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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (6): 844-857.doi: 10.3724/SP.J.1006.2020.94171

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

Cloning and expression analysis of sugarcane Fe/S precursor protein gene ScPetC

ZHENG Qing-Lei1,YU Chen-Jing1,YAO Kun-Cun1,HUANG Ning1,QUE You-Xiong1,4,LING Hui2,3,*(),XU Li-Ping1,4,*()   

  1. 1Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture and Rural Area / Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    2College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    3College of Crop Science, Yulin Normal University, Yulin 537000, Guangxi, China
    4Key Laboratory of Crop Genetics and Breeding and Comprehensive Utilization, Ministry of Education / Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
  • Received:2019-11-10 Accepted:2020-01-15 Online:2020-06-12 Published:2020-02-17
  • Contact: Hui LING,Li-Ping XU E-mail:linghuich@163.com;xlpmail@126.com
  • Supported by:
    National Natural Science Foundation of China(31801424);China Agriculture Research System(CARS-17)

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Abstract:

Cytochrome b6f complex Rieske Fe/S precursor protein (PetC) is encoded by the nuclear PetC gene, and its mature protein involved in the formation of the cytochrome b6f complex, which is important for electron transfer. Based on our previous transcriptome data of sugarcane (Saccharum spp. hybrids) infected by Sorghum mosaic virus (SrMV), a cytochrome b6f complex reduced iron-sulfur precursor protein gene was cloned from leaves of sugarcane superior elite cultivar ‘ROC22’, and named as ScPetC (GenBank accession number: MH333037.1). Bioinformatics analysis showed that the ScPetC gene was 824 bp in length, containing a 678 bp open reading frame (ORF), and encoding a peptide of 226 amino acids. ScPetC belongs to the PRK13473 superfamily and has a typical Rieske domain at its C-terminus of the amino acid chain. ScPetC is a stable and hydrophilic protein with pI 8.19. Most of the secondary structural elements in ScPetC were random coil. Compared to the PetC from the other plants, ScPetC contained one more fragment of helix in its third dimensional structure. The transient expression of YFP-fused protein in Nicotiana benthamiana leaves showed that ScPetC was located in the chloroplast, cytoplasm and cell membrane. Although the previous study indicated that the expression of ScPetC was affected by SrMV infection in sugarcane, different to the interaction between Pinellia ternata PetC and SMV-P1, without interaction between ScPetC and the SrMV-P1, it did interact with SCYLV-P0, i.e. the P0 protein of Sugarcane yellow leaf virus (SCYLV). Real-time quantitative PCR analysis showed that ScPetC gene was expressed constitutively in different tissues of sugarcane, and the highest level of its expression was found in leaves. When sugarcane plant was exposed to abscisic acid stress for 3 h, the expression of ScPetC was significantly up-regulated, but the expression level was then inhibited with longer treatment time. Under the stress of methyl jasmine, salicylic acid, copper chloride, cadmium chloride and sodium chloride, the expression of ScPetC was significantly down-regulated. The study on biological function, expression pattern and interaction of ScPetC with the proteins of sugarcane pathogenic virus will improve the understanding of the happening of yellow leaf symptom, which caused by SCYLV.

Key words: sugarcane, cytochrome b6f complex Rieske Fe/S precursor protein, bioinformatics, subcellular location, protein interaction, real-time flourescent quantitative PCR

