Cloning And Preliminary Characterization Of Genes In Response To Lead Stress In Nicotiana Benthamiana

With the development of industrial,lead?Pb?pollution becomes one of underestimated environmental issues.Pb and its compounds contaminate air,soil,water,plants and animals when connected to them.It is reported that plant will regulate related genes expression to keep normal metabolism via epigenetic modification,such as DNA methylation modification in response to stresses.Unfortunately it is little known that which genes are modified on DNA Methylation status and the roles of these genes in response to Pd stress in plant.Methylation-sensitive amplified polymorphism?MSAP?was previously applied to analyze alteration of genome DNA methylation status under different concentration Pd²⁺stress in Nicotiana benthamiana in our lab,and some genes related to DNA methylation modification were cloned.In this thesis,seven above genes,DNA-directed RNA polymerase subunit beta?NbDDRP?,EH domain-containing protein 1?NbEF?,Homogentisate phytyltransferase?NbHPT?,NADH dehydrogenase?NbNADH?,Transmembrane protein?NbTM?,V-type proton ATPase subunit F?NbVAPF?,WRKY transcription factor?NbWRKY?,were selected for further analysis on the roles in response to Pb²⁺stress in plant by quantitative real-time PCR?qRT-PCR?,bisulfite Sequencing PCR?BSP?,virus-induced Gene Silencing?VIGS?and confocal imaging.the results were listed as below:?1?The expression level of DNA methylase coding genes?CMT3,MET1 and DRM2?,demethylase?ROS1?and selected genes were estimated by qRT-PCR after 15-day exposure of different concentration Pb²⁺in N.benthamiana.Pb²⁺stress can induce changes in the expression levels of methylase,demethylase and 7 selected genes in N.benthamiana?2?Treatment of N.benthamiana with different concentrations of Pb²⁺caused changes in DNA methylation staus in selected regions of selected genes.?3?Silencing of selected genes led to restarted growth,and worse to be lethal.Silencing of NbHPT led to decrease on concentration of chlorophyll pigments compare to control plants.While concentration of chlorophyll pigments was increased in NbTM VIGS plants.VIGS plants showed stunt growth and worse to death after 11-day exposure of 800mg·L^-1 Pb²⁺.To undermine the accumulation of Pb²⁺in different parts of plants,root,stem and leaves were collected separately from NbHPT,NbWRKY,NbTM VIGS and control plants.The results indicated that silencing of NbHPT and NbWRKY resulted in reduction of Pb²⁺accumulation in root,while knock-down of NbTM had no impact on Pb²⁺accumulation in root compared with control plants.Silencing of NbHPT led to higher accumulation of Pb²⁺in stem,the content of Pb²⁺in stem of NbWRKY silencing plants were lower than in control plants.And the content of Pb²⁺in stem of NbTM silencing plants had no obvious difference with control plants.The contents of Pb²⁺in leaves of NbHPT and NbWRKY silencing plants were higher than in control plants,while contents of Pb²⁺in leaves of NbTM silencing plants had no obvious difference with control plants.In sum,silencing of NbTM had no obvious effects on accumulation of Pb²⁺,while silencing of NbHPT and NbWRKY affected the accumulation of Pb²⁺in different organs of plants.It indicated that the expression of the selected genes was down-regulated,which led to the growth and development of the corresponding plants,the change of chlorophyll content,the weak tolerance to Pb²⁺and the disorder of Pb²⁺uptake.?4?To analyze the cellular sublocalization of selected proteins by confocal imaging,the results indicated that NbDDRP was located in chloroplast,plasma membrane and nucleus.NbEF and NbNADH were located in nucleus.NbVAPF-GFP fluorescence signal was failed to be detected.The reasons that NbVAPF was predicted locating in vacuole where pH was not suitable for GFP florescence.