水稻种子特异性谷蛋白GluB1启动子在水稻愈伤组织中驱动外源基因表达

doi:10.16819/j.1001-7216.2018.8036

摘要/Abstract

摘要：

【目的】水稻谷蛋白启动子GluB1(GluB1 promoter,pGluB1)常用于外源基因在种子中特异性高效表达的研究,也是研究种子储藏蛋白基因表达调控机制的模型。前人研究表明,pGluB1只在水稻胚乳中表达,而在根、茎、叶片、叶鞘、颖壳等组织中均无表达活性。研究的目的是为了克服种子特异表达启动子筛选周期长的缺点。【方法】将由pGluB1驱动的霍乱毒素B亚单位和重组胰岛素原组成的融合基因(a fusion gene of the cholera toxin B subunit and human proinsulin, CTBIN)表达载体pCAMBIA1302-pGluB1sig-CTBIN-NOS经农杆菌介导法转化水稻成熟胚愈伤组织。通过RT-PCR和蛋白质印迹杂交试验检测融合基因CTBIN在水稻愈伤组织中的转录和翻译表达。【结果】获得的7个转基因愈伤克隆中,有6个克隆的融合基因CTBIN在转录水平上表达。选取其中4个克隆进一步进行蛋白质印迹杂交试验检测证实融合基因CTBIN均在翻译水平上表达,而且从分子量大小推断融合蛋白包含的谷蛋白GluB1的N-端信号肽序列(24个氨基酸残基)在所测的愈伤组织细胞中均被成功切除。【结论】水稻种子特异性启动子pGluB1在愈伤组织中具有驱动外源基因表达活性,种子蛋白体亚细胞定位信号肽序列可在愈伤组织细胞中被切除。这为在愈伤组织细胞中快速检测种子特异表达启动子活性和探索愈伤组织中蛋白质的亚细胞分拣机制奠定了基础。

关键词: 谷蛋白B1启动子, 信号肽, 水稻, 成熟胚愈伤组织, 表达活性

Abstract:

【Objective】The promoter of rice glutelin B1(pGluB1) has been intensively used to study high seed-special expression of exotic genes, and as a model to understand regulation mechanism of seed-storage protein genes. Former researchers reported that pGluB1expressed only in rice seed endosperm with no expression activity in other tissues such as root, stem, leaf, sheath and glume etc. The present study is aiming to overcome the shortcoming of time consuming for screening of seed-special expression promoters.【Methods】The expression vector pCAMBIA1302-pGluB1sig- CTBIN-NOS, in which a fusion gene of the cholera toxin B subunit and human proinsulin (CTBIN) was driven by the 2.3 kb promoter sequence of rice glutelin GluB1 with its signal peptide (pGluB1sig), was transformed into the rice calli from mature embryo via Agrobacterium-mediated method. The expression of fusion gene CTBIN at both transcription and translation level was tested by RT-PCR and Western-blotting assay.【Results】Among the seven transgenic calli clones, six clones of the fusion gene CTBIN were expressed at transcription level. The selected four clones subjected to Western-blotting assay were all fatherly verified in their expression at translation level. Additionally, according to the molecular weight, we speculated that the signal peptide (24 aa) of GluB1 at N-terminus of the fusion protein CTBIN has been excised successfully from callus cells of all the test clones.【Conclusion】The rice seed-specific expression promoter pGluB1 can drive an exotic gene expression in callus, and the seed protein body subcellular-targeted signal peptide could be excised from callus cells. The results lay a foundation of the quick detection of expression activity of plant seed-specific expression promoters in callus and exploiting the mechanism of protein subcellular sorting selection in callus cells.

Key words: glutelin B1 promoter, signal peptide, rice, mature embryo calli, expression activity

中图分类号:

Q755
S511.01

赵艳, 唐湧洲, 史玉倩. 水稻种子特异性谷蛋白GluB1启动子在水稻愈伤组织中驱动外源基因表达[J]. 中国水稻科学, 2019, 33(1): 28-34.

Yan ZHAO, Yongzhou TANG, Yuqian SHI. Rice Seed-specific Glutenin GluB1 Promoter Drives Exogenous Gene Expression in Rice Callus[J]. Chinese Journal OF Rice Science, 2019, 33(1): 28-34.

图/表 6

图1 表达载体pCAMBIA1302-pGluB1sig-CTBIN-NOS的T-DNA结构 T BORDER(L)―左边界; T BORDER(R)―右边界; 35S―烟草花椰菜病毒35S启动子; hpt―潮霉素B磷酸转移酶基因; NOS―胭脂碱合成酶终止子; pGluB1―水稻 2.3 kb谷蛋白GluB1启动子; signal―水稻谷蛋白GluB1信号肽序列; CTBIN―人工设计合成的胰岛素原与霍乱毒素B亚单位融合基因,C端附加KDEL ER滞留信号基因序列; mGFP―修饰的绿色荧光蛋白基因。

Fig. 1. Sketch map of T-DNA of pCAMBIA1302-pGluB1sig-CTBIN-NOS vector. T BORDER(L), The left boundary; T BORDER(R), The right boundary; 35S, Cauliflower mosaic virus 35S promoter; hpt, The hygromycin B phosphotransferase gene; NOS, Nopaline synthase terminator; pGluB1, Rice glutelin GluB1 promoter of 2.3 kb; signal, Rice glutelin GluB1 signal peptide gene sequence; CTBIN, Artificially designed and synthesized fusion gene sequence of cholera toxin B subunit and human proinsulin, with the KDEL ER retention signal gene sequence at the C terminus; mGFP, Modified green fluorescent protein gene.

图2 筛选中的抗性愈伤组织(A)和继代中的抗性转基因愈伤组织(B)

Fig. 2. Resistant rice calli on screening medium(A) and resistant transgenic calli on subculture medium(B).

图3 PCR鉴定水稻转基因愈伤组织克隆中的CTBIN基因 M-DL 2000 DNA 标记; P-质粒阳性对照; CK-非转基因愈伤组织阴性对照; 1~7分别代表转基因水稻愈伤不同克隆。

Fig. 3. PCR analysis of CTBIN gene in rice transgenic callus clones. M, DL2000 DNA marker; P, Plasmid(positive control); CK, Nontransgenic callus clone(negative control); 1-7, Number of different transgenic rice calli clones.

图4 绿色荧光蛋白基因gfp在水稻转基因愈伤组织中表达的荧光显微镜观察

Fig. 4. Fluorescence microscope detection of the gfp gene expression in rice transgenic calli.

图5 RT-PCR检测水稻转基因愈伤组织中CTBIN基因的转录表达上图为目的基因CTBIN扩增条带,下图为水稻内参基因β-actin扩增条带。M-DL2000 DNA标记;CK-非转基因愈伤克隆阴性对照;1~7代表转基因水稻愈伤不同克隆。

Fig. 5. Detection of transcriptional expression of CTBIN gene in transgenic rice calli by RT-PCR. The upper part shows the amplified bands of purpose gene CTBIN, the lower part the amplified bands of rice inner control gene β-actin. M, DL2000 DNA marker; CK, Nontransgenic rice calli clone(negative control); 1-7, Number of different transgenic rice calli clones.

图6 Western blot检测水稻转基因愈伤组织中CTBIN基因的翻译表达 CK―非转基因愈伤克隆阴性对照;1, 2, 4, 5―不同转基因愈伤克隆编号。

Fig. 6. Detection of translational expression of CTBIN gene in transgenic rice calli by Western blot. CK, Nontransgenic rice calli clone (negative control); 1, 2, 4 and 5, Number of different transgenic rice calli clones.

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