Study On The Pathway And Mechanism Of Microspore Embryogenesis Of Different Temperature Pretreatment In Broccoli (Brassica Oleracea L. Var. Italica)

Broccoli (Brassica oleracea L.var.italica) is a variant of cabbage，which belongs to brassica faimaly.Broccoli is a cross-pollinated plant and possesses heterosis. Cross breeding is the main way of Broccolibreeding. In order to obtain the genetically stable and pure inbred lines, it s necessary to do continuousself-fertilize at bud stage，and it would be6to8times. That is a long and tedious process. The long timecan be shorten by microspore culture. The materials in theory can be obtained in2years. The breedingprocess can be speeden up by microspore culture.The yield of embryo can be effected by the gene type, which limits the application of thistechnology. It is meaningful to learn the pathway of the microspore embryogenesis and the differencebetween the4transcriptomes, and this is of use for understanding of the cellular and molecularmechanisms of microspore embryogenesis.In the study, B908and B1092were used to be the experimental materials.The fluorescencemicroscopy, qPCR, RACE and transciptome analysis were used. The study on the pathway andmechanism of microspore embryogenesis of different temperature pretreatment were investigated fromthe two levels of cellular and molecular.Some conclusions are as follows:1．The results showed that the pathways of embryogenesis were much the same of differentpretreatment. The B pathway was the main way under4℃、32.54℃、44℃+32.54℃（combinationtreatment),that two identical or similarly size cells were formed from the symmetrical division, anddivided repeatedly to form an embryo. Some state of E and D pathway can be be observed. It wasdifficult to survey the sampe with25℃. Only some globular embryos and some tissues can be seen,when it was visible. The combination of cold temperature at4℃and heat shock at32.5℃can inducethe highest embryo yield.2．The expression patterns of TDF4, TDF9, TDF20, TDF25and BBM were different at differenttemperature pretreatment. Among the5TDFs, the expression pattern of TDF19was similar to BBM,and the correlation coefficient was0.76.3．A gene related to microspore embryogenesis was cloned. The gene was2283bp in length, andencode760amino acid residues with a estimated molecular mass of85.5KD and an isoelectric point of6.11. There were helix，-turn，random coil in the secondary structure. There were3models in thethertiary structure，one of which was bond by Zn. The result of Blast showed that the protein maybelong to the kinase and took an important role in the process of the signal transduction process ofmicrospore embryogenesis.4．3.56784407,52339892,68744711, and73345307high quality reads were obtained from the4samples (the ck，the sample with4℃pretreatment, the sample with32.5℃pretreatment，the samplewith combination pretreatment) by sequencing. Between different samples, there were31isoformsexpressed differently between the samples4℃and and the control.It was1281between the sample with32.5℃and the control. It was1673between the sample with combinant pretreatment and the control.5．KEGG metabolic pathway enrichment analysis were performed, and there were more KEGG pathways between high temperature and combinant temperature to the control. Some metabolic pathwaywere involved in the process of microspore embryogenesis, such as signal transduction and cell signaltransduction, secondary metabolite synthesis and metabolism, salicylic acid and jasmonic acidstimulation, abscisic acid signal transduction.