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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (7): 1683-1696.doi: 10.3724/SP.J.1006.2022.14126

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

Identification and expression analysis of uncoupling protein gene family in sweetpotato

CHEN Lu(), ZHOU Shu-Qian, LI Yong-Xin, CHEN Gang, LU Guo-Quan, YANG Hu-Qing()   

  1. School of Food and Health, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China
  • Received:2021-07-15 Accepted:2021-10-19 Online:2022-07-12 Published:2021-11-01
  • Contact: YANG Hu-Qing E-mail:13097686588@yeah.net;yanghq@zafu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(31871857);China Agriculture Research System(CARS-10-B19);Natural Science Foundation of Zhejiang Province(LQ20C200002)

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Abstract:

The objectives of this study are to identify and analyze the uncoupling protein (UCPs) gene family members in sweetpotato (Ipopoea batatas (L.) Lam) and to investigate its expression specificity in different tissues of sweetpotato and its response to low temperature (4℃), high salinity (NaCl) and drought (PEG-6000) stresses. The results showed that the UCP (IbUCP) gene family of sweetpotato included five genes, which were named IbUCP1 (GenBank accession number: MW753000), IbUCP2 (GenBank accession number: MW753004), IbUCP3 (GenBank accession number: MW753001), IbUCP4 (GenBank accession number: MW753002), and IbUCP5 (GenBank accession number: MW753003), respectively. IbUCP contains 261-375 amino acid residues with the theoretical isoelectric point from 8.53 to 9.86. IbUCP were mainly located in mitochondria. IbUCP was a hydrophilic protein belonging to the superfamily of mitochondrial. The secondary structure of IbUCP was mainly composed of α-helix and random coils, which was consistent with the prediction of tertiary structure. IbUCP did not contain transmembrane helix structure and signal peptide. IbUCP family members were divided into five branches, which were closely related to Ipomoea triloba and Ipomoea nil, and was conserved to a certain extent. Promoter prediction revealed that IbUCPs family genes not only has basic transcription elements, but also some signal response elements, transcription factor recognition binding elements, and stress response cis-acting elements. Expression analysis showed that the IbUCPs gene family were tissue-specific. The IbUCP4 expressed the highest in stem, and the other IbUCPs genes were found expressed the highest in tuberous root. IbUCP1, IbUCP4, and IbUCP5 responded to low temperature stress.All members of the IbUCPs gene family all responded to high salinity stress. Additionally, under drought stress, IbUCP1, IbUCP4, and IbUCP5 all responded and reached the peak at different time, respectively. Various stresses can regulate the expression of IbUCPs, and this study provides a theoretical basis for the function mining of UCP gene in sweetpotato and the selection of sweetpotato stress-resistant varieties.

Key words: sweetpotato (Ipomoea batatas (L.) Lam), uncoupling protein, bioinformatics, relative expression analysis, stress

Table 1

Primers used for this study"

引物用途
Primer function		 引物名称
Primer name		 引物序列
Primer sequence (5′-3′)
qRT-PCR扩增
qRT-PCR amplification		 IbUCP1-F GGAAGGGCGTTATACCTGGC
IbUCP1-R TGGGATTAGCCACAGCGATT
IbUCP2-F GGAAGGGCGTTATACCTGGC
IbUCP2-R TGGGATTAGCCACAGCGATT
IbUCP3-F AAACGGCTTTTCGGGGCTAT
IbUCP3-R GGAGAGAAAGTGGCCGATCA
IbUCP4-F ACGATGAGATCAAGGAGGCG
IbUCP4-R TATCACATCCACCGGGTTCG
IbUCP5-F CGCAACTACAAAAGCGTGGT
IbUCP5-R AGCCCGTCCTTCATGATTCC
内参基因
Internal reference genes		 α-Tubulin-F CAACTACCAGCCACCAACTGT
α-Tubulin-R CAAGATCCTCACGAGCTTCAC

Table 2

Sequence and physicochemical properties analysis of UCP in sweetpotato"

基因名称 Gene name 编码氨基酸数量
Amino acids number		 分子式
Formula		 分子量
Molecular
weight		 等电点
pI		 不稳定指数
Instability index		 脂溶指数
Aliphatic
index		 总平均
亲水性
GRAVY		 亚细胞定位
Subcellular
localization
IbUCP1 308 C1485H2370N406O419S11 32,968.28 9.48 29.16 95.32 0.096 线粒体Mitochondria
IbUCP2 375 C1860H2868N476O512S15 40,571.01 8.53 38.33 93.25 0.261 线粒体Mitochondria
IbUCP3 261 C1254H2016N356O363S9 28,176.51 9.62 42.00 96.05 0.017 线粒体Mitochondria
IbUCP4 291 C1347H2182N384O396S14 30,541.32 9.82 30.88 88.80 0.087 线粒体Mitochondria
IbUCP5 319 C1511H2452N420O426S20 33,959.85 9.86 26.78 89.00 0.104 线粒体Mitochondria

