Cloning And Functional Analysis Of SnRK1 ??-subunit Gene In Prunus Persica

SnRK1 is considered to be crucial element of transcriptional,metabolic and developmental regulation in plant.Moreover,SnRK1 complexes were trimeric complexes,which included the catalytic subunit ? and the regulatory subunits ? and ?.The atypical ??-subunit only existed in the plant kingdom,as the canonical ?-subunit.Many studies on the ??-subunit focused on Arabidopsis,maize(Zea mays),tomato(Lycopersicon esculentum)and alfalfa(Medicago truncatula)and other herbaceous plants.However,to data,there was no research about the gene identification and expression analysis of the ??-subunit on peach.So,the aim of this study was to clone full-length cDNA of peach the ??-subunit,to investigate its sequence characteristics,to analyze its expressions in different organs,to assay its function by genetic engineering.The main results are as follows:1.In Prunus persica genome,ppa004800 m and ppa004966 m are the ??-subunit in SnRK1,homologous with AMPK of mammal and SNF1 of yeast.The reading frame of PpSnRK1??1 and PpSnRK1??2 possesse 1476 bp and 1452 bp,respectivtly,and encodes 492 and 484 amino acid residues deduced from the DNA sequence.Sequence analysis showed that they contain CBM and four CBS domain.This indicated PpSnRK1??1/2 may participate in carbohydrate metabolism.Phylogenetic tree analysis indicated that PpSnRK1??1 was very closely related to SnRK1?? of GuoMei,while PpSnRK1??2 was Malus sieversii.Homology analysis showed that the deduced PpSnRK1??1/2 protein was highly homologous to other SnRK1?? proteins from different species,which recorded 79.40%.Real-time PCR results showed that the PpSnRK1??1/2 expressed in root,stalk and leaf of peach seedling and leaf,flower and fruit of ‘LuXing 'peach.,which showed that PpSnRK1??1 /2 was constitutive expression.2.We construct the recombinant plasmid p35S::PpSnRK1??1,successfully,and obtain the transgene plant.Compared to WT,the SnRK1 activity in the transgenic plants increased by 77.13 %.Through statistical studies,we found that transgenic plant flowered 2.19 days later than WT,and produced around 1.17 rosette leaves than WT.The contents of chlorophyll,soluble sugar,and soluble proteins of transgenic plant were significantly higher than WT,which increased by 13.7%,12.9% and 23.3%,respectively.There is no difference between transgenic plant and WT in starch content.3.Compared to the wide type plants,PpSnRK1??1-overexpressing plants showed better seed germination rate and relatively longerroot length under the oxidative stress.After treated with 2 ?mol·L-1 methyl viologen(MV)3 h,compared with WT,transgenic plants had 40.7% lower the MDA contents,and the activity of CAT,SOD,GSTs and POD was higher,which indicated that overexpression PpSnRK1??1 could improve the oxidative stress tolerance of Arabidopsis.Besides,under the oxidative stress condition,the activity of SnRK1 in transgenicplants was more than two times higher than WT.Moreover,the relative expression ofAtHSPRO1 and AtHSPRO2,the stress-responsive genes,was 2.56 and 3.12 times higher than WT.Therefore,the overexpression PpSnRK1??1 could improve the oxidative stress tolerance ofArabidopsis via participating in regulating the expression of HSPRO.