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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (2): 414-425.doi: 10.3724/SP.J.1006.2023.24022

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

Identification and expression analysis of PIN-LIKES gene family in sugarcane

PAN Jie-Ming1(), TIAN Shao-Rui3, LIANG Yan-Lan2, ZHU Yu-Lin1, ZHOU Ding-Gang4, QUE You-Xiong2, LING Hui1,*(), HUANG Ning1,*()   

  1. 1College of Agriculture, Yulin Normal University, Yulin 537000, Guangxi, China
    2Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian), Ministry of Agriculture and Rural Affairs / Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    3College of Plant Protection, Southwest University, Chongqing 400700, China
    4College of Life Science, Hunan University of Science and Technology/ Key Laboratory of Economic Crops Genetic Improvement and Integrated Utilization, Xiangtan 411201, Hunan, China
  • Received:2022-01-17 Accepted:2022-05-05 Online:2022-05-12 Published:2022-05-12
  • Contact: LING Hui,HUANG Ning E-mail:jiemingpan@163.com;linghuich@163.com;hning2012@126.com
  • Supported by:
    National Natural Science Foundation of China(31801424);National Natural Science Foundation of China(32160435);Scientific Research Foundation of Yulin Normal University(G2020ZK03)

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Abstract:

PILS (PIN-LIKES) is a newly reported auxin efflux carrier, which is involved in the polar transport of auxin. In this study, bioinformatics analysis indicated that 11 SsPILS genes and 4 ScPILS genes were identified from the Saccharum spontaneum genomic and the sugarcane cultivars transcriptomic data. 11 SsPILS gene family members were located at 6 chromosomes, and had 5-11 introns, respectively. Phylogenetic tree analysis showed that these PILS from S. spontaneum was clustered into 3 different branches and had highly homologous with PILS from Oryza sativa. The transcriptomic data revealed that, ScPILS1c, the ortholog of SsPILS1c, was differentially expressed in the sugarcane cultivars under Sorghum mosaic virus and Sporisorium scitamineum stress. ScPILS1c (NCBI accession number: OM258732), cloned by RT-PCR, had 1332 bp in length, containing a 1233 bp complete open reading frame and encoding 410 amino acids residues. The isoelectric point, instability coefficient, and average hydrophobicity value of ScPILS1c were 6.17, 39.31, and 0.689, respectively. The ScPILS1c was predicted as a stable acid hydrophobicity protein. The secondary structure of ScPILS1c protein was mainly composed of α-helix (48.54%) and random coils (35.37%) and was highly consistent with the prediction of tertiary structure. The qRT-PCR demonstrated that the relative expression of ScPILS1c was tissue-specific and the highest expression was observed in epidermis while the lowest in pith. ScPILS1c was significantly induced by H2O2 treatment and Sporisorium scitamineum infection. Transient expression on Nicotiana benthamiana leaves suggested that ScPILS1c was localized on cell membrane. This study provides a reference for further research on the structure and function of PILS gene in sugarcane.

Key words: sugarcane, PILS gene, bioinformatics, qRT-PCR, gene family

Table 1

Primers used in this study"

引物
Primer name		 引物序列
Primer sequence (5'-3')
PILS1c-F GCGCGGTTGTTGAGGATAAG
PILS1c-R ACGACAATACAACACTTCGGC
YFP-PILS1c-F CGAACGATACTCGAGGTCGACATGGATCTCGTACAACTCTTCATC
YFP-PILS1c-R GCTCACCATACTAGTGGATCCCGACAGCGTCCACATGAAGAAG
q-PILS1c-F AGCTCTTTGCTCCTTCGACC
q-PILS1c-R GACAACTATGACGCCGGCTA
CUL-F TGCTGAATGTGTTGAGCAGC
CUL-R TTGTCGCGCTCCAAGTAGTC
CAC-F ACAACGTCAGGCAAAGCAAA
CAC-R AGATCAACTCCACCTCTGCG

Fig. 1

Chromosomal distribution (A) and syntenic analysis (B) of PILS gene family in Saccharum spontaneum The red line indicates synteny within PILS and the grey line indicates synteny within all genes in Saccharum spontaneum genome."

Table 2

Analysis of physicochemical properties and subcellular localizations prediction of PILS proteins in Saccharum spontaneum"

蛋白名称Protein name 基因编号
Genetic code		 氨基酸个数Number of amino acids 等电点
pI		 分子量
Molecular weight (kD)		 亚细胞定位
Subcellular localization
SsPILS1a Sspon.02G0011550-3C 847 9.76 89.11 质膜 Plasma membrane
SsPILS1b Sspon.02G0011610-1A 436 7.47 46.48 质膜 Plasma membrane
SsPILS1c Sspon.02G0011610-2B 422 7.63 45.10 质膜 Plasma membrane
SsPILS1d Sspon.02G0011610-3C 436 8.02 46.47 质膜 Plasma membrane
SsPILS1e Sspon.02G0011550-1A 412 8.02 44.08 质膜 Plasma membrane
SsPILS5a Sspon.01G0050600-1C 339 7.59 36.61 质膜 Plasma membrane
SsPILS5b Sspon.01G0001780-4D 431 7.01 47.33 质膜 Plasma membrane
SsPILS6a Sspon.03G0003950-1P 433 8.52 46.92 质膜 Plasma membrane
SsPILS6b Sspon.03G0003950-2C 433 8.56 46.97 质膜 Plasma membrane
SsPILS7a Sspon.02G0008260-1A 337 8.10 36.37 质膜 Plasma membrane
SsPILS7b Sspon.02G0008260-2B 301 6.12 32.59 质膜 Plasma membrane

Fig. 2

Phylogenetic tree of PILS in Saccharum spontaneum (Ss), Arabidopsis thaliana (At), and Oryza sativa (Os)"

Fig. 3

Conserved motifs (A)and gene structures (B) of S. spontaneum PILS gene family Different colored boxes indicate different conserved motifs, and black lines indicate amino acid sequences in Fig. 3-A. Different colored boxes indicate different gene structures, and black lines indicate introns in Fig. 3-B."

Fig. 4

Relative expression level of ScPILS genes under different biotic stresses A: Sorghum mosaic virus infection; B: Sporisorium scitamineum infection."

Fig. 5

Bioinformatics analysis of ScPILS1c protein A: the open reading frame of SsPILS1c gene and its encoded protein; B: the conserved domain of ScPILS1c protein; C: the transmembrane helices of ScPILS1c protein; D: the comparison of third structure model of ScPILS1c with 5aymA; E: the third structure model of ScPILS1c, and red color indicates transmembrane domain."

Fig. 6

Predicted hydrophobicity/hydrophilicity of the amino acid sequence of ScPILS1c protein"

Fig. 7

Relative expression level of ScPILS1c genes in different tissues Different lowercase letters above the bars indicate significant difference at the 0.05 probability level."

Fig. 8

Relative expression of ScPILS1c genes under different stresses A: H2O2 stress; B: Sporisorium scitamineum stress. Different lowercase letters above the bars indicate significant difference at the 0.05 probability level."

Fig. 9

Subcellular localization of ScPILS1c in the lower epidermal cells of Nicotiana benthamiana"

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