Fig. 1

Hydrophilic and hydrophobic analysis of UCP in sweetpotato"

Table 3

Secondary structure prediction of UCP in sweetpotato (%)"

蛋白名称
Protein name		 α-螺旋
Alpha helix		 β-转角
Beta turn		 延伸链
Extended strand		 无规则卷曲
Random coil
IbUCP1 49.35 6.82 13.96 29.87
IbUCP2 44.53 7.73 17.33 30.40
IbUCP3 46.46 8.81 13.03 31.80
IbUCP4 47.08 6.53 10.65 35.74
IbUCP5 46.08 7.84 12.54 35.54

Fig. 2

Secondary and tertiary structure of UCP in sweetpotato"

Fig. 3

Conserved structural domain of UCP in sweetpotato"

Fig. 4

Conserved protein motif of UCP family in sweetpotato"

Fig. 5

Transmembrane structure analysis of UCP in sweetpotato"

Fig. 6

Prediction of signal peptide of UCP in sweetpotato"

Fig. 7

Amino acid sequence alignment between Ipomoea batatas UCP and Arabidopsis thaliana UCP"

Fig. 8

Phylogenetic tree of UCP family AtPUMP1, AtUCP2, AtUCP3, AtUCP4, AtUCP5: Arabidopsis thaliana PUMP1 (NP_190979.1), UCP2 (NP_974962.1), UCP3 (NP_172866.1), UCP4 (BAH56967.1), UCP5 (NP_179836.1); FvUCP4: Fragaria vesca subsp. Vesca UCP4 (XP_004299717.1); SpUCP1, SpUCP2-like isoform X1, SpUCP2-like isoform X2, SpUCP3, SpUCP5: Solanum pennellii UCP1 (XP_015086211.1), UCP2-like isoform X1 (XP_015086155.1), UCP2-like isoform X2 (XP_015086156.1), UCP3 (XP_015074974.1), UCP5 (XP_015058445.1); HbUCP1-like, UCP2-like, UCP3, UCP5-like: Hevea brasiliensis UCP1-like (XP_021664307.1), UCP2-like (XP_021647687.1), UCP3 (XP_021691868.1), UCP5-like (XP_0216 35248.1); ZjUCP1, ZjUCP2 isoform X1, ZjUCP2 isoform X2, ZjUCP2 isoform X3, ZjUCP3, ZjUCP5: Ziziphus jujuba UCP1 (XP_015897075.1), UCP2 isoform X1 (XP_015873942.1), UCP2 isoform X2 (XP_024926760.1), UCP2 isoform X3 (XP_024926761.1), UCP3 (XP_015895 050.1)、UCP5 (XP_015890603.1); LeUCP, LeUCP2, LeUCP3, LeUCP5: Lycopersicon esculentum UCP (NP_001234584.1), UCP2 (XP_004246961.1), UCP3 (XP_004238805.1), UCP5 (XP_0042 50140.1); TaUCP1a: Triticum aestivum UCP1a (BAB16384.1); OsUCP1, OsUCP2, OsUCP3, OsUCP5: Oryza sativa UCP1 (XP_015616794.1), UCP2 (XP_015622201.1), UCP3 (XP_015636356.1), UCP5 (XP_01565 0890.1); StUCP, StUCP3, StUCP5-like: Solanum tuberosum UCP (CAA72107.1), UCP3 (XP_006355129.2), UCP5-like (XP_006360391.1); HaUCP, HaUCP1, HaUCP3, HaUCP5: Helianthus annuus UCP (XP_022001748.1), UCP1 (XP_021988906.1), UCP3 (XP_021997212.1), UCP5 (XP_022009806.1); InUCP1, InUCP2-like, InUCP3, InUCP5, InUCP5-like: UCP1 (XP_019186835.1), UCP2-like (XP_019167977.1), UCP3 (XP_019165923.1), UCP5 (XP_019153217.1), UCP5-like (XP_019 18563 7.1); ItUCP1, ItUCP2-like isoform X1, ItUCP2-like isoform X1, ItUCP2-like isoform X2, ItUCP2-like isoform X3, ItUCP2-like isoform X4, ItUCP3, ItUCP5: Ipomoea triloba UCP1 (XP_03 1102145.1), UCP2-like isoform X1 (XP_031110514.1), UCP2-like isoform X2 (XP_031110 515.1), UCP2-like isoform X3 (XP_031110516.1), UCP2-like isoform X4 (XP_031110517.1), UCP3 (XP_031115656.1), UCP5 (XP_031109433.1); IbUCP1, IbUCP2, IbUCP3, IbUCP4, IbUCP5: Ipomoea batatas UCP1 (MW753000), UCP2 (MW753004), UCP3 (MW753001), UCP4 (MW753002), UCP5 (MW753003). "

