Genome-Wide Identification and Expression Analysis of nsLTP Gene Family in Rapeseed (Brassica napus) Reveals Their Critical Roles in Biotic and Abiotic Stress Responses

Abstract

Non-specific lipid transfer proteins (nsLTPs) are small cysteine-rich basic proteins which play essential roles in plant growth, development and abiotic/biotic stress response. However, there is limited information about the nsLTP gene (BnLTP) family in rapeseed (Brassica napus). In this study, 283 BnLTP genes were identified in rapeseed, which were distributed randomly in 19 chromosomes of rapeseed. Phylogenetic analysis showed that BnLTP proteins were divided into seven groups. Exon/intron structure and MEME motifs both remained highly conserved in each BnLTP group. Segmental duplication and hybridization of rapeseed's two sub-genomes mainly contributed to the expansion of the BnLTP gene family. Various potential cis-elements that respond to plant growth, development, biotic/abiotic stresses, and phytohormone signals existed in BnLTP gene promoters. Transcriptome analysis showed that BnLTP genes were expressed in various tissues/organs with different levels and were also involved in the response to heat, drought, NaCl, cold, IAA and ABA stresses, as well as the treatment of fungal pathogens (Sclerotinia sclerotiorum and Leptosphaeria maculans). The qRT-PCR assay validated the results of RNA-seq expression analysis of two top Sclerotinia-responsive BnLTP genes, BnLTP129 and BnLTP161. Moreover, batches of BnLTPs might be regulated by BnTT1 and BnbZIP67 to play roles in the development, metabolism or adaptability of the seed coat and embryo in rapeseed. This work provides an important basis for further functional study of the BnLTP genes in rapeseed quality improvement and stress resistance.

Keywords: expression analysis; genome-wide identification; nsLTP; phylogenetic analysis; rapeseed (Brassica napus).

Figure 1

Sequence logos for the eight-cysteine motif (8CM) of BnLTP proteins. The height of each amino acid residue represents the degree of conservation. The numbers on the x-axis represent the positions in the 8CM. The y-axis shows the information content measured in bits.

Figure 2

Phylogenetic relationships of nsLTP proteins among B. napus, B. rapa, B. oleracea and A. thaliana. This tree was generated using the protein sequences of their 8CM domains. BnLTP, BrLTP, BoLTP and AtLTP are shown with the red (Bn), green (Br), blue (Bo) and yellow (At) squares, respectively.

Figure 3

Genome-wide synteny analysis for nsLTP genes in B. napus. BnA01–10 and BnC01–09 represented chromosomes in A and C sub-genomes in B. napus, respectively. Red, green and blue lines linked the syntenic gene pairs within BnA-BnA, BnC-BnC and BnA-BnC subgenomes of B. napus, respectively.

Figure 4

Expression patterns of BnLTP genes in 42 various tissues and organs. The abbreviations combinations of 42 samples of B. napus cultivar ZS11 are listed in supplementary Table S11. The corresponding heatmap containing BnLTP gene names is shown in supplementary Figure S3.

Figure 5

Expression patterns of BnLTP family genes in the leaves of susceptible (Westar) and tolerant (ZY821) genotypes of rapeseed infected with S. sclerotiorum at 24 h post-inoculation (24 hpi).

Figure 6

Expression patterns of BnLTP family genes in the cotyledons of rapeseed resistant (DF78) and susceptible (Westar) lines at 0, 3, 7 and 11 days post-L. maculans inoculation.

Figure 7

The heatmap of the expression levels of BnLTP family genes in 3 week-old plants of rapeseed under multiple abiotic stresses (NGDC project ID CRA001775), including dehydration (1 h and 8 h), ABA (25 μM; 4 h and 24 h), NaCl (200 mM; 4 h and 24 h), and cold (4 °C, 4 h and 24 h).

Figure 8

The heatmap of the expression levels of BnLTP genes at different germination stages of B. napus accessions seeds with representative germination rates (high, medium, or low; C129, C033 and C032, respectively) at 0, 12, 24, 48 and 72 h after imbibition.

Figure 9

The numbers of BnLTP family DEGs in transgenic rapeseeds (Westar cultivar) overexpressing pBAN::BnTT1 (BOE, 20 DAP seeds, seed coat-specific promoter), pNapA::BnTT1 (NOE, 20 DAP seeds, seed embryo-specific promoter) and pNapA::BnbZIP67 (B67O, 25 DAP seeds, seed embryo-specific promoter) (a); Venn diagram of the numbers of BnLTP family DEGs in BOE and NOE (b).

Figure 10

The impact of S. sclerotiorum infection on photosynthesis of rapeseed leaves (a,b); and the qRT-PCR expression level of two top Sclerotinia-responsive BnLTP genes, BnLTP129 and BnLTP161 (c). Standard images of the RGB, Fv/Fm, ΦPSII and NPQ from S. sclerotiorum-infected rapeseed leaves at 20, 30 and 40 hpi. A false color scale is used for each parameter. The values represent the average ± SD of 3 biological replicates. Fv/Fm, maximum quantum yield of PSII; ΦPSII, effective quantum yield of PSII; NPQ, non-photochemical quenching. * p < 0.05 and ** p < 0.01 compared with samples at 20 hpi.