Microspore Culture And Differential Expression Analysis Of Anthocyanin Synthesis Genes Related To In Non-heading Chinese Cabbage(Brassica Rapa Ssp.chinensis Makino)

Non-heading Chinese cabbage(Brassica rapa ssp.chinensis Makino)is a biennial herbaceous plant of the Brassica of the cruciferous family.As a leaf vegetable variety with high potassium and calcium content,the germplasm resources in China are not abundant.The microspore culture method is used to create the Non-heading Chinese cabbage DH line,which can provide homozygous materials for the diversification of new varieties of it.In the identification of the regenerated plants of Non-heading Chinese cabbage,green mutant materials were found.In this study,anthocyanin content determination and RNA-Seq were carried out,and the differentially expressed genes related to anthocyanin synthesis were verified by qRT-PCR,in order to discover the genes related to anthocyanin synthesis in it.1.In this experiment,four commercially available materials were used as test materials,and embryoid were successfully obtained in all four test materials.Among them,the embryo induction rate of ‘18sy028’ was 8.70 embryos/bud,‘18sy026’ is only 5.83 embryos/buds.The experiment found that heat shock is necessary for the culture of microspores of Non-heading Chinese cabbage.The induction rate of embryoid is the highest under the condition of heat shock treatment for 24 h,and heat shock treatment for more than 72 h will reduce the induction rate of embryoid.In the seedling formation test of microspore regeneration plants,the direct seedling formation ability of ‘18sy029’ and the indirect seedling formation ability of callus were the highest,while the direct seedling formation rate of ‘18sy027’ was the lowest,and the indirect seedling formation ability of ’18sy027’ was the worst.The ploidy identification of the regenerated plants found that the natural occurrence rate of double haploid in ‘18sy029’ was the highest,reaching 62.02%,while that of ‘18sy27’ was the lowest at 41.79%.In the process of identifying the regenerated plants of the plant,it was found that the DH lineage of purple leaf Non-heading Chinese cabbage produced natural mutant green leaf individuals.Homozygous genotype materials were screened through selfing,and their anthocyanin content was measured.The results showed that the anthocyanin content of the purple material ’JSCp1-3’ reached the highest 19.40mg/g,while the two green leaf materials had almost no anthocyanin production.Through RNA extraction,sequencing and database construction of mutant,we obtained a large amount of transcriptome data of green and purple Non-heading Chinese cabbage.Through a series of data analysis,we obtained 3665 differentially expressed genes,1636up-regulated differentially expressed genes,and down-regulated There are 2029 differentially expressed genes in the data;analysis of the GO and KEGG enrichment pathways of the datayields 29 significantly enriched GO terms and 5 significantly enriched metabolic pathways.The most significantly enriched involves 231(11.46%)Structural molecular activity of differentially expressed genes and ribosomal pathways involving 213(23.61%)differentially expressed genes.The significant enrichment pathways are the phenylpropane biosynthesis of brp00940 and the flavonoid biosynthesis pathway of brp00941,which are related to the synthesis of anthocyanins.The expressed genes with a difference of more than 2.0(log2Fold Change)were selected for qRT-PCR verification,and their upward and downward trends were basically consistent with RNA-Seq,which further verified the correctness of the sequencing results.