Construction Of Plant Expression Vector Of GbWOX9 Gene And Genetic Transformation Of Embryogenic Cells In Gossypium Barbadense L.

Stem cells of plant meristem are the key to plant morphogenesis and organ regeneration.WUS/WOX family members are specifically expressed in stem cells of plant meristem and play an important role in plant organ development and cell type transition.In this study,sea island cotton“Xinhai 16”as materials,cloned the gene GbWOX9 related members of the WOX/WUS family of transcription factors in plant embryo development,and has carried on the structure prediction analysis,subcellular localization prediction,homology analysis,phylogenetic analysis and expression of different stages of somatic embryos.At the same time,the construction of chemical inducible GbWOX9 sense and antisense plant expression vector by Agrobacterium mediated genetic transformation of GbWOX9 gene into the“Xinhai 16”positive sense and antisense embryogenic cells and the concentration of inducer treated cells in different tissue expression pattern analysis.The main results are as follows:1.Using PCR technology to clone the GbWOX9 gene using online software and DANMAN and MEGA5 on the gene bioinformatics analysis showed that this gene contains 1038 bp open reading frame of encoding 345 amino acids,the online prediction of the molecular weight of the protein is about 38.26 kD,isoelectric point was 6.7.Through amino acid sequence alignment,we found that the sequence contains a conserved DNA binding domain composed of 65 amino acids,that is,homeodomain.Phylogenetic tree analysis showed that the GbWOX9 protein of sea island cotton and Asian cotton GaWOX9 protein distributed on the same branch,which indicated that the two species had closer phylogenetic relationship.Subcellular localization predicts that GbWOX9 may be localized to the nucleus.qRT-PCR showed that the highest expression level of the gene in the globular embryo“Xinhai 16”in the stage of somatic embryos.2.The inducible positive and antisense plant expression vectors pTA7002-GbWOX9 and Anti pTA7002-GbWOX9 were constructed,and the optimization of Agrobacterium tumefaciens genetic transformation system was carried out.The results showed that the embryogenic cells were 10 d OD600?0.5⁰.6 pre culture,infection time was 15 min,24 h of co culture,the concentration of Ceftriaxome and Hygromycin resistance culture medium were 500 mg·L^-1and 20 mg·L^-11 for the better transformation result.After screening two times on an antibiotic medium,transgenic positive cells were obtained.3.The success of the extraction and transformation of island cotton“Xinhai 16”positive and antisense plant expression vector of embryogenic cell total RNA expression by fluorescence quantitative PCR detection of GbWOX9.The experimental results,indicating the antisense strand of embryogenic cells after the induction of DEX concentration of 30?mol·L^-1,the expression of GbWOX9 had significant difference with the control which witnout the induction of Dexamethasone?P<0.01?.