Transcriptome analysis for walnut seed during oil synthesis period

Abstract:【Objective】Walnut seeds accumulate a lot of fat during the oil synthesis period. It is of great importance for us to understand the genes involved in lipid metabolism for genetic manipulation. Transcriptomic analyses were employed to explore the expression pattern of the genes related to lipid metabolism during the course of lipid transition period in the seeds of walnut.【Methods】In this study, Juglans regia‘Xinxin 2'in Xinjiang was used as materials, TIANGEN (DP441) RNA extraction kit was used for extracting m RNA. The seeds were sampled at three different stages of oil synthesis (60 to 70 days after flowering referred to as G1; 90 to 100 days after flowering referred to as G2; 120 to 130 days after flowering referred to as G3) . The sequencing of transcriptome was performed by Beijing Novogene Bioinformatics Technology Company. The sequencing platform was Illumina Hi SeqTM4000. The clean reads were obtained by removal of the reads containing the sequence of the joint, the reads in which the N ratio was over10% and the reads of low quality. There is no reference genome to the walnut, so we used the Trinity software to do the de novo sequencing using the data obtained from each gene as transcriptome, and the longest transcript of each gene was taken as unigene. The sequence features of transcript and the length of the unigene were statistically analyzed respectively for late analysis. Protein Annotation information aboutthe unigene was obtained by sequence alignment in Nr, Nt, KEGG, Swiss-Prot, PFAM, GO and KOG protein databases. The transcripts of Trinity splicing were used as reference sequence (ref) , the RSEM software was used (bowtie 2, the parameter is mismatch 0) for mapping the clean reads and ref of each sample, and accounting for the number of readcount for clean reads to each unigene. The data obtained from the gene expression level of readcount were used as input data for differentially expressed genes and the differentially expressed genes were screened by DESeq software (P< 0.05) .【Results】Through the RNASeq analysis of the seeds of walnut during the oil transformation period, 174 545 unigenes were obtained, the average length of the unigene was 644 bp and the N50 was 1 050 bp, among them, the length of unigene sequence between 1 000 and 2 000 bp accounted for 8.51%, and the length of sequence over 2 000 bp accounted for 6.52%. Annotation analysis of unigenes indicated that 94 133 transcripts were homologous with those of other species in the public protein database; however, 80 412 sequences were not annotated and might be walnut-specific. Walnut seeds with 69 235 unigenes were annotated with Nr protein database. According to the classification of KOG, 27 333 unigenes were divided into functional categories, and the function classification of GO are 51 769 unigenes. In addition, for KEGG pathway classification, 26 946 unigenes were annotated. There were 9 408, 8 316 and 6 398 differential unigenes in G2 vs G1, G3 vs G2 and G3 vs G1 were up-regulated, 11 916, 10 485 and 5 218 differential unigenes in G2 vs G1, G3 vs G2 and G3 vs G1 were down-regulated, respectively, which were annotated to the KEGG pathway at each stage of lipid transformation. It was found that the highest concentration of the genes related to the metabolic pathway of the fatty acid bio-synthesis was mostly obvious at the stages of G1, G2 and G3.There were 34 differentially expressed genes of G2 vs G1, G3 vs G2 and G3 vs G1 of fatty acid biosynthetic pathway. In the fatty acid biosynthetic pathway, the expression of carboxylase subunit gene (acc A) , biotin carboxyl carrier protein gene (acc B) and biotin carboxylase subunit gene (acc C) of acetyl coenzyme A were continuously up-regulated. The expression of β-ketoacyl ACP synthase gene (fab F) and β-ketoacyl ACP reductase gene (fab G) at G1 to G3 period continued to increase. The expression of stearyl-ACP desaturase gene (FAB2) at G3 phase was higher than that at G2 phase. q RT-PCR was used for the quantitative determination of 8 randomly selected genes, the results were consistent with the sequencing data.【Conclusion】Most of the unigene of the walnut seeds had higher matching degree with the known genes in the existing database. The oil synthesis of walnut seeds was mostly concentrated at the G2 stage. The accA, accB, fabF, fabG, fabI and FAB2 genes involved in the lipid biosynthesis were all up-regulated.