Table 1

Primer sequence used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 用途
Usage
ScPetC-F TGGACGCCAGGGAGTGAAC 基因克隆
Gene cloning
ScPetC-R TGTAGCAACAACGCCTTCGG
ScPetC-QF GTACAACAACCAGGGCAAGG 实时荧光定量PCR
qRT-PCR
ScPetC-QR CAGGGACAGGAGGCATACAT
CAC-F ACAACGTCAGGCAAAGCAAA 内参基因
Reference gene
CAC-R AGATCAACTCCACCTCTGCG
CUL-F TGCTGAATGTGTTGAGCAGC 内参基因
Reference gene
CUL-R TTGTCGCGCTCCAAGTAGTC
ScPetC-Subloc-F CGAACGATACTCGAGGTCGACATGGCGACCTCCGCG 亚细胞定位载体
Vector for subcellular localization
ScPetC-Subloc-R GCTCACCATACTAGTGGATCCTGCCTTCCACCACGG
ScPetC-AD-F CCATGGAGGCCAGTGAATTCATGGCGACCTCCGCGGCGCT 酵母双杂交载体
Vector for Y2H
ScPetC-AD-R AGCTCGAGCTCGATGGATCCTGCCTTCCACCACGGGTCCT
SrMV-P1-BK-F TGGCCATGGAGGCCGAATTCATGGCAGGAGCATGGAGCAC 酵母双杂交载体
Vector for Y2H
SrMV-P1-BK-R CGCTGCAGGTCGACGGATCC AAAATAAGTAATTTCATTGA
SCYLV-P0-BK-F TGGCCATGGAGGCCGAATTCATGCTTTTCAACGAATTCTC 酵母双杂交载体
Vector for Y2H
SCYLV-P0-BK-R CGCTGCAGGTCGACGGATCCTATATCATGAGAATAGGTGC
ScPetC-YN-F TCTGAGGAGGATCTTCCCGGGATGGCGACCTCCGCGGCGCT 双分子荧光互补实验载体
Vector for BiFC
ScPetC-YN-R AGGGCATGCCTGCAGGTCGACGTGCCTTCCACCACGGGTCCT
SrMV-P1-YC-F TCTAGGAGCTCGGTACCCGGGATGGCAGGAGCATGGAGCAC 双分子荧光互补试验载体
Vector for BiFC
SrMV-P1-YC-R ATCGTATGGGTACATACTAGTAAAATAAGTAATTTCATTGA
SCYLV-P0-YC-F CTAGGAGCTCGGTACCCGGGATGCTTTTCAACGAATTCTC 双分子荧光互补实验载体
Vector for BiFC
SCYLV-P0-YC-R TCGTATGGGTACATACTAGTTATATCATGAGAATAGGTGC

Table 2

Database and software used for bioinformatics analysis"

工具Tool 网址Website
ORF Finder https://www.ncbi.nlm.nih.gov/orffinder/
ExPaSy http://web.expasy.org/protparam/
Prabi https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_hnn.html
SWISSMODEL http://swissmodel.expasy.org/interactive
SignalP 4.1 Server http://www.cbs.dtu.dk/servers/SignalP/
NCBI conserved domain http: //blast.ncbi.nlm.nih.gov/
WoLFPSORT https://wolfpsort.hgc.jp/
TMHMM 2.0 Server http://www.cbs.dtu.dk/servers/TMHMM/
ProtScale https://web.expasy.org/protscale/
MEME http://meme-suite.org/

Fig. 1

RT-PCR amplification of ScPetC gene in sugarcane and its amino acid sequence (A) M: DNA marker 100 bp; I: RT-PCR product. (B) ScPetC: Sugarcane cytochrome b6f complex reduced iron-sulfur protein precursor sequence; SoCYT: Sugarcane cytochrome b6f complex iron-sulfur subunit amino acid sequence[34]. "

Table 3

Primary structure analysis of ScPetC"

一级结构特性Primary structure characteristic 预测结果Predicted result
编码的氨基酸个数Number of coding amino acids 225
等电点 Theoretical pI 8.19
分子量 Molecular weight (×103) 23.85
分子式 Formula C1054H1668N294O315S11
不稳定系数 Instability index (II) 21.61
平均疏水性Grand average of hydropathicity (GRAVY) -0.146

Fig. 2

Prediction result of sugarcane ScPetC by bioinformatics software A: signal peptide; B: transmembrane domain; C: conservative domain. "

Fig. 3

Predicted third structure of mature PetC in Saccharum spp. hybrids, Oryza sativa, Pinellia ternate, and Spinacia oleracea Yellow arrow is the conservative binding domain [2Fe-2S] of PetC; blue and red arrows represent the N-terminus and C-terminus of the amino acid chain; green arrows represent α helix. White arrow represents the α helix in sugarcane that is different from other plants of tertiary structure in figure A. "

Fig. 4

Amino acid sequence aligenment of sugarcane ScPetC and other plant PetC Red squares indicate the reported conserved motif of amino acid regions within the ScPetC. CAM57108: Pinellia ternate; XP_015647138: Oryza sativa; XP_002441121: Sorghum bicolor; P08980: Spinacia oleracea; P26291: Pisum sativum; CAA45705: Nicotiana benthamiana. "

