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作物学报 ›› 2022, Vol. 48 ›› Issue (3): 608-623.doi: 10.3724/SP.J.1006.2022.14004

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘薯Dof基因家族挖掘及表达分析

靳容1(), 蒋薇1, 刘明1, 赵鹏1, 张强强1, 李铁鑫2, 王丹凤1, 范文静2, 张爱君1, 唐忠厚1,*()   

  1. 1江苏徐淮地区徐州农业科学研究所/江苏徐州甘薯研究中心/农业农村部甘薯生物学与遗传育种重点实验室, 江苏徐州 221131
    2安徽农业大学, 安徽合肥 230036
  • 收稿日期:2021-01-11 接受日期:2021-04-26 出版日期:2021-05-20 网络出版日期:2021-05-20
  • 通讯作者: 唐忠厚
  • 作者简介:E-mail: jinrong_2012@126.com
  • 基金资助:
    国家农业现代产业技术体系建设专项(CARS-11-B-13);农业农村部薯类作物生物学与遗传育种综合性重点实验开放课题(NYBSL201802)

Genome-wide characterization and expression analysis of Dof family genes in sweetpotato

JIN Rong1(), JIANG Wei1, LIU Ming1, ZHAO Peng1, ZHANG Qiang-Qiang1, LI Tie-Xin2, WANG Dan-Feng1, FAN Wen-Jing2, ZHANG Ai-Jun1, TANG Zhong-Hou1,*()   

  1. 1Xuzhou Institute of Agricultural Sciences of Xuhuai District of Jiangsu Province/Xuzhou Sweetpotato Research Center of Jiangsu Province/Key Laboratory of Sweetpotato Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, Xuzhou 221131, Jiangsu, China
    2Anhui Agricultural University, Hefei 230036, Anhui, China
  • Received:2021-01-11 Accepted:2021-04-26 Published:2021-05-20 Published online:2021-05-20
  • Contact: TANG Zhong-Hou
  • Supported by:
    China Agriculture Research System(CARS-11-B-13);Open Project of Ministry of Agriculture and Rural Affairs: Key Laboratory of Biology, Genetics and Breeding of Potato Crops(NYBSL201802)

摘要:

单锌指蛋白超家族Dof (DNA binding with one finger)转录因子广泛参与植物的各种生命活动。以甘薯泰中6号基因组为参照挖掘得到46个甘薯Dof基因, 每个家族成员均具有C2C2-Dof锌指结构, 按照其在染色体上的位置, 命名为IbDof1~IbDof46。该基因家族可分为4个亚家族(A~D), 且不同亚族的基因结构和基序的分布差异显著。基序1和基序2存在于所有亚家族中; 基序5和基序9只存在于亚家族A中; 基序6、基序7、基序8和基序10只存在亚家族D中。IbDof的进化极为保守, 共有12对染色体复制事件和5对串联重复序列事件(IbDof2/IbDof3IbDof12/IbDof13IbDof9/IbDof10IbDof28/IbDof29IbDof32/IbDof33), 分歧时间平均是3552万年前和186万年前, Ka/Ks比值范围从0.07 (IbDof12/IbDof13)到0.68 (IbDof6/IbDof25), 且与甘薯野生近缘种Ipomoea trifida同一染色体上的Dof同源基因对有38对。组织特异性分析表明, IbDof各亚族在各个组织器官中表达强度不同, 且亚族内部呈现不同的表达趋势。IbDof启动子序列中包含逆境和激素响应等相关元件, 实时荧光定量PCR进一步分析表明, IDof基因受低温、干旱、高盐和H2O2等非生物胁迫诱导表达。比如大部分IbDof受低温调控表达; IbDof10IbDof14受干旱胁迫诱导表达; IbDof2IbDof14IbDof37IbDof41IbDof43受高盐诱导表达; 而IbDof8IbDof10IbDof25IbDof41则受H2O2诱导表达, 这说明IbDof家族成员协同控制了甘薯的生长发育及参与非生物胁迫过程。

关键词: 甘薯, Dof基因家族, 系统进化, 表达分析

Abstract:

