Differential Expression Analysis Of Lignin Synthesis Related Genes In The Seed Coat Of Brassica Napus L.

Brassica napus L.?2n=38,AACC?is a Brassica plant of the Cruciferae.This species is one of the most important oil crop species and sources of protein for animal feed worldwide.Lignin is a large molecular aromatic polymer that is one of the major components of the cell wall.However,an excessive lignin content in the seed coat reduces the availability and restricts the development of rapeseed cake.To reduce both the content of lignin and the hull rate,and increased the feeding value of the cake,and high-lignin?H-lignin?and low-lignin?L-lignin?materials of recombinant inbred lines?RILs?were selected in our study.The aim of our study was to explore the key genes or regulation factors causing different lignin contents in the seed coat of B.napus.The main conclusions are as follows:1.Screening of H/L-lignin materials of B.napusH-lignin?ADL:2.49%⁴.70%?and L-lignin materials?ADL:0.97%¹.34%?were screened from two years of near-infrared scanning data of 172 different RIL varieties.The seeds of the H-lignin material were black gray?degree of yellow:47.45^.67.35?,and the seeds of the L-lignin material were ginger yellow gray?degree of yellow:116.58¹44.71?.2.Analysis of quality traits of H/L-lignin seeds in B.napusThe hull content of the H-lignin material seed ranged from 15.26%¹8.76%,while that of the L-lignin material ranged from 10.94%¹2.26%.The hull content of the H-lignin material seed was significantly greater than that of the L-lignin material.We speculated that the seed coat of the H-lignin material was thicker than that of the L-lignin material.The fat content of H-and L-lignin materials was 36.70%⁴0.99%,37.60%⁴2.63%,respectively.The protein content of H-and L-lignin materials was25.58%²8.25%,27.80%³0.89%.The fat and protein content of the L-lignin material was slightly higher than that of the H-lignin material.3.Sectional observations of the seed coat of H/L-lignin material of B.napusMature seed coat sections of H/L-lignin material were observed microscopically.The results showed that the seed coat of the H-lignin material was thicker than that of the L-lignin material,especially the palisade layer,indicating that the difference in seed coat thickness may be due to the content of lignin.The frozen slices of 40 DAF and mature seeds were observed by pyrogallol staining.It was found that the cell wall of endothelial layer and the palisade of the H-lignin material were stained with phloroglucin in red,and L-lignin material may not stain significantly due to low lignin content.4.Transcriptome analysisThe seed coats and embryos at 35,40,and 46 days after flowering?DAF?were subjected to RNA sequencing?RNA-Seq?.Differentially expressed genes?DEGs?were screened based on an|log₂?fold change?|?1 and a false discovery rate?FDR?<0.01.A total of 15030 DEGs were screened.Gene Ontology?GO?enrichment analysis revealed that both upregulated and downregulated DEGs were enriched in cells,organelles,transport activities,and metabolic processes.DEGs were enriched mainly in cell walls,secondary metabolic processes,the cinnamic acid biosynthesis process,and phenylalanine ammonia-lyase activity according to a TopGO analysis.The general phenylpropanoid pathway was located upstream,and DEGs encoding PAL,C4H and 4CL were highly expressed.In the specific lignin synthesis pathway,BnCCoAOMT and BnCOMTs expression levels were high,and it was speculated that lignin in the seed coat was composed of mainly G-type and S-type lignin.In the three stages?35,40 and 46 DAF?of seed coat development,the expression of the DEGs BnPAL and BnC4H was significantly higher in the H-lignin seed coat than in the L-lignin,and most members of the Bn4CL gene family also presented this trend.MiRNAs that regulated BnPAL,BnC4H and Bn4CL were predicted,and the results showed that most DEGs were regulated by bna-miR172c,bna-miR396a and bna-miR172b.The proteins encoded by the DEGs of the PAL,C4H and 4CL families were predicted to determine their interactions,and the number of BnC4H-4 was found to be the highest.The expression of BnGA20OX3,which encodes gibberellin 20-oxidase 3,and BnJMT,which encodes jasmonic acid carboxyl methyltransferase,was upregulated,and the expression of BnAuxin,which encodes a dormancy/auxin-associated family protein,was downregulated,which may be the cause of the difference in lignin content of seed coat.It was found that the expression of 8 BnNACs was upregulated in the three DEG sets,especially BnNAC080 and BnNAC083.The expression of BnMYB9,BnMYB9-1,BnMYB60 and BnMYB60-1 was upregulated,and the expression of BnMYB91 was downregulated,suggesting that these genes may regulate structural genes downstream of lignin biosynthesis.The 23 key genes were verified by qRT-PCR and consistent with the results of RNA-Seq.