Isolation And Functional Analysis Of BlOFPs Genes In Betula Luminifera

Betula luminifera is not only a high-quality timber species with a priority to the promotion,but also an ideal material for forest genetic study. Improvement of wood properties is one of theimportant contents of its breeding nowadays. Deciphering the molecular mechanism of woodformation and gene expression during the same stage, is the premise of achieving efficientbreeding of Betula luminifera.On the basis of transcriptome data, seven full-length BlOFP cDNAs from Betula luminiferawere isolated using RACE. The length of sequences of cDNA are ranging from665to1328bp.the Open Reading Frame(ORF) are ranging from345to1062bp, the identity of they are rangingfrom24.5%to61.5%. The predicted proteins encoded by these genes are composed of115to354amino acid residues, seven predicted proteins share limited amino acid identity with each other,ranging from11to50%, and each of which contains a OVATE domain that is characteristic ofplant OFP proteins.The phylogenetic analysis showed that BlOFP1and BlOFP2belonged to thesame clade, BlOFP3, BlOFP5and BlOFP7belonged to the same clade, and BlOFP4and BlOFP6belonged to the same clade.Gene expression analysis using qRT-PCR indicated that transcripts of BlOFP1have aincreasing trend during the development of stem, and were most abundant in mature leaf. TheBlOFP2expression levels in the lignified stem (Stem3-Stem5) were obviously higher than theother samples. The BlOFP1, BlOFP2expression levels in the outside tissues of stem were higherthan the inside tissues of stem. BlOFP3, BlOFP4,BlOFP6and BlOFP7were most abundant in leafand famale inflorescence, and presented the similar expression patterns. The expression patterns ofBlOFP5shown no rule in the organs. BlOFP3, BlOFP4, BlOFP5and BlOFP6were mostabundant in phloem, and presented the similar expression patterns in4tissues of stem(phloem,cambium, outer xylem, inner xylem). In4tissues of stem, BlOFP7were most abundant in phloemand cambium, while less in outer xylem and inner xylem. The analysis of qRT-PCR implied thatBlOFP1, BlOFP2are relevant to the development of secondary cell wall synthesis, while BlOFP3,BlOFP4, BlOFP5, BlOFP6and BlOFP7relevant to development of leaf and form of inflorescence.Gene expression analysis using in situ mRNA hybridization clarified that the same expressionpattern of BlOFP1, BlOFP2in4tissues of stem, when they were most abundant in cambium and little in pith, further more they were considerable amount in phloem and xylem. IAA has acooperative restraining action with GA to expression of BlOFP1, BlOFP2in stem. The BlOFP1expression levels declined in response to mechanical stress, tension wood was lower than oppositewood. The BlOFP2expression levels rose in tension wood,while fell in opposite wood.In order to elucidate the functions of BlOFP in the molecular mechanism of wood formationin Betula luminifera, we conducted the Yeast Two-Hybrid. The7BlOFPs were cloned into a preyvector pGADT7and also a bait vector pGBKT7, The5BlSTMs were cloned into a bait vectorpGBKT7. pGBKT7-BlSTM2, pGBKT7-BlSTM did autonomously activate the reporter genes inY2HGold, in the absence of a prey protein, while others did not.The result of Yeast Two-Hybrid,there are7out of70groups showed the interaction, they are OFP3-KNOX2, OFP3-KNOX4,OFP3-KNOX5, OFP4-KNOX2, OFP6-KNOX2, OFP6-KNOX4and OFP6-KNOX5. weconcluded that BlOFP forms a functional complex with BlKNOX protein to regulate a certainmetabolic pathway.The7BlOFPs were cloned into a over-expression vector pCAMBIA13011. theover-expression vector with BlOFP1, BlOFP2were transform into Betula luminifera respectively,and we got transgenic plants. Plants overexpressing BlOFP2displayed very unique phenotypes.The characteristic phenotypes of these plants were obscure bough, ramose, smaller leaf. Accordingto the grobacterium-mediated transformation of cambial issue in the stem, we found that thecoalescence of wound site was relevant to the time of tissue exposure to grobacterium, andtransformation efficiency was involved. The area of tissue exposure to grobacterium was also afactor to transformation efficiency.The study will not only lay the foundation of process of understanding wood formationmechanism in Betula luminifera, but provide important information of sequence and gene resourcein subsequent molecular-breeding.