DNA-binding One Zinc Finger (Dof) transcription factors are widely involved in various life activities of plants. Forty-six IbDof genes from sweetpotato cv. Taizhong 6 with a highly conserved Dof domain structured as a C2C2-type zinc finger were identified and named from IbDof1 to IbDof46 according to their position on the chromosomes. IbDof family could be divided into four subgroups (A-D), which shared the similar motifs and gene structures. Motif 1 and Motf 2 occurred in all of the identified IbDofs, Motif 5 and Motif 9 only occurred in subgroup A, and Motif 6-Motif 8 and Motif 10 only occurred in subgroup D. Twelve segment duplicated gene pairs and five tandem duplicated gene pairs of IbDofs (IbDof2/IbDof3, IbDof12/IbDof13, IbDof9/IbDof10, IbDof28/IbDof29, and IbDof32/IbDof33) contributed to the expansion of IbDof family in sweetpotato. The average divergence times of segmental duplication gene pairs and tandem duplicated gene pairs seemed to have emerged 35.22 MYA and 1.86 MYA, and the Ka/Ks ratios of the paralogous IbDofs were range from 0.07 (IbDof12/IbDof13) to 0.68 (IbDof6/IbDof25). Tirty-eight orthologous IbDof gene pairs between sweetpotato and their wild relative species Ipomoea trifida were involved in duplicated genomic blocks based on synteny analysis. Transcriptome analysis indicated different subgroups expressed specifically in various tissues, and IbDofs in the same subgroup also revealed different expression tends. Various hormones and stresses response element were identified in the promoters of IbDof genes, and qRT-PCR demonstrated specific IbDof genes responded to various environmental stresses, including cold, drought, salt, and H2O2. Most IbDof genes were regulated by cold treatment; IbDof10 and IbDof14 were up-regulated by drought treatment; IbDof-2, -14, -37, -41, -43 were up regulated by high salt stress; and IbDof-8, -10, -25, -41 were up regulated by H2O2 treatment. In summary, our result indicated that IbDof family genes coordinately regulated the growth and development of sweetpotato and been involved in the various abiotic stresses process.

Key words: sweetpotato, Dof gene family, evolution analysis, gene expression profiling

表1

实时荧光定量PCR所用的引物"

基因名称
Gene name
正向引物序列
Forward sequence (5'-3')
反向引物序列
Reverse sequence (5'-3')
Dof2 GCTGTTGTTGTTGGATTATC CGTTGTTAGGTAGAGTTGTC
Dof5 CTCATTGCCGTCATATTACC TCCTGCCATAGAACTTGTAG
Dof8 GCTGGAGATAATGGAGATGA AACTGCGGTCAATGGATAA
Dof10 GGTTGTGGAGAATGTTGTG GAGGAAGAAGATGATAAGGAAG
Dof14 GAAGGTGATGATGACAACAG CCATGCCAAGAAGAACCA
Dof15 GCTTCATCTCCTCCTCCT GTTCCTTCATCCTCTTCACT
Dof17 CAACTATGGCGACTGATAATC GATGTTCCTCCTCCTCCT
Dof21 TTACTGTCGAACATGCAGACGC GTGGTTGGAGTTGAGGTAGTGA
基因名称
Gene name
正向引物序列
Forward sequence (5'-3')
反向引物序列
Reverse sequence (5'-3')
Dof24 CGACGAGATATTGGAACCTT GGCTGGCTCTGTTGAATC
Dof25 CTACAACAACTACAACCTCTC AGCAATGACGTGAAGGAG
Dof33 TCCGTTCCTCGATCCAAT TGATGGTGTGATTGTTGTTG
Dof37 CGACCAAGACTGAAGATGA GGTGATGTGGCAATGAGA
Dof40 CTCCATCATCATCATCATCATC GGCAGCAGTAGTCATTCC
Dof41 CATCATCATCATCATCTCCATC GGCAGCAGTAGTCATTCC
Dof43 TTCTGTTACGACACCTTCC TACTGCTTCTACCGACTCT

表2

IbDof基因家族相关信息"

