菠菜磷酸丙糖转移蛋白在烟草中正义及反义表达及其对碳水化合物转运的影响

Sense and Antisense Expression of Spinach Triose Phosphate Translocator in Tobacco and Its Effects on Carbohydrate Partitioning

以菠菜 (SpinaciaoleraceaL .)为材料 ,取幼叶分离mRNA ,反转录合成cDNA ,以cDNA第一链为模板 ,通过PCR扩增 ,获得菠菜磷酸丙糖转移蛋白 (Triosephosphatetranslocator,TPT)cDNA目的片段。对其进行序列分析 ,结果表明 ,分离的目的片段核苷酸序列与文献报道相比同源率为 99 9% ,只有 1个碱基发生改变。将得到的菠菜tptcDNA与CaMV 35S启动子和NOS 3′终止子融合 ,分别构建了正义及反义表达载体。通过根癌土壤杆菌(Agrobacteriumtumefaciens)介导转化烟草 (NicotianatabacumL .) ,在含有卡那霉素的抗性筛选培养基上再生的植株经PCR及Southern杂交检测 ,证明tpt基因已整合到烟草基因组上 ,菠菜丙糖磷酸转移蛋白基因导入烟草后使叶绿体中TPT的含量表现出明显差异 ,并导致烟草光合作用产物分配的变化 ,表现在叶绿体中淀粉含量的改变。

mRNAs were isolated from the young leaves of spinach ( Spinacia oleracea L.) and cDNAs were synthesized from reverse transcription.The triose phosphate translocator(TPT)cDNA was amplified by PCR using cDNA first strains as templates.Sequence analysis indicated that the cloned cDNA fragment was showing 99 9% homology to the sequence reported before.Only one base is different.The sense and antisense expression vector with TPT full length cDNA under the control of CaMV 35S promoter were constructed respectively and were transformed into tobacco ( Nicotiana tabacum L.)by leaf disc transformation.The transformed tobaccos were selected in the medium containing kamamycin,and were identified by PCR and Southern blotting analysis.The carbon partitioning of photosynthates is affected in tpt transgenic tobacco resulting in the alternation of the TPT content and the starch accumulation in the chloroplasts.