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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (5): 882-893.doi: 10.3724/SP.J.1006.2021.04128

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

Identification, localization and expression analysis of beta-carotene isomerase gene family in sugarcane

HUANG Ning2,*(), HUI Qian-Long1, FANG Zhen-Ming2, LI Shan-Shan2, LING Hui2, QUE You-Xiong1, YUAN Zhao-Nian1,*()   

  1. 1National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
    2College of Crop Science, Yulin Normal University, Yulin 537000, Guangxi, China
  • Received:2020-06-15 Accepted:2020-09-13 Online:2021-05-12 Published:2020-09-30
  • Contact: HUANG Ning,YUAN Zhao-Nian E-mail:hningch@ylu.edu.cn;yzn05@sina.com
  • Supported by:
    China Agriculture Research System(CARS-170208)

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Abstract:

Strigolactones (SLs), a class of plant hormones, exists widely in plants and involves in the regulation of plant tillering and environmental adaptation. β-carotene isomerase (D27) is a key enzyme for SLs synthesis, but there are few reports about the identification and analysis of D27 gene family in sugarcane. In this study, five members of D27 gene family from one of the original parent of modern sugarcane cultivars, Saccharum spontaneum, were analyzed. Phylogenetic tree analysis showed that these D27s from S. spontaneum was clustered into 3 different branches and were highly homologous to sorghum D27s. Conserved domain prediction revealed that D27s contained a typical domain of β-carotene isomerase, Pfam: DUF4033. The results of cis-element analysis showed that D27s mainly involved in the regulation of hormone response, plant growth and development, and stress response. Based on the transcriptomic data of the modern cultivated varieties, the expression analysis of the homologous transcripts of Saccharum spontaneum D27s showed that Sspon.06G0012830-1A was regulated at tillering stage and under Sporisorium scitamineum infection. Besides, the cDNA sequence of Sspon.06G0012830-1A was cloned from sugarcane cultivar ROC22 and named as ScD27.1 (GenBank accession number: MT499895). Bioinformatics analysis indicated that ScD27.1 encoded an unstable protein of 266 amino acids with an isoelectric point of 8.91 and a molecular weight of 30.00 kD, and might be located in the chloroplast, containing chloroplast transit peptides, 4 ubiquitination sites and 18 phosphorylation sites. Its secondary structure mainly included alpha helix and random coils. qRT-PCR analysis demonstrated that ScD27.1 was significantly induced by ABA and H2O2 and did not respond to MeJA and SA. Subcellular localization revealed that ScD27.1 might be located in cell membrane and chloroplast and involved in the vesicle sorting or transported by sorting vesicle in plant cells. The results suggested that ScD27.1 may involve in the tillering induced by S. scitamineum and signaling pathway of ABA and H2O2. The present study provides a basic understanding of intracellular transport and tillering in sugarcane and the involvement of ScD27.1 during sugarcane-S. scitamineum interaction.

Key words: β-carotene isomerase, gene family, bioinformatics, tillering, Sporisorium scitamineum

Table 1

List of primers"

引物名称 Primer name 引物序列 Primer sequence (5°-3°)
ScD27.1-F CTCGCAGCCTCACAAACGCGGGCGG
ScD27.1-R TCTCAAGCCAACGGTCTCCTA
G-ScD27.1-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGCCGTGCTTCCGCGACC
G-ScD27.1-R GGGGACCACTTTGTACAAGAAAGCTGGGTCCGCCTGAAGTTTGGGACAGA
q-ScD27.1-F GGGTGCTCAAGAAAT
q-ScD27.1-R CTCGTTGGTGAAGAAGTC
CAC-F ACAACGTCAGGCAAAGCAAA
CAC-R AGATCAACTCCACCTCTGCG
CUL-F TGCTGAATGTGTTGAGCAGC
CUL-R TTGTCGCGCTCCAAGTAGTC

Fig. 1

Genome wide identification of D27 gene family in Saccharum spontaneum A: chromosomal location of D27s in Saccharum spontaneum; B: amino acid sequence similarity of D27s in Saccharum spontaneum."

