Isolated Microspore Culture Of Non-heading Chinese Cabbage And Cloning And Expression Analysis Of Embryogenesis Related Genes BcSERKl And BcCYP78A5

Non-heading Chinese cabbage(Brassica campestris ssp.chinensis Makino)is one of the most important vegetable crops in the Brassica genus.Breeding by conventional cross breeding requires multiple generations of selfing to obtain excellent inbred parent,while homozygous plants can be obtained within 1-2 years by isolated microspore culture.In this paper,the effect of antioxidant glutathione on microspore embryogenesis of non-heading Chinese cabbage was studied and the regenerated plants were ploidy identified.The application of cell-penetrating peptides on microspore of non-heading Chinese cabbage was explored.BcSERKl and BcCYP78A5 genes were cloned from non-heading Chinese cabbage and related bioinformatics analysis and expression analysis were performed.The main findings are as follows:1.Three cultivars of non-heading Chinese cabbage were used as experimental materials to study effects of different concentrations of glutathione on microspore embryogenesis and the ploidy of the regenerated plants were identified.The results showed that when the concentration of glutathione reached 10 mg·L-1,the microspore embryogenesis frequency of ’Qinggeng’ and ’Kuaicai’ were the highest,reaching 42 embryo/bud and 36 embryo/bud,respectively;when the concentration of glutathione is 20 mg·L-1,the microspore embryogenesis frequency of’Erzhuangbai’ is the highest,reaching 20 embryos/bud.With increasing glutathione concentration,the microspore embryogenesis frequency of non-heading Chinese cabbage was inhibited.The experiment of cell-penetrating peptides showed that the cell penetrating peptide can protect the plasmid and the optimal ratio of the plasmid to the plasmid is 4:1;the cell penetrating peptide can enter the microspores separately and can mediate the plasmid into the microspores.But embryoid bodies obtained without fluorescence were in this study.2.BcSERK1 had an open reading frame(ORF)of was 1878 bp encoding 625 amino acids.BcSERK1 protein contained two transmembrane domains and a signal peptide,and was localized on the cell membrane;phylogenetic analysis indicates that the protein BcSERKl is closely related to Brassica napus and Brassica oleracea var.oleracea,with homology of 99.68%and 99.04%,respectively and highly conserved in the evolutionary process.The PC1300-BcSERK1-YFP vector was constructed by double enzyme digestion and transiently transferred into onion epidermal cells by particle bombardment.The results showed that BcSERKl protein was localized on the cell membrane.The results showed that the microspore embryogenesis frequency of ’Erzhuangbai’ was higher,reaching 6.67 embryos/bud and ’Sijiucaixin’ did not produce embryos.Real-time quantitative PCR(RT-qPCR)was used to analyze the expression characteristics of BcSERKl in different tissues and the early stage of microspore embryogenesis of two cultivar.It was found that BcSERKl expressed in different tissues of two varieties,but the expression level of each tissue except for root in ’Sijiucaixin’ was lower than that in Erzhuangbai’,and in the early stage of microspore embryogenesis,the expression level of BcSERK1 gene ’Erzhuangbai’was higher than that in the ’Sijiucaixin’.In conclusion,the expression level of BcSERK1 in the different tissues except the root of embryogenic material cabbage was higher than that of the non-embryogenic material,the expression level in the early stage of microspore embryo development in higher embryogenic material was higher than that in non-embryogenic material,and in the early stage of microspore embryo development,its expression decreased.3.BcCYP78A5 had an open reading frame(ORF)of 1551 bp encoding 516 amino acids;BcCYP78A5 protein had transmembrane region at 20-42 of the amino acid position,did not contain signal peptide and belonged to hydrophilic protein;amino acid sequence alignment and phylogenetic evolution indicated that it has the closest relationship with Brassica napus and Brassica oleracea var.oleracea with homology of 98.85%and 97.69%,respectively and highly conserved in the evolutionary process.Real-time quantitative PCR(RT-qPCR)was used to analysis the expression patterns of BcCYP78A5 during microspores embryogenesis.It was found that the expression level of this gene was extremely low at 0 d,1 d and 21 d after isolated microspore culture and the expression level increased during 7-14 d.The function of BcCYP78A on microspore embryogenesis was analyzed by VIGS(Virus-induced gene silencing)technique and the results showed that there was no significant difference in the microspore embryogenesis frequency between VIGS plants and control plants.