Screening and functional prediction of differential expression genes at lignification stage of endocarp in ‘Zhipi' walnut

Abstract: 【Objective】The differential expression genes from the three representative samples were obtained by synthetically analyzing the expression of transcripts in the endocarp of walnuts, which laid a foundation for the comprehensive and systematic study on the molecular mechanism of lignification in walnut endocarp.【Methods】Walnut fruit was divided into two parts after sampling, one for observing lignin deposition in endocarp and the other for extracting RNA. With the former fruit, it was cut transvwersely and then longitudinally into thin slices. Finally, all the flakes were washed with flowing water and soaked in the modified Wiesner reaction solution for 5 minutes, and the image was photographed.With the later fruit, it was cut into pieces with a knife, and only the endocarp was preserved by removing the green peel (husk) and seed, and the endocarp (shell) was frozen with liquid nitrogen and preserved at-80 ℃. The extraction of total RNA from walnut endocarp was carried out with pBIOZOL plant tissue RNA extraction kit (BioFlux) . Specific operation was carried out according to the instruction sheet. The samples of RNA extraction were sent to Beijing Genomics Institute in Shenzhen, China for quality and integrity detection. After qualified detection, mRNA was enriched with the magnetic beads of Oligo (dT) and then sequenced library was constructed before it was sequenced on computer.The sequenced image data was transformed into sequence data (raw reads) by base calling software, filtered by filter fq software to obtain the final needed data (clean reads) , and then the clean reads was spliced and assembled. Finally, the required Unigene was used to screen out the differential expression genes among samples by bioinformatics and software and to predict the biological function of the Unigene.【Results】By Wiesner method, we found that the lignifization of the endocarp began at 44 days after full bloom, and the process of lignification was basically completed by 80 days after full bloom.The lignin deposition was on the endocarp at the same time, but the amount of lignin deposition in the top of endocarp of the fruit was higher than that in other regions. A total of 76 814 Unigenes were obtained by using Trinity software to assemble reads, and 1 077 differential expression genes were obtained using FDR≤0.001 and |log2 Ratio|≥1 F as screening conditions. Three statistical significant gene expression profiles were obtained by further analysis of 1 077 selected differential expression genes in The Short Time-series Expression Miner (STEM) software. The gene expression profile was No.1 (0, -2, 1) , No.6 (0, -1, 1) , and No.10 (0, 1, 0) in turn. Through expression profile analysis, we finally selected 609 differential expression genes to be assigned into the salient model. In order to explore the biological function of 609 Unigenes, we first selected GO database to annotate 609 differential expression Unigenes. The results showed that Unigenes were significant enrichment in 42 functional terms under three major Gene Ontology, and in their biological process there were 17 terms, mainly including some cellular process, metabolic process, biological regulation, developmental process, signaling and reproduction and growth; there were 8 terms for cell components, including cell, cell part, organelle, membrane, membrane part, organelle part and cell junction and symplast, and there were only 11 enriched term numbers in molecular function. Secondly, KEGG annotation showed that differential expression genes were significantly enriched in protein processing in endoplasmic reticulum, ABC transporters, endocytosis, plant-pathogen interaction, phenylpropanoid biosynthesis, spliceosome, phenylalanine metabolism, plant hormone signal transduction, ubiquinone and other terpenoid-quinone biosynthesis. Among them, transcription factor BIM1 gene CL169.Contig7＿All, serine/threonine-protein kinase SAPK3 gene Unigene28134＿All, ethylene receptor 2 gene Unigene18324＿All, auxin response factor 3 gene CL447.Contig4＿All, histidine kinase 2 gene CL4024.Contig3＿All and so on were involved in plant hormone signal transduction pathway, the gene CL2824.Contig3＿All was involved in the synthesis of ATP-binding cassette transporter, the gene CL9312.Contig1＿All, Unigene27245＿All, and CL3278.Contig2＿All were involved in the synthesis of ABC transporter A family member 2, ABC transporter B family member 11 and ABC transporter C family member 3 in the ABC transporter pathway and gene CL2405.Contig5＿All, Unigene24904＿All, CL2983.Contig6＿All and Unigene7110＿All were involved in phenylpropane biosynthesis pathway, phenylalanine catabolism pathway, phenylalanine lyase, coenzyme A ligase, acyltransferase, acyl-coenzyme A synthase and peroxidase.【Conclusion】We screened out the main metabolic pathways of differential expression genes in the endocarp of walnut, which laid a good foundation for the systematic study on the molecular mechanism of lignification in the endocarp of walnut in the future.