拟南芥AKIN11基因的过表达及其功能分析

Over-expression and function analysis of AKIN11 gene in Arabidopsis

将蔗糖非发酵基因相关蛋白激酶AKIN11克隆到植物表达载体pEGAD的35S启动子下游与GFP融合,基因枪法导入洋葱表皮细胞进行瞬时表达,发现集中在细胞核内表达.农杆菌介导花序浸泡法转化拟南芥,Basta筛选和RT-PCR鉴定得到两个遗传稳定的T3代转基因株系.突变体和野生型的表型没有明显差异,但突变体叶片中淀粉含量较野生型增加.RT-PCR分析淀粉合成途径关键酶的mRNA水平,发现突变体中腺苷二磷酸葡萄糖焦磷酸化酶表达量增加,而α-淀粉酶却没有变化.表明AKIN11基因可能在拟南芥淀粉积累途径中起着重要的调节作用.

The sucrose nonfermenting-1-related protein kinase （SnRK1） AKIN11 was inserted the downstream of the 35S promoter in the plant expression vector pEGAD and then was transformed to Onion Epidermal cell by the particle bombardment. We found that the green fluorescent was focused on the nucleus. The recombination vector was introduced into Arabidopsis thaliana by Agrobacterium tumefaciens through the floral dip method. The two independent homozygous transgenic lines and T3 progenies were obtained by the Basta screening and RT-PCR method. Mutants have no distinguishable phenotype from that of the wild type. However, the starch content shows an increase in leaves of transgenic lines. RT-PCR analyses find that the AKIN11 gene affects the expression of the AGPase, which is a key enzyme in the pathway of the starch synthesis. In contrast, there are no changes in expression of the AMY3. The results indicate that the AKIN11 gene may play an important role in the regulation of pathway of the starch accumulation in Arabidopsis thaliana.