成员启动子区域含有不同数量的光响应元件、激素响应元件及胁迫响应元件, 表明MePEPC基因家族成员可能参与不同的生长发育调节过程。表达谱显示, MePEPC1、MePEPC4和MePEPC5具有较高表达, 且在不同光照时间、不同发育时期、干旱及ABA胁迫下呈现出不同的表达模式, 而MePEPC2和MePEPC3表达量极低, 几乎不表达。以上研究为深入研究MePEPC基因家族在木薯中的功能提供了基础数据, 为木薯高光效育种提供候选基因。

关键词: 木薯, 磷酸烯醇式丙酮酸羧化酶, 表达谱, 干旱, ABA, 昼夜节律

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

表1

试验所用引物"

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

表2

木薯PEPC蛋白基本理化性质"

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

图1

木薯PEPC家族在染色体上的分布"

表3

木薯PEPC家族蛋白二级结构分析"

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

图2

木薯、玉米、谷子、水稻、拟南芥、大豆PEPC蛋白家族成员系统进化树"

图3

MePEPC基因结构及保守基序 A: MePEPCs基因结构; B: MePEPCs的保守基序。"

表4

木薯PEPC基因家族成员启动子区域顺式元件分析"

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

图4

MePEPC1、MePEPC4和MePEPC5的亚细胞定位"

图5

木薯PEPC家族在不同组织中的相对表达量 每个处理设置3个生物学重复, 相对表达量的数值是3个生物学重复的平均值±STDEV。不同小写字母表示同一处理中基因表达量间存在显著性差异(P ≤ 0.05)。"

图6

不同光照条件下MePEPC的相对表达量 横坐标第1行是时间点, 第2行是时间点对应的光强。每个处理设置3个生物学重复, 相对表达量的数值是3个生物学重复的平均值±STDEV。不同小写字母表示同一处理中基因表达量间存在显著性差异(P ≤ 0.05)"

图7

干旱胁迫下木薯MePEPC家族在叶与根中的相对表达量 A: SC124叶片; B: Arg7叶片; C: SC124根; D: Arg7根。每个处理设置3个生物学重复, 相对表达量的数值是3个生物学重复的平均值±STDEV。不同小写字母表示同一处理中基因表达量间存在显著性差异(P ≤ 0.05)。"

图8

ABA胁迫下MePEPC1、MePEPC4、MePEPC5在叶与根中的相对表达量 A: 叶; B: 根。每个处理设置3个生物学重复, 相对表达量的数值是3个生物学重复的平均值±STDEV。不同小写字母表示同一处理中基因表达量间存在显著性差异(P ≤ 0.05)。"

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