Cloning And Functional Analysis Of Small G Protein Gene StRab5b Regulation Potato Resistance Against Late Blight

RAB proteins are widely distributed in plants,animals and microorganisms.They regulate the opening and closing of signaling pathways through guanylate exchange factors(GEFs)and GTPase activating proteins(GAPs).Previous studys have shown that RAB GTPases were involved in plant growth and development,biotic and abiotic stresses,especially in the establishment of plant resistance.In this study,the small G protein gene StRab5b was cloned from potato,and its biological information,expressing profile and induced expressing level were analyzed.Through overexpression and gene silencing,the function of StRAB 5b in regulating potato resistance to Phytophthora infestans and salt tolerance were studyed.The results were as follows:1.The StRab5b(607bp)and StRab(673bp)genes were cloned by specific primers.The StRAB 5b protein structure prediction results showed that the primary structure of StRAB 5b protein contained 200 amino acids with molecular weight of 21.5 kDa.The secondary structure analysis found that StRAB 5b included 1 GGT2 Rab catalytic sites,1 REP and 3 palmitoylation sites,16 phosphorylation sites and no signal peptide.The phylogenetic tree analysis showed that the homology of StRAB 5b was 94%between potato and tobacco,and shared the conservative domain:Rab F2(83?87),Rab F4(YYRGA,102?106)and Rab F5(131?135).In tertiary structure,StRAB 5b contained 5?-helices and 5?-folds.2.Expressing profile analysis indicated that the level of StRab5b gene was significantly different in young leaves,stem,old leaves and root in descending order.The relative expression level of StRab5b gene in different potato varieties from high to low was Zihuabai(ZHB),Shepdy(XPD),Longshu No.7(L7),Desiree(DXR),Zhongshu No.4(ZS4)and Atlantic(DXY).The induced expression of StRab5b gene in Jizhangshu No.8 and Desiree showed an initial increase which peaked at 72hpi and then decreased.3.After transient expression of and gene in potato and tobacco respectively,the lesion area was significantly lower than that of GFP and H2O at different inoculation times.The lesion area of leaves with transient expression of StRab5b gene in tobacco were lower than that of StRab transient gene expression,however the lesion area on potato leaves were higher with StRab5b transient gene expression than that of StRab gene.4.The overexpression and silencing vectors of StRab5b and StRab were constructed,and these yielded 9 StRab5b and 5 StRab positive transgenic plants respectively.The lesion area in transgenic plant StRab5b-L8 and StRab-L5 were significantly smaller than that of the EV after inoculation.At the same time,DAB staining confirmed that the accumulation of H2O2 in transgenic plant leaves was higher than that of the EV after inoculation.Trypan blue staining showed that the number of necrotic cell in the leaves of EV was significantly higher than that of transgenic plants.5.The transcriptional level of resistance genes showed that the expression level of LOX and NPR1,the key enzyme genes of JA and SA signaling pathway,were significantly higher in StRab5b-L8 and StRab-L5 than those in EV respectively.Meanwhile,the activities of SOD,POD,CAT and APX in transgenic leaves of StRab5b-L8 and StRab-L5 were higher than those in EV(except for SOD activity at 72h and POD activity at 24 h).6.VIGS technique was established and used to silence the homologous genes in potato.The silencing efficiency of StPds,StRab5b and StRab genes were 100%,61%and 59%respectively.The lesion area in StRab5b and StRab gene silencing leaves was significantly larger than that of the mock plants.The results of coomassie brilliant blue staining also showed that the number of mycelia in StRab5b and StRab gene silencing leaves was more than that of the mock.7.The results of transgenic plants under salt stress showed that overexpression of StRab5b and StRab genes could positively regulate the salt tolerance of potato.