【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。

【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.