水稻<i>Osp39</i>基因的组织表达和亚细胞定位分析

doi:10.7668/hbnxb.20193511

摘要/Abstract

摘要：

为研究Osp39基因在水稻叶绿体蛋白质输入调控过程中的作用,采用生物信息学方法对水稻的Osp39基因进行了序列搜索和启动子上游元件分析,对其编码的OsP39蛋白进行了同源性比对、结构和性质预测,并分别通过定量PCR和瞬时表达进行了基因的组织表达分析和蛋白质亚细胞定位分析。结果显示,Osp39基因位于水稻5号染色体,基因编码区含11个外显子,无可变剪切方式。Osp39启动子中含有I box、ATCT-motif等多种光响应和叶绿体调节元件,转录分析也显示,在苗期和扬花期,Osp39基因在水稻叶、叶鞘等绿色组织中的转录水平均极显著高于根、花等非绿色组织。OsP39蛋白稳定性好,耐热,分子量约38.7 ku,理论等电点pH值 8.64,共含有361个氨基酸残基,富含甘氨酸,第245-271氨基酸残基形成高度保守的L6 loop区,为OMP85蛋白家族的特征性结构域,空间结构预测显示,OsP39蛋白是富含β-折叠片的β-桶状膜蛋白,亚细胞定位结果显示,OsP39蛋白定位于水稻叶绿体膜。上述结果表明,Osp39是一个叶绿体膜功能相关基因,参与水稻的光响应和叶绿体功能调节。

关键词: 水稻, Osp39基因, 生物信息学, 组织表达, 亚细胞定位

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

腾海艳. 水稻Osp39基因的组织表达和亚细胞定位分析[J]. 华北农学报, 2023, 38(3): 10-17. doi: 10.7668/hbnxb.20193511.

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.

http://www.hbnxb.net/CN/Y2023/V38/I3/10

图/表 13

表1 PCR引物序列

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

图1 Osp39基因结构下括号位置为 L6 loop区编码位点。

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

表2 Osp39启动子中的上游元件

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

图2 P39蛋白的系统进化树 At.拟南芥;Os.水稻;Sb.高粱;Zm.玉米;Ac.菠萝;Ao.芦笋;Bo.甘蓝;Gm.大豆;Nn.荷花;Nt.烟草;Sl.番茄;St.马铃薯;Vv.葡萄。图3同。

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.

图3 P39蛋白的序列比对

Fig.3 Sequence alignment of P39 proteins

图4 OsP39蛋白的信号肽分析

Fig.4 Analysis of signal peptide of OsP39 protein

图5 OsP39蛋白的跨膜螺旋区分析

Fig.5 Analysis of transmembrane helical region of OsP39 protein

图6 OsP39蛋白的二级结构元件

Fig.6 Secondary structure elements of OsP39 protein

图7 OsP39蛋白的三级结构黑色箭头指示L6 loop区。

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

图8 OsP39蛋白与其他水稻蛋白的互作关系预测直线的粗细程度表示可信程度。

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

图9 OsP39蛋白的亚细胞定位预测

Fig.9 Prediction of subcellular localization of OsP39 protein

图10 OsP39-GFP融合蛋白和GFP在水稻原生质体中的定位

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

图11 Osp39基因在不同时期和组织中的表达水平叶、叶鞘和根取自14 d水稻苗;旗叶、茎和花取自扬花期水稻。不同的大写字母表示差异的极显著性(P<0.01)。

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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