Fig. 2

Phylogenetic tree and domain analysis based on the amino acid sequences of D27s in Saccharum spontaneum and other 5 species The horizontal axis indicates the amino acid length of the proteins; the color line represents the protein from the different species; the color bar code represents the different domain of protein."

Fig. 3

Prediction of cis-elements in Saccharum spontaneum D27s The horizontal axis indicates region nucleotide length of gene promoter; the color bar code represents the different cis-element in the promoter region."

Table 2

Sequence similarity between D27s from Saccharum spontaneum and sugarcane cultivar"

基因名称
Gene ID		 6-BA和DA-6共同诱导甘蔗栽培种ROC22
分蘖转录组
Transcriptome of sugarcane cultivar ROC22
treated with 6-BA and DA-6		 甘蔗栽培种ROC22和YC05-179应答黑穗病菌
侵染转录组
Transcriptome of sugarcane cultivar ROC22 and YC05-179 responding to Sporisorium scitamineum infection
c116894.
graph_c0		 c112688.
graph_c0		 c161113.
graph_c0		 Sugarcane_
Unigene.66941		 Sugarcane_
Unigene.58014		 Sugarcane_ Unigene.87626
Sspon.06G0012830-3C 94.57 94.96
Sspon.06G0012830-1A 97.93 98.34
Sspon.07G0014180-2B 82.33 98.31
Sspon.07G0014180-3C 80 82.42
Sspon.06G0016140-2C 97.06 95.83

Fig. 4

Expression of D27s gene in different transcriptome in sugarcane cultivar A: the transcriptomic data from 6-BA and DA-6 induced tillering in sugarcane cultivar ROC22; B: the transcriptomic data from sugarcane cultivar ROC22 and YC05-179 in response to Sporisorium scitamineum infection. X-axial represents the accession of transcript presenting in the sugarcane transcriptomic data, while Y-axial represents the relative expression of transcript in the treatment sample comparing to the control and the significantly different expression data (P < 0.05) was marked by the asterisk (*)."

Fig. 5

Cloning and sequencing of ScD27.1 gene in sugarcane cultivar A: agrose electrophoretic analysis of RT-PCR product of ScD27.1; M: 100 bp-III DNA marker (Generay, Shanghai, China). B: the open reading frame of ScD27.1 (*, stop codon) and its aa sequence; the sequence fragment complementary to primer is bolded."

Table 3

Result of bioinformatics analysis"

生物信息学分析 Bioinformatics analysis 结果Result
一级结构特性
Primary structure characteristics		 氨基酸长度 Length of amino acids 266
等电点 Theoretical pI 8.91
分子量 Molecular weight (kD) 30.00
不稳定系数 Instability index (II) 56.40
二级结构类型
Secondary structure types		 α-螺旋 Alpha helix 30.08%
延伸链 Extended strand 12.41%
无规则卷曲 Random coil 57.52%
蛋白亚细胞定位
Protein subcellular location		 Plant-Ploc工具 Plant-Ploc tool 叶绿体Chloroplast
ProtComp 9.0工具 ProtComp 9.0 tool 膜结合叶绿体Membrane bound Chloroplast
WoLFPSORT工具 WoLFPSORT tool 叶绿体Chloroplast
功能位点
Function sites		 靶向肽类型 Type of targeting peptide 叶绿体转运肽Chloroplast transit peptide
泛素化位点 Ubiquitination sites 4
磷酸化位点 Phosphorylation sites 18

Fig. 6

Expression of ScD27.1 gene under different stresses A: the amplification efficiency curve of the primer pairs of ScD27.1; B: expression of ScD27.1 gene under different stress of ABA, MeJA, SA, and H2O2. *: significant difference at the 0.05 probability level; the error bars represent the standard error of the group (n = 3)."

Fig. 7

Identification of the subcellular location of ScD27.1 protein The figures from A to F indicate the cellular location of the protein under natural condition and the bar scale was 10 μm; the figures from G to L indicate the cellular location of the protein under wortmannin (16.5 μmol L-1) treatment and the bar scale was 15 μm."

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