水稻TAF12b基因cDNA克隆及其分子特性鉴定

doi:10.16819/j.1001-7216.2023.230108

摘要/Abstract

摘要：

【目的】 明确水稻通用转录因子TFⅡD复合物组分中的OsTAF12b的选择性剪接形式并鉴定其亚细胞定位及其表达模式，为深入研究OsTAF12b功能提供基础性信息。【方法】 利用5＇-/3＇-RACE技术扩增并克隆了OsTAF12b基因的全长cDNA；通过生物信息学进行了多重序列比对和进化树构建；利用激光共聚焦显微镜观察OsTAF12b的亚细胞定位；通过qRT-PCR技术分析了该基因在非生物逆境下的表达模式。【结果】 发现OsTAF12b基因有4个选择性剪接转录本，其在编码区内仅存在一个赖氨酸的差异。OsTAF12b与其他禾本科植物成员高度同源且在进化树中聚在一个分支上。在本氏烟叶片细胞和水稻原生质体中融合GFP标签的OsTAF12b蛋白均与细胞核标记蛋白H2B共定位。OsTAF12b转录本在水稻叶片中的积累水平较高，而且在多种非生物逆境胁迫下显著上调表达。【结论】 水稻OsTAF12b基因存在4种选择性剪接产物，可编码2个仅相差1个赖氨酸残基的细胞核蛋白。表达模式分析表明OsTAF12b可能参与水稻多种非生物逆境胁迫响应过程。

关键词: TAF12b, RACE, 亚细胞定位, 非生物胁迫, 表达模式

Abstract:

【Objective】 In order to determine the alternative splicing forms of TATA-box binding protein (TBP) associated factor 12b (OsTAF12b), a component of the general transcription factor ⅡD (TFⅡD), in rice (Oryza sativa), characterize their subcellular localization and expression patterns, and provide basal information for further functional studies. 【Method】 The full-length cDNA of OsTAF12b was amplified and cloned by rapid amplification of cDNA 5’-/3’-ends (RACE). Multiple sequence alignment and a phylogenetic tree were conducted by bioinformatic analysis. The subcellular localization of OsTAF12b was observed with a laser confocal microscopy and its expression patterns under abiotic stresses were analyzed by qRT-PCR. 【Results】 Four alternative splicing forms of OsTAF12b were identified and there was only one lysine difference within their coding regions. OsTAF12b was highly homologous to those of other gramineous plants and they were grouped together in a clade in the phylogenetic tree. The GFP-fused OsTAF12b protein was colocalized with H2B, a marker labeling nuclear localization, in both cells of N. benthamiana leaves and rice protoplasts. Its transcript level was higher in leaves and significantly up-regulated when exposed to different abiotic stresses. 【Conclusion】 The OsTAF12b gene has four alternative splicing forms of transcripts and encodes two nuclear proteins with only a single lysine residue difference. Its expression patterns suggest that OsTAF12 could be involved in response to a variety of abiotic stresses.

Key words: TATA-box binding protein (TBP) associated factor 12b (TAF12b), rapid amplification of cDNA ends (RACE), subcellular localization, abiotic stress, expression pattern

齐盼盼, 郭留明, 李静, 吕明芳, 袁正杰, 张恒木. 水稻TAF12b基因cDNA克隆及其分子特性鉴定[J]. 中国水稻科学, 2023, 37(6): 577-586.

QI Panpan, GUO Liuming, LI Jing, LÜ Mingfang, YUAN Zhengjie, ZHANG Hengmu. cDNA Cloning and Molecular Characterization of OsTAF12b Gene in Oryza sativa[J]. Chinese Journal OF Rice Science, 2023, 37(6): 577-586.

图/表 8

表1 本研究用到的引物信息

Table 1. Information of primers used in the study.

