Mechanism Of DELLA (GbGAI4) Protein In Somatic Embryogenesis Of Gossypium Barbadense L.

Southern Xinjiang because of the unique climate to become China’s island cotton main planting.In order to improve the production development and international competitiveness of Xinjiang island cotton,it is necessary to improve the varieties of island cotton.Cotton somatic embryogenesis and genetic engineering technology can be combined without seasonal restrictions.Efficient and directional improved germplasm can shorten the time to obtain cotton materials with improved traits.This study centered on the somatic embryogenesis of sea-island cotton,based on bioinformatics analysis of differentially expressed genes during the somatic embryogenesis of sea-island cotton.With the help of promoter activity detection technology,the GbGAI4 gene was detected in sea-island cotton embryos.Activity in sex cells;using RT-PCR technology to detect the correlation between the differentially expressed gene Gb LEC2 of sea island cotton and somatic embryogenesis;through the yeast two-hybrid test,verify the GbGAI4 protein and somatic embryo development in the process of somatic embryogenesis in sea island cotton.The interaction relationship of related protein Gb LEC2,preliminary elucidation of the mechanism of GbGAI4 protein and its interaction protein in the embryonic development of the sea island cotton plant,provide a technical and theoretical basis for the molecular breeding of the sea island cotton in the later stage.The main findings are as follows:1.With the help of cell observation method,the method of screening cotton somatic embryogenic callus with high differentiation rate is summarized,which is helpful for the subsequent selection and acquisition of genetically modified materials.The p CAMBIA1304-Pro _GbGAI4 recombinant vector was constructed by seamless cloning technology,and p CAMBIA1304-Pro _GbGAI4 was transformed into the embryogenic callus of sea island cotton by means of Agrobacterium-mediated method.After antibiotic screening,the transgenic positive cells were obtained.Through GUS staining analysis,the expression of GbGAI4 gene in embryonic cells was studied.2.Using RT-PCR technology,the c DNA of the four stages of somatic embryo development of sea island cotton was used as a template to detect the correlation between Gb LEC2 gene and sea island cotton somatic embryogenesis.The results showed that the Gb LEC2 gene was expressed in four important stages of cotton somatic embryogenic callus,and the expression reached the highest in the cotyledon embryo stage,showing significant differences.It is preliminarily inferred that the Gb LEC2 gene is involved in the somatic embryogenesis of island cotton.3.A seamless cloning method was used to construct two yeast two-hybrid vectors,p GBKT7-GbGAI4and p GADT7-Gb LEC2 are bait protein and target protein,respectively.Yeast two-hybrid results confirmed the interaction between Gb LEC2 protein and GbGAI4 protein,which was revealed from the molecular level.The molecular mechanism of DELLA protein in the somatic embryogenesis of sea island cotton provides a theoretical basis for the induction of embryogenic callus of sea island cotton and shortening the plant regeneration cycle.