Table 4

Predictive analysis of cis-acting elements of IbUCPs promoters"

顺式元件
Cis-element		 典型序列
Typical sequence		 特性
Characteristic		 基因
Gene
AE-box AGAAACAA, AGAAACTT 光响应元件的一部分
Part of a light responsive element		 IbUCP1, IbUCP2, IbUCP5
G-box CAGCAC 光响应的顺式作用元件
Cis-acting regulatory element involved in light responsiveness		 IbUCP1, IbUCP2, IbUCP3, IbUCP5
ARE AAACCA 厌氧诱导的顺式作用元件
Cis-acting regulatory element essential for the anaerobic induction		 IbUCP1, IbUCP4, IbUCP5
AuxRR-core GGTCCAT 生长素响应有关的元件
Cis-acting regulatory element involved in auxin responsiveness		 IbUCP3, IbUCP5
ERE AAATTTTA 乙烯响应
Ethylene responsive element		 IbUCP1, IbUCP3, IbUCP4, IbUCP5
CGTCA-motif, TGACG-motif ACTGC, TGACG MeJA响应
MeJA-responsiveness		 IbUCP5
TCA-element TTTTTTCTACC 水杨酸响应
Salicylic acid response element		 IbUCP3
as-1 TGACG 水杨酸响应
Salicylic acid response element		 IbUCP5
GARE-motif TATGTTG 赤霉素响应
Gibberellin-responsive element		 IbUCP4, IbUCP5
ABRE AACCCGG, ACGTG 脱落酸响应
Cis-acting element involved in the abscisic acid responsiveness		 IbUCP2, IbUCP3
MYB ACCAAC, TAACCA, CAACCA MYB 结合位点相关元件
Elements associated with MYB binding sites		 IbUCP1, IbUCP2, IbUCP3, IbUCP4, IbUCP5
顺式元件
Cis-element		 典型序列
Typical sequence		 特性
Characteristic		 基因
Gene
MRE AACCTAA MYB 结合位点相关元件
Elements associated with MYB binding sites		 IbUCP1, IbUCP4, IbUCP5
MBS CAACTG MYB 结合位点
MYB binding site		 IbUCP2, IbUCP3
W-box TTGACC WRKY转录因子结合位点
WRKY transcription factor binding site		 IbUCP1, IbUCP4, IbUCP5
CAT-box GCCACT 分生组织相关的顺势调节元件
Cis-acting regulatory element related to meristem expression		 IbUCP2, IbUCP5
circadian CTATAGAAAT 参与昼夜节律调控的顺式调控元件
Cis-acting regulatory element involved in circadian control		 IbUCP3
TC-rich repeats GTTTTCTTA 参与防御和胁迫响应的顺式调控元件
Cis-acting element involved in defense and stress responsiveness		 IbUCP1, IbUCP2
O2-site GGTGGAGTAG 参与玉米醇溶蛋白代谢调节的顺式作用调节元件
Cis-acting regulatory element involved in zein metabolism regulation		 IbUCP1
GCN4_motif TGAGTCA 参与胚乳表达的顺式调控元件
Cis-regulatory element involved in endosperm expression		 IbUCP1

Fig. 9

Tissue specific expression analysis of IbUCPs genes Different lowercase letters above the bars indicate significant difference at the 0.05 probability level. "

Fig. 10

Roots and seedlings of sweetpotato after stress treatment A: sweetpotato roots were treated at low temperature for 0, 1, 2, 3, 7, 14, 21, and 28 days respectively from left to right; B: sweetpotato seedlings were treated at low temperature for 0, 1, 3, 6, 12, 24, and 48 hours respectively from left to right; C: sweetpotato seedlings were treated at high salinity for 0, 1, 3, 6, 12, 24, and 48 hours respectively from left to right; D: sweetpotato seedlings were treated at drought for 0, 1, 3, 6, 12, 24, and 48 hours respectively from left to right. "

Fig. 11

Relative expression analysis of IbUCPs gene family in response to abiotic stress *, **, and *** mean significant differences among different treatments of same gene at the 0.05, 0.01, and 0.001 probability levels, respectively. "

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