基因名称
Gene name
基因编号
Gene ID
染色体定位
Chromosome
CDS长度
Coding domain sequence (bp)
氨基酸长度
Amino acid length
等电点
Isoelectric point (pI)
分子量
Molecular weight (kD)
亚细胞定位
Subcellular location
分组
Group
IbDof1 g1970.t1 1 891 297 6.35 31.96 细胞核 Nucleus D3
IbDof2 g5563.t1 1 807 269 4.94 29.78 细胞核 Nucleus D3
IbDof3 g5605.t1 1 807 269 4.94 29.76 细胞核 Nucleus D3
IbDof4 g6131.t1 1 600 200 9.16 20.99 细胞核 Nucleus C2.2
IbDof5 g9564.t1 2 1164 388 9.33 42.57 细胞核 Nucleus B1
IbDof6 g10705.t1 2 561 187 9.60 21.15 细胞核 Nucleus B2
IbDof7 g11533.t1 3 1542 514 5.28 55.81 细胞核 Nucleus B1
IbDof8 g11556.t1 3 1494 498 5.09 53.53 细胞核 Nucleus B1
IbDof9 g15022.t1 3 759 253 8.15 27.43 细胞核 Nucleus D1
IbDof10 g15141.t1 3 732 244 8.90 26.49 细胞核 Nucleus D1
IbDof11 g17470.t1 4 330 110 9.66 12.60 细胞核 Nucleus B2
IbDof12 g21747.t1 4 960 320 9.20 33.86 细胞核 Nucleus A
IbDof13 g21785.t1 4 957 319 9.20 33.80 细胞核 Nucleus A
IbDof14 g21856.t1 4 846 282 6.78 30.99 细胞核 Nucleus D2
IbDof15 g26198.t1 5 1587 529 6.19 58.14 细胞核 Nucleus B1
IbDof16 g26413.t1 5 786 262 9.18 27.65 细胞核 Nucleus C1
IbDof17 g26723.t1 5 825 275 7.17 29.90 细胞核 Nucleus D1
IbDof18 g27204.t1 5 807 269 5.03 29.57 细胞核 Nucleus D3
IbDof19 g28361.t1 6 498 166 9.73 19.31 细胞核 Nucleus B2
IbDof20 g28392.t1 6 606 202 9.32 22.91 细胞核 Nucleus B2
IbDof21 g28584.t1 6 606 202 8.45 22.54 细胞核 Nucleus B2
IbDof22 g28692.t1 6 687 229 8.79 25.43 细胞核 Nucleus B2
IbDof23 g31777.t1 6 630 210 8.16 22.06 细胞核 Nucleus C2.2
IbDof24 g32164.t1 6 1164 388 9.05 42.28 细胞核 Nucleus C2.2
IbDof26 g34747.t1 7 786 262 8.64 28.71 细胞核 Nucleus D2
IbDof27 g34761.t1 7 786 262 8.49 28.58 细胞核 Nucleus D2
IbDof28 g38905.t1 8 657 219 8.84 23.95 细胞核 Nucleus D1
IbDof29 g38982.t1 8 1038 346 8.83 38.71 细胞核 Nucleus D1
IbDof30 g42311.t1 9 939 313 9.33 34.08 细胞核 Nucleus C3
IbDof31 g42323.t1 9 1014 338 9.14 36.42 细胞核 Nucleus C3
IbDof32 g46228.t1 9 972 324 9.60 33.39 细胞核 Nucleus A
IbDof33 g46266.t1 9 1032 344 9.30 35.94 细胞核 Nucleus A
IbDof34 g48623.t1 10 861 287 6.19 31.08 细胞核 Nucleus D3
IbDof35 g50844.t1 10 1050 350 8.90 37.73 细胞核 Nucleus C3
IbDof36 g50864.t1 10 837 279 6.15 30.23 细胞核 Nucleus B1
IbDof37 g53865.t1 11 1398 466 5.76 50.71 细胞核 Nucleus B1
基因名称
Gene name
基因编号
Gene ID
染色体定位
Chromosome
CDS长度
Coding domain sequence (bp)
氨基酸长度
Amino acid length
等电点
Isoelectric point (pI)
分子量
Molecular weight (kD)
亚细胞定位
Subcellular location
分组
Group
IbDof38 g55460.t1 11 1050 350 6.97 38.44 细胞核 Nucleus D2
IbDof39 g57153.t1 12 1002 334 9.63 35.93 细胞核 Nucleus A
IbDof40 g58686.t1 12 1008 336 8.24 36.37 细胞核 Nucleus D1
IbDof41 g59490.t1 12 1011 337 8.21 36.31 细胞核 Nucleus D1
IbDof42 g59977.t1 12 939 313 9.48 33.42 细胞核 Nucleus A
IbDof43 g65472.t1 14 1317 439 7.22 47.43 细胞核 Nucleus B1
IbDof44 g65553.t1 14 1053 351 8.99 39.00 细胞核 Nucleus B2
IbDof45 g68289.t1 14 804 268 8.24 29.58 细胞核 Nucleus D2
IbDof46 g68388.t1 14 882 294 9.17 30.81 细胞核 Nucleus A

