Studies On The Mechanism Of Somatic Embryogenesis Regulated By Uniconazole And NF-YB Gene

Cotton is an important economic crop that provides the natural fiber for human living.Upland cotton is the most widely cultivated allotetraploid with high yield in modern times,however,the genetic background of upland cotton is very narrow.Genetic engineering is especially important in the research of upland cotton for broadening the diversity of germplasm.Genetic transformation of upland cotton is realized by somatic embryogenesis,stepped through callus formation,embryogenic callus,somatic embryo stage and then transformed into seedling.And so,improving the culture system and understanding the molecular mechanism of somatic embryogenesis will be helpful to enhance the transformation efficiency in upland cotton.1.Uniconazole promotes somatic embryogenesis by regulating the homeostasis of endogenous gibberellin and abscisic acid.Somatic cells start the process of embryogenesis under some specific conditions.In order to improve the culture system,plant growth regulator uniconazole was added into the callus induction medium.Uniconazole inhibited the proliferation of callus,and promoted the transformation of callus into embryogenic callus,which promoted the time of transformation from callus to embryogenic callus and also the transformation of the whole callus into embryogenic callus.The contents of endogenous gibberellin and abscisic acid in leaves of hydroponic seedlings treated with uniconazole were detected.Compared with the control,the content of gibberellin in leaves was decreased,while the content of abscisic acid was increased significantly.The expressions of AGL15,BBM,FUS3,LEC1 and LEC2 in callus were detected by q RT-PCR,the results showed that uniconazole promoted the expression of these genes.Transcriptional analysis of callus treated with uniconazole for 0 h,0.5 h,1 h and 3 h,we found that uniconazole affected the biosynthesis and metabolism of gibberellin,promoted the up-regulation of gibberellin3-oxidase and down-regulation of gibberellin 2-oxidase in the process of callus development.And then all of these affected the gene expression homesstasis in gibberellin signaling pathway.Meanwhile,uniconazole inhibited abscisic acid metabolism pathway by down-regulating ABA 8’-hydroxylase.This result is consistent with the level of endogenous hormone in cotton seedlings treated with uniconazole.At the same time,the effects of synthesized gibberellin inhibitos(paclobutrazole and ancymidol)and abscisic inhibtor(fluridone)were verified and the results showed that none of these three substances could promote somatic embryogenesis.It was concluded that the change of gibberellin or abscisic acid content seperately could not promote somatic embryogenesis.The results suggested that uniconazole promoted somatic embryogenesis by regulating the homeostasis of endogenous gibberellin and abscisic acid.2.Effect of NF-YB Gene on somatic embryogenesis in Gossypium hirsutum L.The NF-YB transcription factor gene family members play important roles in many physiological processes,such as seed development,drought tolerance,and flowering time.However,the function of NF-YB genes in cotton remains unclear.A total of 23,24 and 50NF-YB genes were identified in Gossypium arboreum,Gossypium raimondii,and Gossypium hirsutum respectively.Phylogenetic analysis was carried out among Gossypium arboreum,Gossypium raimondii,Gossypium hirsutum,rice,sorghum,and cacao to predict functional conservatives,and divided 150 NF-YB genes into five clades.Of these clades,αis the largest one,whileδis the least.γclade contains LEC1 type NF-YB proteins.The syntenic analysis revealed that paralogues of NF-YB genes in G.hirsutum had a good collinearity.Due to the segmental duplication within A sub-genome(A_t)and D sub-genome(D_t),NF-YB genes had expanded in G.hirsutum.Furthermore,we investigated the structures of exons,introns,and conserved motifs of NF-YB genes in upland cotton.The expression pattern of NF-YB gene in upland cotton showed that most of the NF-YB genes were generally expressed,and only a few genes were highly expressed in specific tissues in accordance with the results of q RT-PCR detection.Gh A/DNF-YB6,Gh A/DNF-YB18 and Gh A/DNF-YB22 genes were highly expressed in embryogenic calli,and the somatic embryogenesis of cotton was promoted by transgenic Gh DNF-YB22.This study was the comprehensive identification of the Gh NF-YB gene family in cotton.This work provides a basis for further understanding the role of NF-YB genses on somatic embryogesis in cotton.