The Functional Research Of Developmental Regulators In Gossypium Somatic Embryogenesis

Gossypium barbadense,with the best quality cotton fiber in the world,has high economic value.In China,G.barbadense only could be planted in Xinjiang southern area.At present,there are some problems in the production of G.barbadense,such as low yield,poor stress resistance,and fiber quality.Therefore,the strains need to be improved.Somatic embryogenesis is an efficient and operable transgenic technology.However,the establishment of somatic embryogenesis is difficult and highly genotype-dependent,which limits the process of molecular breeding in Xinjiang G.barbadense.Developmental regulators could promote somatic cells to develop into regenerated plants directly,thus bypass the tissue culture process and break the restriction of genotype dependence on plant regeneration system.In this study,overexpressed GbBBM8-A and other developmental regulators(WUS/DELLA/IPT)in G.barbadense somatic cells to verify whether these regulators can induce somatic cell differentiation into regenerated plant.In addition,for developmental malformation in developmental regulator-mediated regenerated plants,this study also focused on research a promoter that could drive developmental regulators expressing in young leaves,embryos,callus,but not in roots,meristems and reproductive tissues.The results are shown as follows:(1)The BBM gene GbBBM8-A was obtained by RT-PCR.The open reading frame of GbBBM8-A gene was 2019 bp,encoding a protein of 672 amino acids with two AP2 protein domains.Fluorescence quantitative PCR results displayed that the expression of GbBBM8-A in torpedo embryos is significantly higher than other stages of somatic embryogenesis,indicating that the physiological role of GbBBM8-A gene was involved in somatic embryogenesis.To verify whether development regulators could induce somatic cell differentiation into regenerated plant,the plant expression recombinationation vector pCAMBIA3301-GbBBM8-A was constructed by the seamless cloning technology,and the hypocotyls of Gossypium barbadense were infected with the Agrobacterium-mediated method,and then transferred on the medium without any phytohormone.The results showed that there was no regenerated bud but serious embolus and some callus in the hypocotyl section at 20 d after infected with the control group,GbBBM8A/GbIPT group,and GbBBM8-A/GbDELLA group.By contrast,the regenerated bud appeard in in hedpocotyls at 20 d after infected with GbBBM8-A group and GbBBM8-A/Gb WUS group.However,these bud evtually developmentd into abnormal leaves and shoots.(2)Semi-quantitative PCR results displayed that GbLEC2 was expressed in globular embryo,torpedo embryo and cotyledon embryo,but not in root,stem,leaf and embryogenic callus.And bioinformatics analysis demonstrated that there were two seed-specific cis-elements,Skn-1 element at-300 bp and RY repeat element at-403 bp respectively in GbLEC2 promoter.To further verify the promoter activity of GbLEC2,four plant expression vectors with different fragments GbLEC2-1(2000 bp),GbLEC2-2(1500 bp),GbLEC2-3(1000 bp)and GbLEC2-4(500 bp)were constructed and transferred into the different tissues and cells in G.barbadense with the transient transfection method.The results of GUS activity assay showed that GUS activities in the genotypes infected with all the GbLEC2 promoter fragments could be detected in somatic embryogenesis including embryogenic callus,globular embryos,torpedo embryos and cotyledon embryos,but not in leaves.However,there was a slight blue GUS staining in stem tissues of GbLEC2-l and GbLEC2-2 genotypes;and only a faint of GUS staining in root tissues(including main roots and lateral roots)could be dected in the GbLEC2-1 genotype.(3)These results indicate that the 500 bp and 1000 bp fragments in GbLEC2 promoter display transcription activities in somatic embryogenesis,which fit to the characters for promoters to drive the expression of developmental regulators.Therefore,GbLEC2 promoter is expected to be a new candidate promoter to accurately regulate the expression of developmental regulators,so as to be applied in developmental regulators-mediated transgenic technology in G.barbadense.