Fig. 5

Phylogenetic tree analysis of sugarcane ScPetC and PetC in other species and their corresponding motif prediction The orange part of the phylogenetic tree represents the cytochrome b6f complex cluster, the blue part represents the cytochrome bc1 complex cluster; the red cycle is labeled as ScPetC; the different color boxes in the figure represent different groups (I, II, III, IV, V, VI, VII). NP_001237648.2: Glycine max; XP_015647138.1: Oryza sativa; XP_0202013301.1: Triticum aestivum; Q9ZR03.1: Arabidopsis thaliana; P26291: Pisum sativum; AAD55565.1: Volvox carteri f nagariensis; CAA27194: Rhodobacter sphaeroides; CAA45705: Nicotiana tabacum; CAA70823: Phormidium laminosum; CAB72244: Trichormus variabilis; CAM57108.1: Pinellia ternata; CAM57109.1: Zantedeschia aethiopica; O49078: Fritillaria agrestis; P05417: Paracoccus denitrificans; P08067: Saccharomyces cerevisiae; P08980: Spinacia oleracea; P14698: Desmonostoc sp. PCC; P23136: Rhodospirillum rubrum; P26290: Synechocystis sp. PCC; P26291: Pisum sativum; P26292: Synechococcus sp. PCC; P37841: Solanum tuberosum; P49728: Chlamydomonas reinhardtii; Q9ZR03: Arabidopsis thaliana; XP_002441121: Sorghum bicolor; ACG28308: Zea mays; XP_004951312: Setaria italica. motif 1: X6NAXCTHLGCVVPX3ENKFXCPCHGSX10GPAP; motif 2: NLX11PDX3RX3NLX22PX3GX3GXXAKGXXGNDX6LX7LXXGLKGDPTYX2V; motif 3: X7WXETDFRTX3PWW; motif 4: XGDX3 GWFCPCHGSHYDX2GRIRXGPAPXNLX15; motif 5: X7MX4DX2AX5 VX7GX6 WXGKPVFXRXRX4IX5VX4LXDX10; motif 6: X2AXSIX ADXV; motif 7: XWLX3GXCTHLGCX; motif 8: X9QLX10; motif 9: LVVEXDXT; motif 10: FX5FX3DDSSX2RSXSPSLXSXFLX3RGFS SNSVSPAHX2GLVXDLPXTVAAIKNPXSKIVYDX2 NHERYPPGDPSKRAFAYFVLTGGRFVY. "

Fig. 6

Results of subcellular localization of sugarcane ScPetC Subcellular localizations of the Agrobacterium tumefaciens mediated transformation of ScPetC and empty vector in Nicotiana benthamiana leaves after 48 h infiltration; the epidermal cells of Nicotiana benthamiana were used for taking images of visible light, yellow fluorescence, chloroplast autofluorescence, and merged visible light. Blue arrows 1 and 2 indicate nucleus and plasma membrane. The area in the red rectangle is magnified. Bar=50 μm; 35S::YFP: the Agrobacterium tumefaciens strain carrying the empty vector pCAMBIA2300-YFP; 35S:: ScPetC::YFP: the Agrobacterium tumefaciens strain carrying the recombinant vector pCAMBIA2300-ScPetC-YFP."

Fig. 7

The interaction assay of sugarcane ScPetC with virus proteins of Sorghum mosaic virus (SrMV) or Sugarcane yellow leaf virus (SCYLV) A: ScPetC was verified by yeast double-hybrid interaction with SrMV-P1 protein. B: ScPetC was verified by yeast double-hybrid interaction with SCYLV-P0 protein; SD/-Ade/-His/Leu/-Trp: synthetic dropout medium plate without adenine, histidine, leucine, tryptophan; SD/-Ade/-His/Leu/-Trp/X-α-Gal: synthetic dropout medium plate without adenine, histidine, leucine, tryptophan (plus 5-bromo-4-chloro-3- indoxyl-α-D-galactopyranoside); SD/-Ade/-His/Leu/-Trp/X-α-Gal/AbA: synthetic dropout medium plate without adenine, histidine, leucine, tryptophan (plus 5-bromo-4-chloro-3-indoxyl-α-D-galactopyranoside and aureobasidin A). C: the bimolecular fluorescence complementation (BiFC) assay of the interaction between ScPetC and SCYLV-P0 protein; YC, YN, YN-ScPetC, YC-P0 represent the plasmids pCAMBIA2300S-YC, pCAMBIA2300S-YN, pCAMBIA2300S-YN-ScPetC, pCAMBIA2300S-YC-P0, respectively[42]."

Fig. 8

Transcriptional expression pattern analysis of sugarcane ScPetC gene A: the tissure-specific expression analysis of ScPetC gene in sugarcane; B and C: relative expression of ScPetC in sugarcane under different exogenous stresses. Bars with different superscripts (a, b, c, d) represent differ significantly (P ≤ 0.05) and error bars represent the standard error of each treating group (n = 3)."

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