Cloninq Expression And Function Analysis Of The SBP-box Family Genes In Grape

The SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box gene family is specific toplants and encodes a class of zinc finger-containing transcription factors with a broad range offunctions. Although SBP-box genes have been identified in numerous plants including greenalgae, moss, silver birch, snapdragon, Arabidopsis, rice and maize, there is little informationconcerning SBP-box genes function in grapevine. On the basis of the preliminary study aboutSBP5from Vitis pseudoreticulata, we first systematically performed a genome-wideidentification of SBP-box genes in the V. vinifera genome and then conducted further geneclassification through an examination of exon–intron structure, gene phylogeny and syntenyanalysis using grape genome database. We also endeavored to investigate expression patternsof grape SBP-box genes under various abiotic, biotic stresses, phytohormone treatments and invarious grape organs and at different stages of fruit development. Furthermore, we alsoparticularly study the functions of VpSBP5,VpSBP11and VpSBP16. Results obtained are asfollows:1. Eighteen SBP-box gene family members were identified in V.vinifera, twelve of whichbore sequences that were complementary to miRNA156/157. Phylogenetic reconstructiondemonstrated that plant SBP-domain proteins could be classified into seven subgroups, with theV. vinifera SBP-domain proteins being more closely related to SBP-domain proteins fromdicotyledonous angiosperms than those from monocotyledonous angiosperms. In addition,synteny analysis between grape and Arabidopsis demonstrated that homologs of several grapeSBP genes were found in corresponding syntenic blocks of Arabidopsis. Expression analysis ofthe grape SBP-box genes in various organs and at different stages of fruit development revealeddistinct spatiotemporal patterns. While the majority of the grape SBP-box genes lacking amiR156/157target site were expressed ubiquitously and constitutively, most genes bearing amiR156/157target site exhibited distinct expression patterns, possibly due to the inhibitory roleof the microRNA. Furthermore, microarray data mining and quantitative real-time RT-PCRanalysis identified several grape SBP-box genes that are potentially involved in the defenseagainst biotic and abiotic stresses.2. Five SBP-box genes (VpSBP3，VpSBP5，VpSBP11，VpSBP12，VpSBP16) were isolatedfrom Chinese wild Vitis pseudoreticulata clone ‘Baihe-35-1’ using ReverseTranscription-Polymerase Chain Reaction (RT-PCR). The VpSBP3cDNA sequence included a1170bp open reading frame (ORF) which encodes a polypeptide of390amino acids; theVpSBP5cDNA sequence included a3090bp ORF which encodes a polypeptide of1030amino acids; the VpSBP11cDNA sequence included a510bp ORF which encodes apolypeptide of170amino acids; the VpSBP12cDNA sequence included a1137bp ORFwhich encodes a polypeptide of379amino acids; the VpSBP16cDNA sequence included a1674bp ORF which encodes a polypeptide of558amino acids They all contains a highlyconserved SBP-domain bearing two zinc-binding sites of the C2HCH type and a nuclearlocalization signal (NLS). We further confirmed that they are indeed targeted to the nucleusand possesses transcriptional activation activity by subcellular localization andtrans-activation assay.3. The overexpression vector pCAMBIA2300-35S-VpSBP16was constructed andtransformed into Arabidopsis thaliana by floral dip methods. Over-expression VpSBP16inArabidopsis,the transgenic lines showed enhanced resistance to high osmoticum,dehydration, long-term drought, and salt stress compared with the wild type. Theaccumulation of reactive oxygen species (ROS) in the transgenic lines were appreciablydecreased under the stresses.In addition, the transgenic lines had longer roots and highergermination rate than the wild type under the stresses. These results indicate thatoverexpression VpSBP16in Arabidopsis can improve the toleration to salt stress and droughtstress.4. The transcript levels of VpSBP5increased faster and stronger in the E.necator-resistant V. pseudoreticulata clone ‘Baihe-35-1’ than the susceptible clone ‘Hunan-1’upon infection with E. necator, which significantly increased until it reached the maximumlevel at24-48hpi in ‘Baihe-35-1’. However, the induction peak of VpSBP5was delayed andobserved at72-96hours post infection (hpi) in the susceptible ‘Hunan-1’. Moreover, theexpression of VpSBP5was mainly induced by salicylic acid (SA) and methyl jasmonate(MeJA) in ‘Baihe-35-1’. Together, our results indicate that VpSBP5is likely to participate inthe regulation of the resistance to E. necator by inducing SA, MeJA molecular signals ingrape, and the degree of disease resistance of the grapevine genotypes may correlate with thetime of the peak appearing.5. The overexpression vector pCAMBIA2300-35S-VpSBP11was constructed andtransformed into Arabidopsis thaliana by floral dip methods.Overexpression VpSBP11inArabidopsis can promote Arabidopsis flowering in long-day,short-day, normal light. At thesame time, the transgenic lines had more little rosette leaves, leaf curl and petiole elongation.