小麦淀粉分支酶基因SBE-Ⅱb植物表达载体的构建及表达鉴定

Construction of Plant Transformation Vector of Wheat Starch Branching Enzyme SBE-Ⅱb and Its Expression Identification

从花后15 d的小麦(Triticum aestivum L.)胚乳中提取RNA,根据GeneBank上已经公布的小麦淀粉分支酶基因(SBE-Ⅱb)序列(AY740401.1)设计引物,经RT-PCR扩增得到一条2 511 bp的条带,测序结果与公布的序列一致。将克隆得到的基因构建到了植物表达载体pPZP35S上,通过农杆菌叶盘转化法转化烟草叶片,经卡那霉素筛选、PCR扩增鉴定证明,SBE-Ⅱb基因已成功转入烟草细胞中,获得4个转基因株系。转基因烟草叶片与野生型比较,颜色变暗,植株较小。RT-PCR半定量和实时荧光定量RT-PCR分析表明,转基因烟草株系的叶片中有SBE-Ⅱb基因的表达,与对照植株相比,转SBE-Ⅱb基因的植株中的转录本达到了显著性的变化,但是其淀粉分支酶活性没有达到显著性变化。

The total RNA was isolated from wheat（Triticun aestivum L.） endosperm 15 days after pollination.One fragment of 2 511 bp of SBEⅡb gene was reversely transcribed and amplified from its mRNA using primer pairs designed according to the known SBEⅡb gene sequences（AY740401.1） in GeneBank,and the results of sequence analysis showed that the nucleotide sequence was the same with the known.The plant expression vector for pPZP35S was constructed.The plasmid was transformed into tobacco by leaf dish transformation,and four transgenic plants were obtained by kanamycin selecting and PCR analysis,and the results demonstrated that SBEⅡb gene was transformed into tobacco cells,successfully.The differences between the wild plants and the transformed plants were obvious in the original plant morphology,and the transformed plants leaves turned dark and the plants were smaller,and SqRT-PCR and qRT-PCR analysis results indicated that the SBEⅡb gene was expressed in the leaves of all the transformed plants.Compared with the check plants,the transcriptional level of the transformed plants changed significantly,however,the starch branching enzyme activity showed no difference between them.