Function Analysis Of Apple MdPIP1;2 Gene Under Low Temperature Stress And Identification Of It’s Upstream Regulators

Aquaporin(Aquaporin,AQP)is a channel protein for transmembrane water transport in organisms.Plasma membrane aquaporins(Plasma membrane intrinsic protein,PIP),constituting the largest plant AQP subfamily,exist in the plasma membrane and were widely distributed in various tissues and organs.It plays an important role in maintaining plant water balance and defense responses against biotic and abiotic stresses.In this study,we used MdPIP1;2 transgenic Arabidopsis thaliana and apple callus as materials to analyze the function of apple MdPIP1;2 gene under low temperature stress.The MdPIP1;2 promoter sequence was cloned from Malus domestica genome,fused to GUS to generate fusion constructions.Then the fusion constructions was transformed into Arabidopsis thaliana and apple callus to determine the specific expression of GUS driven by MdPIP1;2 promoter and its response to low temperature stress.Finally,screening and verification of potential transcriptional regulators binding to MdPIP1;2 promoter.The results obtained are as follows:1.Ectopically expressing of MdPIP1;2 in transgenic Arabidopsis thaliana could improve the cold tolerance of transgenic plants.The results showed that at 4℃,the germination rate and root elongation of transgenic seeds were significantly higher than those of wild type.When 12-18 day old and 21-28 day old seedling were treated at-20℃ for 1.5 h and 2 h,respectively,the survival rate of transgenic plants was significantly higher than that of wild type,and the membrane damage of transgenic plants was less than that of wild type.2.Overexpression of MdPIP1;2 gene could improve the cold tolerance of transgenic apple callus.MdPIP1;2 transgenic apple callus was growing better with significantly higher fresh weight compared to that of the wild type at 4℃.3.The 1645 bp upstream promoter of MdPIP1;2 was cloned from the first translational start codon.Searching the cis-element of MdPIP1;2 promoter using Plantcare and PLACE,the cis-acting elements mainly include: Hormone-responsive elements ABRE CGTCA-motif and TGA-element;Anaerobic response elements ARE;Light response elements G-BOX chs-CMA1 a AE-BOX Pc-CMA2 c GT1-motif BOX 4 and BOX Ⅱ;Stress response elements MBS and CBFHV.The promoter was truncated into four fragments: P1645,P940,P561 and P204,fused to GUS separeately to generate pro MdPIP1;2-GUS fusion constructs for GUS histochemical staining.The results showed that all the four fragments could efficiently drive the expression of GUS reporter gene while the GUS was highestly driven by P561.It indicated that P561 fragment might be the core region regulating the transcription of MdPIP1;2 gene.4.The transgenic Arabidopsis thaliana harborins GUS drived by MdPIP1;2 promoter(1645 bp)was obtained and the stable T2 generation lines were used for further analysis.The results showed that GUS driven by MdPIP1;2 promoter was highly expressed in roots,stems,and flowers,while a small amount was expressed in pods and seeds.In addition,a large amount of GUS was expressed in apical meristem and trichomes of leaves.We also obtained transgenic apple callus containing MdPIP1;2 promoter fused to GUS gene through genetic transformation.Grown at 4℃,the expression of GUS in the transgenic callus was significantly higher than that of the control group,indicating that low temperature could upregulate the GUS reporter activity driven by MdPIP1;2 promoter.5.The cadidate transcription factors bingding to MdPIP1;2 promoter was obtained by yeast one-hybrid screening,while the one of the candidate related transcription factor MdCBF-l1 was further verified.The results showed that MdCBF-l1 could bind to the promoter sequence of MdPIP1;2,and up-regulate the expression of MdPIP1;2 gene.