Tissue Expression and Subcellular Localization of Osp39 Gene in Rice

doi:10.7668/hbnxb.20193511

Abstract

Abstract:

To explore the role of Osp39 gene in the regulation of rice chloroplast protein import, the sequence of Osp39 gene and upstream elements in its promoter were searched and analyzed by using bioinformatics assay,and the homology,properties and structure of the OsP39 protein were also carried out.The tissue expression of Osp39 gene and subcellular localization of OsP39 protein were analyzed by quantitative PCR and transient expression in protoplasts,respectively.The results showed that the Osp39 gene was located on chromosome 5 of rice.There were 11 exons in the coding region of Osp39 gene,and its transcription product had no alternative splicing mode,so there was only one type of transcription product.The Osp39 promoter contained a variety of light-responsive and chloroplast regulatory elements such as I box and ATCT-motif.Transcriptional analysis of Osp39 gene also showed that the transcription level of Osp39 gene in green tissues such as leaf and leaf sheath was significantly than that in non-green tissue such as root and flower during seeding and flowering stages.OsP39 protein was stable and heat-resistant,which molecular weight was about 38.7 ku and theoretical isoelectric point was pH 8.64.There were a total of 361 amino acid residues in OsP39 protein,of which the content of glycine was the highest.The 245-271 amino acid residues formed a highly conserved L6 loop region,which belonged to the characteristic domain of the OMP85 protein family.Spatial structural analysis revealed that OsP39 was a β-barrel membrane protein rich in β-sheets.Subcellular localization analysis showed that OsP39 protein was localized at the rice chloroplast membrane.The above results indicated that Osp39 gene was a chloroplast membrane related gene,which was involved in the regulation of light response and chloroplast function in rice.

Key words: Rice, Osp39 gene, Bioinformatics, Tissue expression, Subcellular localization

TENG Haiyan. Tissue Expression and Subcellular Localization of Osp39 Gene in Rice[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(3): 10-17. doi: 10.7668/hbnxb.20193511.

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Figures/Tables 13

Tab.1 Primer sequences for PCR

引物名称 Name of primer	序列(5'-3') Sequence(5'-3')	用途 Application
Osp39-F	GGTAGCGTACGGGGTTATGG	定量PCR
Osp39-R	ACCAGGGACATGACAAGCAG	定量PCR
OsActin1-F	CTTCATAGGAATGGAAGCTGCGGGTA	定量PCR
OsActin1-R	CGACCACCTTGATCTTCATGCTGCTA	定量PCR
Osp39-EcoR Ⅰ-F	TGCAGAATTCATGGGAGCTCAGCAGAGC	片段扩增
Osp39-Hind Ⅲ-R	TCAGAAGCTTTGAACCGCCACCACTGTT	片段扩增

Fig.1 Structure of Osp39 gene The position of the lower bracket is the coding site of L6 loop.

Tab.2 Upstream elements in Osp39 promoter

元件名称 Name of element	序列(5'-3') Sequence(5'-3')	元件类型 Type of element	数量 Amount
I box	GATAA	光调节保守序列	5
ATCT-motif	AATCTAATTC	光响应元件	2
G-box	CACGTC	光响应元件	1
TCT-motif	TCTTAC	光响应元件	1
Box 4	ATTAAT	光响应元件	1
CPBCSPOR	TATTAG	细胞分裂素、叶绿素、叶绿体调控相关位点	4
TGACG-motif	TGACG	MeJA(茉莉酸甲酯)响应元件	5
ARFAT	TGTCTC	生长素响应元件	1
ABRE	ACGTG	脱落酸响应元件	1
MYB recognition site	CCGTTG	MYB转录因子识别位点	1
MYB binding site	CAACAG	MYB转录因子结合位点	3
CCAAT-box	CAACGG	MYBHv1结合位点	1
ARE	AAACCA	厌氧诱导元件	2
ANAERO1	AAACAAA	厌氧诱导元件	2
LTR	CCGAAA	低温响应元件	2

Fig.2 Phylogenetic tree of P39 proteins At.Arabidopsis thalianan;Os.Oryza sativa;Sb.Sorghum bicolor;Zm.Zea mays;Ac.Ananas comosus;Ao.Asparagus officinalis;Bo.Brassica oleracea var.Oleracea;Gm.Glycine max;Nn.Nelumbo nucifera;Nt.Nicotiana tabacum;Sl.Solanum lycopersicum;St.Solanum tuberosum;Vv.Vitis vinifera.The same as Fig.3.

Fig.3 Sequence alignment of P39 proteins

Fig.4 Analysis of signal peptide of OsP39 protein

Fig.5 Analysis of transmembrane helical region of OsP39 protein

Fig.6 Secondary structure elements of OsP39 protein

Fig.7 Tertiary structure of OsP39 protein Black arrows indicate the L6 loop region.

Fig.8 Prediction of interactions of OsP39 protein with other rice proteins The thickness of the straight line indicates the degree of confidence.

Fig.9 Prediction of subcellular localization of OsP39 protein

Fig.10 Subcellular localization of OsP39-GFP fusion protein and GFP in rice protoplasts

Fig.11 Expression level of Osp39 gene in different stages and tissues Leaves,leaf sheaths and roots were taken from 14-day rice seedlings;flag leaves,stems and flowers were taken from flowering rice.Different capital letters indicate extremely significant differences(P<0.01).

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