Research On Cloning, Expression Analysis And Transformation Of MpSnRK2.4Gene

SnRK2(Sucrose non-fermenting-related protein kinase2) has been identified as anindispensable positive regulation factor in ABA signal transduction and plays a key role inplant-stress-signaling pathways. In this study, we cloned the full-length ORF sequence ofMpSnRK2.4from Malus prunifolia (willd.) Borkh. Then we investigated its expressionprofiles by qRT-PCR, structured the overexpression vector of pGWB411-SnRK2.4byGateway technique for plant transformation and transformed it into the tomato cultivar ofMicro-Tom by the Agrobacterium mediated transformation method successfully. We alsoobtained the Km resistant plants of M.9. These works preliminarily revealed the expressionpatterns of MpSnRK2.4under adversity stresses and laid a foundation for the further researchon SnRK2gene family. The main findings are as follows:1A sequence of1220bp of MpSnRK2.4gene was cloned in this study. This sequencecontained a full-length of ORF sequence (1026bp) which encoding341amino acids. Wefound the gDNA sequence contained8introns through online software analysis and thepredict molecular weight is38.475KD. The phylogenetic tree indicated that MpSnRK2.4ismost homologous with Oryza sativa SAPK3and amino acid sequence alignment showed thatMpSnRK2.4is highly homologous with other known SnRK2s protein sequences.2qRT-PCR showed that MpSnRK2.4expresses in roots, stems, leaves, flowers and fruitsof Malus prunifolia. Among these, green fruits have the highest expression abundance, whichis about6-fold larger than that in leaves and24-fold larger in roots. Among different tissues,the expression pattern of MpSnRK2.4is: green fruits> stems> mature leaves> flowers> roots.3The result of expressions experiments of MpSnRK2.4under different stress treatmentsshowed that the gene is induced by drought, salt, ABA and low temperature. On the6thd ofdrought treatment, MpSnRK2.4was significantly induced by about78folds in3-month leaves.The expression of MpSnRK2.4increased dramatically to about70folds after6h treatment,and then decreased rapidly under salt stress. The expression level at12h is almost the same as3h’, and then remained at the original level. With the exogenous ABA treatment, theexpression of MpSnRK2.4has increased about120times in the first8h, after which itrecovered to the4h’ level at12h, and then dropped to the original level gradually. Undercold condition (4°C), the expression of MpSnRK2.4showed a completely opposite trend of salt treatment: at4h it dropped to about1/5of0h’ level and then rebounded slowly as timeextension, finally got close to the initial level after12h.4Successfully structured the overexpression vector of MpSnRK2.4using Gatewaytechnique, and transformed it into Micro-Tom by the Agrobacterium mediated transformationmethod. PCR test indicated that we obtained10overexpression strains. qRT-PCR analysisshowed that there are expression differences between different transgenic lines. All transgeniclines show higher expression level compared with CK plant, especially line1.5We got a satisfactory bud multiplication rate and controlled the vitrification degree ofM9by using two kinds of culture medium to subculture alternately. We also obtained the bestrooting medium of rootstock M9: first step’ medium of the two-step rooting method:1/2MS+2.4mg/L IBA+0.21g/L Phloroglucinol+30g/L Sucrose+7.5g/L Agar, then transferring tothe medium of the second step after5-day darkness:1/2MS+30g/L Sucrose+7.5g/L Agar,with a rooting rate of86%. The optimum Kanamycin selection pressure of leaf regenerationand rooting were5mg/L and was40mg/L for plantlets. The cefotaxime sodiumconcentration was200mg/L. We finally obtained the Kanamycin resistant apple plantlets.