Analysis Of Genes Expression In The Early Stage Of Microspore Embryogenesis In Chinese Cabbage(Brassica Rapa L. Ssp.Pekinensis)

The mechanism of microspore embryogenesis remains unclear till now, which limits the application of isolated microspore culture technology in plant breeding and genetic engineering research. The project of whole genome sequencing for Chinese cabbage has been completed, and the cultivation system of Chinese cabbage microspore was matured. In order to gain more insights into the mechanism of microspore embryogenesis, we performed transcriptome sequencing of microspores in the early stage of embryogenesis, the main conclusions are as follows:1. We chose "jihong82" as the donor plant and five samples as the sequencing material according to screening. The quality of RNA extracted by RNA-pure kit could meet the requirements of transcriptome sequencing.2. High-throughput Illumina RNA sequencing by paired-end sequencing were conducted, according to reference transcriptome and Data Analysis, and approximately141.60M high quality (HQ) reads,28.6Gb of sequence data were generated. For each of the samples, more than80percent of Q30were aligned on the reference genome.3. Six alternative splicing including ES, IR, AFE, ALE, Alt5’and Alt3’were studied, and the result showed that the number of IR was the most.In addition, SSRs were systematically tested, and478new SSRs were searched out.4.2228new transcripts were found by high throughput sequencing since present databases may be incomplete. The sequences of new genes were compared to the database of Nr, SwissProt, GO, COG and KEGG, and1602of them have annotation information.5. Based on RPKM method, we analysed differential expression genes between different samples. According to Hierarchical Cluster Analysis, a number of differentially expressed genes were gained in each group.6. Gene Ontology functional enrichment analyses revealed that the DEGs were primarily distributed in three cellular component functional classifications:cell part, cell and organelle, and two molecular function classifications:binding and catalytic activity, and the classifications in biological process were the most.7. Clusters of Orthologous Groups enrichment analyses showed that the DEGs were primarily associated with General function prediction only, Signal transduction, Transcription and Replication, recombination and repair.8. KEGG pathway enrichment analyses revealed that the DEGs were primarily associated with plant hormone signal transduction, starch and sucrose metabolism pathways, and so on. The expression of related enzymes and genes in the pathways rised significantly under heat shock condition, which suggesting that genes related with these pathways may play important roles in the early stage of microspore embryogenesis.