Development of full-length cDNAs from Chinese cabbage (Brassica rapa Subsp. pekinensis) and identification of marker genes for defence response

Abstract

Arabidopsis belongs to the Brassicaceae family and plays an important role as a model plant for which researchers have developed fine-tuned genome resources. Genome sequencing projects have been initiated for other members of the Brassicaceae family. Among these projects, research on Chinese cabbage (Brassica rapa subsp. pekinensis) started early because of strong interest in this species. Here, we report the development of a library of Chinese cabbage full-length cDNA clones, the RIKEN BRC B. rapa full-length cDNA (BBRAF) resource, to accelerate research on Brassica species. We sequenced 10 000 BBRAF clones and confirmed 5476 independent clones. Most of these cDNAs showed high homology to Arabidopsis genes, but we also obtained more than 200 cDNA clones that lacked any sequence homology to Arabidopsis genes. We also successfully identified several possible candidate marker genes for plant defence responses from our analysis of the expression of the Brassica counterparts of Arabidopsis marker genes in response to salicylic acid and jasmonic acid. We compared gene expression of these markers in several Chinese cabbage cultivars. Our BBRAF cDNA resource will be publicly available from the RIKEN Bioresource Center and will help researchers to transfer Arabidopsis-related knowledge to Brassica crops.

Figure 1.

Clustering of the BBRAF Chinese cabbage (B. rapa subsp. pekinensis) full-length cDNA clones and homology to Arabidopsis transcripts. (A) Distribution of the number of BBRAF clones in each cluster of the sequence assembly. We clustered a total of 10 000 BBRAF clones into 5476 independent clones. The sequence assembly was performed using the PCAP.REP software. (B) Pie chart of the distribution of sequence homology between the BBRAF clones and Arabidopsis TAIR9 transcripts. The e-value of the BLASTN analyses is shown. (C) Distribution of the number of BBRAF clones homologous to Arabidopsis genes in the TAIR9 transcript database.

Figure 2.

Functional annotation of the BBRAF Chinese cabbage (B. rapa subsp. pekinensis) full-length cDNA clones using the Arabidopsis gene ontology database. Functional annotations are presented in relation to (A) the biological process and (B) the molecular function. The distribution is shown for the total BBRAF clones (total), BBRAF clones with high homology to Arabidopsis genes (≤1e − 150), and the whole-genome Arabidopsis (TAIR9 transcripts genome).

Figure 3.

Comparison of the Chinese cabbage (B. rapa) and Arabidopsis RuBisCo small subunit 2B genes. (A) Sequence comparison of five Chinese cabbage (B. rapa subsp. pekinensis) and four Arabidopsis rbcs genes. Fully conserved bases are shown in yellow. The conserved positions for more than five genes are shown in green. (B) The phylogenetic tree for the five Chinese cabbage and four Arabidopsis rbcs genes. (C) Comparison of the amino acid sequences of the five Chinese cabbage and four Arabidopsis RBCS proteins. Fully conserved bases are shown in yellow. The conserved positions for more than five genes are shown in green.

Figure 4.

Comparison of Chinese cabbage (B. rapa subsp. pekinensis) and Arabidopsis PR1 marker genes. (A) DNA sequence comparison. Conserved base positions are shown in yellow. (B) Amino acid sequence comparison. Conserved base positions are shown in yellow.

Figure 5.

Gene expression analyses of gene homologues for Arabidopsis salicylic acid (SA) and jasmonic acid (JA) marker genes. ET, 1-aminocyclopropane-carboxylic acid treatment. (A and B) Chinese cabbage (B. rapa subsp. pekinensis) homologues to Arabidopsis PR1 and BGL2 marker genes for the salicylic acid pathway. (C and D) VSP2 and LOX2 as marker genes for the jasmonic acid pathway. We used 2-week-old plants grown in a single pot in this experiment (n = 5, with two replicates). The plants were treated with 1 mM salicylic acid, 100 μM jasmonic acid, and 50 μM 1-aminocyclopropane-carboxylic acid for 2, 5, 10, or 24 h. The total RNA was extracted, and first-strand cDNA was synthesized for expression analysis. The expression level of each gene was normalized to the expression of BrACT2, which is constitutively expressed and shown as the reference value.

Figure 6.

Gene expression analyses of Chinese cabbage (B. rapa subsp. pekinensis) marker genes for salicylic acid and jasmonic acid response in major cultivars. Cultivars used in this experiment: Chifu, Chifu hakusai; K-P02, KmP02; Kigokoro, Kigokoro 85; Muso; K-No.3, Kyoto No. 3; Okiniiri. (A and B) BrPR1 and BrBGL2 as marker genes for the salicylic acid pathway; (C and D) BrVSP2 and BrLOX2 as marker genes for the jasmonic acid pathway. We used 2-week-old plants (n = 3, with three replicates), grown in a single pot, and treated them with water, 100 μM jasmonic acid, and 1 mM salicylic acid for 24 h. The total RNA was extracted and first-strand cDNA was synthesized for the PCR analysis. The expression level of each gene was normalized with respect to the expression of BrACT2 (control).