图1

甘薯、拟南芥和水稻Dof家族蛋白系统发育树"

图2

甘薯Dof基因保守序列和基因结构分析"

图3

甘薯Dof启动子顺式作用元件分析"

表3

甘薯Dof基因复制事件类型、同义替换率、非同义替换率及其比值"

基因I
Gene I
基因II
Gene II
复制类型
Type of duplication
同义
替换率
Ka
非同义
替换率
Ks
Ka/Ks 分歧时间(百万年前)
Divergence time (MYA)
IbDof2 IbDof3 串联重复序列 Tandem duplicated gene pairs 0.001611 0.016886 0.095413 0.562876
IbDof4 IbDof25 染色体片段复制 Segmental duplication gene pairs 0.158131 0.233118 0.678330 7.77059
IbDof7 IbDof15 染色体片段复制 Segmental duplication gene pairs 0.275346 0.952055 0.289212 31.73516
IbDof9 IbDof10 串联重复序列 Tandem duplicated gene pairs 0.016325 0.053043 0.307773 1.768089
IbDof10 IbDof17 染色体片段复制 Segmental duplication gene pairs 0.291817 1.563082 0.186693 52.10274
IbDof12 IbDof13 串联重复序列 Tandem duplicated gene pairs 0.006995 0.097434 0.071789 3.247803
IbDof14 IbDof27 染色体片段复制 Segmental duplication gene pairs 0.228799 1.117142 0.204807 37.23806
IbDof28 IbDof29 串联重复序列 Tandem duplicated gene pairs 0.008162 0.020799 0.392429 0.693285
IbDof32 IbDof33 串联重复序列 Tandem duplicated gene pairs 0.030506 0.102014 0.299038 3.400458
IbDof35 IbDof30 染色体片段复制 Segmental duplication gene pairs 0.495625 1.024638 0.483707 34.15461
IbDof39 IbDof12 染色体片段复制 Segmental duplication gene pairs 0.624101 1.595607 0.391137 53.1869
基因I
Gene I
基因II
Gene II
复制类型
Type of duplication
同义
替换率
Ka
非同义
替换率
Ks
Ka/Ks 分歧时间(百万年前)
Divergence time (MYA)
IbDof40 IbDof41 染色体片段复制 Segmental duplication gene pairs 0.006582 0.044195 0.148927 1.47318
IbDof40 IbDof29 染色体片段复制 Segmental duplication gene pairs 0.338989 0.932423 0.363557 31.08075
IbDof45 IbDof14 染色体片段复制 Segmental duplication gene pairs 0.290234 0.953107 0.304514 31.77022
IbDof45 IbDof27 染色体片段复制 Segmental duplication gene pairs 0.287356 1.117214 0.257208 37.24048
IbDof46 IbDof13 染色体片段复制 Segmental duplication gene pairs 0.217848 1.104950 0.197156 36.83168
IbDof46 IbDof12 染色体片段复制 Segmental duplication gene pairs 0.192186 1.127080 0.170517 37.56932

图4

甘薯Dof基因在染色体上的位置及基因复制事件"

图5

甘薯和其近缘野生种Ipomoea trifida同源Dof共线性关系 甘薯染色体(上部)与Ipomoea trifida (下部)之间的连线表示Dof同源基因对。"

图6

甘薯Dof基因在不同组织的表达模式 A: 甘薯Dof基因在徐薯22中不同组织中的表达模式; B: 甘薯Dof基因在徐薯18中不同组织中的表达模式。图中IbDof基因在每个组织中的数值为FPKM值。"

图7

甘薯Dof基因在低温胁迫下相对表达分析 *和**分别表示在0.05和0.01水平上差异显著。"

图8

甘薯Dof基因在干旱胁迫下相对表达分析 **表示在0.01水平上差异显著。"

图9

甘薯Dof基因在盐胁迫下相对表达分析 *和**分别表示在0.05和0.01水平上差异显著。"

图10

甘薯Dof基因在H2O2胁迫下相对表达分析 *和**分别表示在0.05和0.01水平上差异显著。"

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