Identification and Expression Analysis of Cold Shock Protein 3 (BcCSP3) in Non-Heading Chinese Cabbage (Brassica rapa ssp. chinensis)

Abstract

A cold-related protein, cold shock protein 3 (BcCSP3), was isolated from non-heading Chinese cabbage in this study. BcCSP3 can encode 205 amino acids (aa) with an open reading frame (ORF) of 618 base pairs (bp). Multiple sequence alignment and phylogenetic tree analyses showed that BcCSP3 contains an N-terminal cold shock domain and is highly similar to AtCSP2, their kinship is recent. Real-time quantitative polymerase chain reaction (RT-qPCR) showed that the expression level of BcCSP3 in stems and leaves is higher than that in roots. Compared with other stress treatments, the change in BcCSP3 expression level was most pronounced under cold stress. In addition, a BcCSP3-GFP fusion protein was localized to the nucleus and cytoplasm. These results indicated that BcCSP3 may play an important role in response to cold stress in non-heading Chinese cabbage. This work may provide a reference for the identification and expression analysis of other CSP genes in non-heading Chinese cabbage.

Keywords: cold shock protein 3; cold stress; expression analysis; non-heading Chinese cabbage; subcellular localization.

Figure 1

Nucleotide acid and corresponding amino acid sequence of BcCSP3 in non-heading Chinese cabbage.

Figure 2

Secondary structural prediction of the BcCSP3 protein. The successively shorter straight lines represent helix, turn, strand and coil, respectively; the blue, red, green and purple curves represent helix, turn, strand and coil, respectively.

Figure 3

Tertiary structure prediction of the BcCSP3 protein. Random coil, extended strand and beta turn make up most of the main body.

Figure 4

Amino acid sequence alignment of the putative BcCSP3 protein with Arabidopsis CSP proteins. Perfectly matched residues, highly conserved residues, and less conserved residues are represented by a black, dark gray and gray box, respectively.

Figure 5

Phylogenetic and motif analysis of the putative BcCSP3 protein and Arabidopsis CSPs. The unrooted tree is based on an alignment of the full-length protein sequences and was constructed by the neighbor joining method.

Figure 6

RT-qPCR analysis of the expression patterns of BcCSP3 in roots, stems and leaves of Pak-choi. Each column represents the average and standard deviation of three replicates. Each bar value represents the mean ± SD (p < 0.05).

Figure 7

RT-qPCR analysis of the expression patterns of BcCSP3 in Pak-choi (leaves) under abiotic stresses. (A) BcCSP3 expression under ABA treatment; (B) BcCSP3 expression under cold treatment; (C) BcCSP3 expression under salt treatment; (D) BcCSP3 expression under dehydration treatment. Each column represents the average and standard deviation of three replicates. Each bar value represents the mean ± SD (p < 0.05).

Figure 8

Subcellular localization of BcCSP3. (A) Constructs used in the experiments; (B) BcCSP3–GFP was transferred to tobacco leaf cells to test and verify the subcellular localization of BcCSP3 (bar = 20 μm).

Figure 9

Online prediction of protein interactions.