引物名称 Primer name	引物序列 Sequence (5＇-3＇)	注释 Annotation
5＇R1	GCCTGCTGCTGTTTCTGAGGAAGGG	5＇RACE
5＇R2	GATTACGCCAAGCTTTGTGGAGACAGTGTGCCTT	5＇RACE
3＇R1	CAGCCAAGGCAGAAGGGTATGGTGC	3＇RACE
3＇R2	GATTACGCCAAGCTTGGGTGGTATGAGAGGAAATG	3＇RACE
OsTAF12b-F	TCAAACTACCCCACAGGACC	OsTAF12b亚细胞定位载体构建 Construction of subcellular localization vector of OsTAF12b
OsTAF12b-R	GCGAACAATCTGTCAGTCAG
pCV-OsTAF12b-GFP-F	ACTCTAGACCCCTGGGATCCATGGCGGATCCGCCGTCCG
pCV-OsTAF12b-GFP-R	GCCCTTGCTCACCATGTCGACGAAACGGGGCACTTTTGAC
pCV-GFP-OsTAF12b-F	GACGAGCTGTACAAGGTCGACATGGCGGATCCGCCGTCCG
pCV-GFP-OsTAF12b-R	TTCGAGCTCGCCTGGGGATCCTTAGAAACGG GGCACTTTT
pCV-H2B-mCherry-F	ACTCTAGACCCCTGGGATCCATGGCGAAGGCAGATAAGA	核定位标记载体构建Construction of nuclear localization marker vector
pCV-H2B-mCherry-R	GCCCTTGCTCACCATGTCGACAGCAGAACTCGTAAACTTC	核定位标记载体构建Construction of nuclear localization marker vector
qActin-F	GTATCCATGAGACTACATACAACT	内参基因定量引物 Primers of internal reference gene
qActin-R	TACTCAGCCTTGGCAATCCACA	内参基因定量引物 Primers of internal reference gene
qOs12b-F	GCATCCTCATCTCAATACCC	OsTAF12b定量引物 qRT-PCR primers of OsTAF12b
qOs12b-R	CCAGTCATAGTTCCACCTGAT	OsTAF12b定量引物 qRT-PCR primers of OsTAF12b

图1 OsTAF12b cDNA扩增 M，DNA分子量标记；泳道1，中间产物；泳道2，5＇-RACE扩增产物；泳道3，3＇-RACE扩增产物。

Fig. 1. Amplification of OsTAF12b cDNA. M, DNA marker; Line 1, Amplified fragment of central region; Line 2, 5＇-RACE product; Line 3, 3＇-RACE product.

图2 OsTAF12b基因结构 A—OsTAF12b基因在水稻第1染色染色体上位置；B—OsTAF12b的4个转录本结构。白色框代表非编码区；灰色框代表编码区；黑色线条代表内含子；碱基中间的虚线表示在此处发生剪切。

Fig. 2. Schematic graph of OsTAF12b gene structure. A, Location of OsTAF12b gene on rice Chromosome 1; B, Schematic graph of four transcripts of OsTAF12b. The white boxes represent the untranslated regions; the gray boxes represent the coding regions; the black lines represent the introns; the dotted lines indicate the alternative splicing sites.

图3 OsTAF12b蛋白序列相似性分析(A)和蛋白结构(B)

Fig. 3. Sequence similarity (A) and structural diagram (B) of OsTAF12b protein.

图4 基于不同物种TAF12b和TAF12氨基酸序列构建的系统进化树

Fig. 4. Phylogenetic tree based on the amino acid sequences of TAF12 and TAF12b from different species.

图5 OsTAF12b在本氏烟叶片表皮细胞(A)和水稻原生质体(B)中的亚细胞定位 A—OsTAF12b蛋白在本氏烟叶片表皮细胞中的定位，标尺代表的长度为50 μm；B—OsTAF12b蛋白在水稻原生质体中的定位，标尺代表的长度为5 μm。

Fig. 5. Subcellular localization of OsTAF12b in Nicotiana benthamiana leaf epidermal cells(A) and rice protoplasts(B). A, Localization of OsTAF12b protein in Nicotiana benthamiana leaf epidermal cells(scale bar 50 μm); B, Localization of OsTAF12b protein in rice protoplasts(scale bar 5 μm).

图6 OsTAF12b的表达模式分析 A—OsTAF12b基因的组织特异性表达分析；B—高温处理下OsTAF12b的表达模式；C—低温处理下OsTAF12b的表达模式；D和F—PEG处理下OsTAF12b的表达模式；E和G—NaCl处理下OsTAF12b的表达模式。数据是来自三组独立实验的平均值±标准差。顶部的星号表示与0 h相比差异显著（ns，不显著；*P<0.05；**P<0.01）。

Fig. 6. Expression patterns of OsTAF12b. A, Relative expression level of OsTAF12b in different tissues of rice; B, Relative expression level of OsTAF12b under heat treatment; C, Relative expression level of OsTAF12b under cold treatment; D and F, Relative expression level of OsTAF12b under PEG treatment; E and G, Relative expression level of OsTAF12b under NaCl treatment. Data are mean±SD from three independent experiments. The asterisks on the top of the columns indicate significant differences from the value at 0 h (ns, not significant; *P < 0.05; **P < 0.01).

图7 OsTAF12b蛋白疏水性(A)和亚细胞定位(B)

Fig. 7. Hydrophobicity (A) and subcellular localization of OsTAF12b∆K protein.

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