| Potato(Solanum tuberosum L.)is the third most important crop in the world.Low temperature damage has always been one of the main factors restricting the development of the potato industry.Hence it is important to study the molecular mechanisms that potato responds to lower stress.Three genes related to cold response in potato cultivar “Atlantic”,succinyl-CoA ligase subunit α(StSUCLα),aldo-keto reductase(StAKR)and chlorophyll a-b binding protein(StCab)were isolated by MSAP in our previous reports.In order to determine the function of these three genes in the process of potato response to low temperature stress,the study used the mian potato variety “Atlantic” as material to analyze the correlation between the expression of StSUCLα,StAKR and StCab and low temperature stress by quantitative PCR(qPCR);We silenced NbSUCLα,NbAKR and StCab by virus-induced gene silencing(VIGS)to observe the developmental phenotype and temperature tolerance of plants,and preliminarily analyzed the function of these three genes;the subcellular localization of NbAKR and NbCab were determined by confocal;the expression of potato methyltransferase genes under low temperature were analyzed by qPCR.The main results are listed in the following:(1)After low temperature stress “Atlantic”,with the extension of low temperature stress time,the results found: the expression of gens coding enzymes involved in DNA methylation in potato were altered and DNA methyltransferase responded to low temperature stress of potato;Secondly,the expression of the three target genes were also changed.The expression of StSUCLα was up-regulated at first 12 hours of coldness treatment,then down-regulated in the following 24 hours,and was revovered to be upregulated at point-in-time of 36 hour.After 72 hours of coldness treatment,the expression of StSUCLα reached maximum.Compared with the control,the expression level of StSUCLα under low temperature was higher than of the control;The expression of StAKR was upregulated at first 12 hours of coldness treatment,then it dropped down lower than the basal expression of this gene.Compared with the control,the expression of StAKR under low temperature was higher than of the control at 0~12 h,while the expression of StAKR under low temperature was lower than that of the control at the time of 12~72 h;The expression of StCab was down regulated with exposure of coldness in potato.Compared with the control,the StCab expression level under low temperature was always lower than that of the control during the whole treatment period.(2)The study selected four most common genes encoding DNA methyltransferases,mamely NbMET1,NbDRM2,NbCMT3 and NbROSI.The results showed that the downregulation of NbMET1,NbDRM2 and NbROSI led to the significant up-regulation of the expression of NbSUCLα.The expression of NbAKR was up-regulated significantly when NbCMT3 and NbROSI were down-regulated.The down-regulation of NbMET1,NbDRM2,NbCMT3 and NbROSI led to the significant up-regulation of the expression of NbCab.(3)When “Atlantic” under coldness treatment,Methylation status of one site of cytosine was found to be altered(C-T-C-T)in selected genomic sequence of StSUCLα by bisulphite seuqncing PCR(BSP),with methylation occurring at 48 h and demethylation at 60 h.(4)The results of RT-PCR detection of the target genes showed that the expression of NbSUCLα,NbAKR,and NbCab were down-regulated in VIGS plants.Down-regulation of NbSUCLα resulted in retarted growth with hooked leaves.Down-regulation of NbAKR showed shorter stem compared with control plants.Silencing of NbCab led to yellowing of plant leaves and etiolated leaves with reduced contents of chlorophyll and lower photosynthetic efficiency.Later,the low temperature sensitivity analyszed of NbSUCL and NbAKR VIGS plants showed that the down-regulation of NbSUCLα and NbAKR resulted in increased plant sensitivity to coldness,which indicated that NbSUCLα and NbAKR were positive regulatory genes during response to cold stress.(5)StAKR and StCab were both chloroplastic protein which were solidated by confocal imaging.The aim of this research is to explore the roles of StSUCLα,StAKR and StCab in response to low temperature stress in potato,and eventually to benefit the development of potato molecular genetic breeding and production. |
