Studies On Genetic Transformation Of Prunus Mume And Functional Identification Of PmNF-YB Transctriptional Factors

Mei flower（Prunus mume Sieb.et.Zucc.）is one of the tradition famous flowers and has very long cultivation history in China.It has high economic value and outstanding ornamental features,such as trees’pose,flower’s fragrance and color.In this study,we optimized the plant regeneration system of cotyledons and somatic embryogenesis,and established Agrobacterium-mediated transformation systems in Prunus mume,using cotyledons and somatic embryos of P.mume as receptor.Meanwhile,Pm NF-Y genes of Mei flower from the Prunus mume genome were identified through bioinformatics methods.And the expression patterns of Pm NF-Ys under ABA,drought,high salinity,and H₂O₂ conditions were detected.In addition,three PmNF-YB genes whose expression levels were up-regulated under abiotic stresses were functionally analyzed.The main results were as follows:1.Optimization of regeneration systems of P.mume.1)Optimization of regeneration system from mature cotyledons of P.mume.We investigated the effects of different concentrations of ZT and IAA on proliferation and elongation of adventitious buds of Mei flower.The results indicated that ZT and IAA significantly influenced proliferation and elongation of adventitious buds,with interaction existing between them,and higher concentrations of ZT and IAA had negative effects on proliferation and elongation of adventitious buds.Thus,the optimal proliferation and elongation medium of adventitious buds was 1/2MS medium containing 1.0 mg/L ZT and0.1 mg/L IAA,on which,the highest multiplication coefficient and average height of seedling were 3.12 and 2.37 cm after 4 weeks,respectively.2)Optimization of regeneration system of somatic embryogenesis of P.mume.Using immature embryos of‘Xue Mei’as explants,we investigated the effects of carbon sources and plant growth regulators on somatic embryo formation.The results indicated that carbon sources,NAA,2,4-D,and TDZ significantly influenced somatic embryogenesis of Mei flower.Somatic embryogenesis only was observed on the induction medium containing with 3%sucrose.Somatic embryos were induced on the medium supplemented with NAA.But it is hard to get the proliferation and germination of somatic embryos.The higher induction rate was obtained on the medium containing1.0 mg/L 2,4-D.However,when the concentrations of 2,4-D were less than or equal to0.2 mg/L,there was not somatic embryo formation.Addition of 0.005mg/L TDZ had positive effects on embryogenesis.So,the optimal induction medium was 1/2MS medium containing 3%sucrose,0.005mg/L TDZ,1.0mg/L 2,4-D and 0.2 mg/L BA,on which,the highest induction rate of 61.11%.In addition,different concentrations of 2,4-D would affect the proliferation of somatic embryos significantly.The higher concentrations of2,4-D（≥0.1 mg/L）had negative effects on the proliferation of somatic embryos.The highest rate of proliferation（75%）was obtained on 1/2MS+0.01 mg/L 2,4-D+0.1 mg/L BA+0.05 mg/L NAA+0.008 mg/L TDZ.2.Established of genetic transformation in P.mume.Based on the enhanced regeneration system from mature cotyledons,using 2500cotyledons of P.mume as receptor,11 transgenic plantlets were observed by Agrobacterium-mediated transformation.The result of PCR analysis showed that 9positive individuals were obtained and the transformation efficiency was 0.36%.Using somatic embryos of‘Xue Mei’as receptor material,an Agrobacterium-mediated transformation protocol was investigated by assessing GUS gene transient expression rate.The resulted showed that infected for 40min,co-cultured for 3d,supplemented with 150mg/L Km and 300 mg/L Cef were optimal for transgenic system,on which,the highest transient GUS expression efficiency（81.48%）was obtained.3.Expression patterns of Pm NF-Y in response to abiotic stress.A total of 6 Pm NF-YA,13 PmNF-YB,and 8 Pm NF-YC genes in the Prunus mume genome were identified and annotated by bioinformatics procedures.Based on Real-time PCR analyses,we investigated the gene expression patterns of all Pm NF-Y genes in ABA,osmotic,salt,ROS stresses.Pm NF-YA1/2/4/5/6,PmNF-YB3/4/8/10/11/13,and Pm NF-YC1/2/4/5/6/8 were found to be up-regulated expression under ABA and osmotic treatment.The expression levels of Pm NF-YA1/2/3/4/5/6,PmNF-YB1/3/8/10/11/13,and Pm NF-YC1/2/5/6/8 were obviously induced by ROS stress.In addition,only 2 Pm NF-Ys（Pm NF-YA2 and PmNF-YB3）also enhanced expression in salt stress.These findings suggested that Pm NF-Y genes may be involved in multiple abiotic stresses response.4.Functional analysis of three PmNF-YB transcription factors.Based on the gene expression patterns analysis of PmNF-YB genes under abiotic stresses,three PmNF-YB genes（PmNF-YB3,PmNF-YB8 and PmNF-YB13）,whose expression levels were increased significantly under abiotic stresses,were cloned from P.mume‘Xue Mei’and overexpression vectors were constructed.Then these genes were transformed to Arabidopsis to investigate their functions in plant stress response.We found that transformed Arabidopsis with over-expressing PmNF-YB3 or PmNF-YB8exhibited higher seed germination and faster root elongation under salt and osmotic treatment compared to wild type.So,the result showed that transgenic Arabidopsis overexpressing PmNF-YB3 or PmNF-YB8 improved tolerance to salt and osmotic,while overexpression of PmNF-YB8 or PmNF-YB13 in Arabidopsis showed higher survival rate than wild type under drought stress.which hinted that these PmNF-YB genes were involved in response to abiotic stresses.