异源表达盐单胞属(Halomonas)周质磷酸盐结合蛋白基因PBP降低转基因烟草砷积累研究

Reduction of As Accumulation by Heterologous Expression of Halomonas Peripheral Phosphate-binding Protein Gene PBP in Transgenic Tobaccos

为探讨PBP基因在异源烟草中的生物学功能,筛选出低砷(As)积累亲本烟草种质。以转PBP基因烟草T2代种子和野生型烟草(Nicotiana tabacum K326)种子为材料,利用Real-time PCR分析外源PBP的时空表达模式,测定As胁迫下转基因烟株抗氧化酶(AOEs)活性变化及分析转基因烟草株系中As积累差异。AOEs活性测定表明,As胁迫第5天时CK植株中SOD和POD酶活性达到极值,分别为80.25 U/g和152.02 U/g,显著高于转基因植株;在胁迫后期(3~5 d)CK植株中H2O2和MDA含量也显著高于转基因植株。另外,在第7天和14天时转基因植株中As含量比CK植株均显著降低。离子转运蛋白基因表达分析表明,As胁迫不仅诱导转基因植株PBP基因表达显著上调,而且也引起转基因和CK植株中HAK1和PHT4等通道蛋白基因的显著上调,且CK植株中HAK1和PHT4的平均表达水平为转基因植株的1.5倍和3.75倍。外源PBP基因导入可有效降低转基因烟草对As的积累。

In order to explore the biological function of PBP gene in transgenic tobaccos and select the low arsenic(As)accumulation parent tobacco germplasm,taking T2 generation seeds of transgenic tobacco with PBP gene and wild-type tobacco(Nicotiana tabacum K326)seeds as materials,Real-time PCR was used to study the spatial and temporal expression profile of exogenous PBP in transgenic tobaccos,and the activities of antioxidant enzymes(AOEs)in transgenic tobacco lines under As stress were also determined.AOEs activity showed that SOD and POD activities reached the maximum value of 80.25 U/g and 152.02 U/g in CK plants at 5 d after As stress,which were significantly higher than those in transgenic plants.Also,H2O2 and MDA contents in CK plants were significantly higher than those in transgenic plants at the later stage of As stress(3-5 d).Additionally,at 7 d and 14 d after stress,the content of As in transgenic tobacco plants was significantly reduced.Based on the expression profiling of ion transporter genes,the results showed that As stress not only induced significant up-regulation of PBP in transgenic plants,but also caused significant up-regulation of channel protein genes such as HAK1 and PHT4 in transgenic and CK plants,and the average expression levels of HAK1 and PHT4 in CK were 1.5 and 3.75 times of that in transgenic plants.The introduction of exogenous PBP gene can effectively reduce the accumulation of As in transgenic tobaccos.