Analysis Of Broccoli Embryogenic Callus By CDNA-AFLP System And Clongling Of EF-1a、LEC1

Using Broccoli somatic embryogenesis not only can be rapid propagation,but also providepremium receptor environment for genetic variation and genetic engineering.This study founddifferential gene of development stage of broccoli SE by cDNA-AFLP, cloned the EF-1α and LEC1cDNA, in order to provide a basis for the study of the molecular mechanisms of somaticembryogenesis.1Research on broccoli embryogenesis plant regenerationCotyledon explants of ‘TaiWangqingnong65’ were cultured induce SE and then the SEdeveloped into shoot. By studying the effect of plant growth regulation agents(PGRs) onBroccoli SE, selected the best broccoli for embryonic callus proliferation medium and thegermination medium.The best medium to induce somatic embryogenesis was Murashige andSkoog (MS) medium supplemented with0.5mg·L-12,4-D+0.1mg·L-1NAA, inducing rate up to97.3%,cuting without veins squares.Secondary somatic embryos were proliferated on MSmedium containing0.2mg·L-12,4-D. Studies have shown that maturation of SE were welldeveloped on1/2MS with IBA(mg·L-1). The mature somatic embryos were germinated and32.6%of them could develope into plantlets by culturing on MS medium containing6-BA(0.5mg·L-1) and IAA(0.5mg·L-1).2Analysis of differential gene of broccoli embryogenic callus usingcDNA-AFLP techniqueIn this study, the loose embryogenic callus and compact embryogenic callus material wereused as materials to analyze the differentially expressed genes which related to EC development.Then optimized selective amplification of cDNA-AFLP system, and the results showed that maincomponents of20ul system as follows:template cDNA(30ng), dNTP(300mol/L),primers(0.5mol/L), Extaq enzyme(0.5U).Selective amplification products were separated by polyacrylamide gel electrophoresis. Theresult showed that43positive TDFs were high homology with relative genes. Then the function of TDFs refered to cell proliferation, energy metabolism, translation-related factor, stress responsegene silencing, regulation of enzyme catalysis, hormone response, and antioxidant restore, etc.3Cloning of EF-1α gene from broccoli embryogenic callusEF-1α gene (Registry Number KC508634) was cloned from broccoli embryogenic callus,contained1705base pair. The putative protein was consisted of449amino acids. By analysis ofblast, the EF-1α gene respectively had95%、91%、89%、88%、87%、87%identity with Brassicanapus,Arabidopsis thaliana, Rosa multiflora Nicotiana tabacum, Litchi chinensis. It wasspeculated that the protein was stable hydrophilic and non-secreted proteins. Secondary structureof the protein was mainly composed of22.72%alpha helix,31.40%β-folded and45.88%irregular curl.There is a small amount of irregular crimp in three-dimensional structure whichconsistency was74.21%, compared to the reference protein.4Cloning of LEC1gene from broccoli embryogenic callusBy RT-PCR and RACE technique,LEC1gene (Registry Number KC541558) was isolated bycompact embryogenic callus of broccoli.The full length of gene was926bp, which encoded239amino acids. The putative amino acid sequence homology was above70%, compared withBrassica napus,Arabidopsis thaliana, longyan, Zea mays, Peanut.Isoelectric point of the proteinwas5.52, simultaneously instability coefficient was46.11.The protein was unstable protein, whichcontained the three CBF/NFYB/HMF conserved domains, without signal peptide and thetransmembrane domain. Secondary structure of the protein was mainly composed of alpha helix,β-folded and irregular curl.