Identification and Functional Characterization of a Cold-Related Protein, BcHHP5, in Pak-Choi (Brassica rapa ssp. chinensis)

Abstract

In plants, heptahelical proteins (HHPs) have been shown to respond to a variety of abiotic stresses, including cold stress. Up to the present, the regulation mechanism of HHP5 under low temperature stress remains unclear. In this study, BcHHP5 was isolated from Pak-choi (Brassica rapa ssp. chinensis cv. Suzhouqing). Sequence analysis and phylogenetic analysis indicated that BcHHP5 in Pak-choi is similar to AtHHP5 in Arabidopsis thaliana. Structure analysis showed that the structure of the BcHHP5 protein is relatively stable and highly conservative. Subcellular localization indicated that BcHHP5 was localized on the cell membrane and nuclear membrane. Furthermore, real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed that BcHHP5 was induced to express by cold and other abiotic stresses. In Pak-choi, BcHHP5-silenced assay, inhibiting the action of endogenous BcHHP5, indicated that BcHHP5-silenced might have a negative effect on cold tolerance, which was further confirmed. All of these results indicate that BcHHP5 might play a role in abiotic response. This work can serve as a reference for the functional analysis of other cold-related proteins from Pak-choi in the future.

Keywords: Pak-Choi (Brassica rapa ssp. chinensis); cold-related; heptahelical protein 5; virus-induced gene silencing.

Figure 1

Multiple sequence alignments corresponding to the encoded amino acid sequences on the 11 heptahelical protein (HHP) genes. (A) In this study, HHPs in Pak-choi (BcHHPs) were isolated and other crop proteins were also selected from GenBank; (B) BcHHP5 and Arabidopsis thaliana heptahelical proteins (AtHHP5) were isolated in the study and selected from GenBank. Less conservative, highly conservative, and perfectly matched residues are represented by green boxes, pink boxes, and black boxes, respectively.

Figure 2

Conserved motifs in HHP. (A) Phylogenetic tree, conserved motifs, and their distribution in each HHP gene and the corresponding combined P-values; (B) the amino acid sequence of each motif. The font size means the frequency of the corresponding amino acid.

Figure 3

Secondary structural analysis of the BcHHP5 Protein. (A) Comparison of the secondary structures of BcHHP5 protein; (B) helix, turn, strand, and coil were represented with blue, red, green, and purple lines, respectively.

Figure 4

3D structural analysis of BcHHP5 Protein. (A) The predicted 3D structure of BcHHP5 protein; (B) local quality estimate of BcHHP5 protein; (C) global quality estimate of BcHHP5 protein; (D) a comparison of non-redundant sets with the Protein Data Bank (PDB) structural model.

Figure 5

Related information of the BcHHP5 protein. (A) Transmembrane domain of the BcHHP5 protein; (B) signal peptide of BcHHP5 protein; (C) hydrophilicity of the BcHHP5 protein; and (D) subcellular localization prediction analysis of the BcHHP5 protein.

Figure 6

Localization analysis of the BcHHP5 protein. (A) Schematic representation of the construction of 35S: GFP and 35S: BcHHP5-GFP; (B) schematic representation of the transient expression of 35S: GFP and 35S: BcHHP5-GFP fusion proteins in tobacco leaves (20 μm scale).

Figure 7

The expression patterns of BcHHP5 in Pak-choi under abiotic stress were analyzed by RT-qPCR. (A) Cold treatment, BcHHP5 expression; (B) salt treatment, BcHHP5 expression; (C) ABA treatment, BcHHP5 expression; (D) salicylic acid (SA) treatment, BcHHP5 expression. *: 0.01 < P < 0.05, **: P < 0.01.

Figure 8

BcHHP5 silencing in Pak-choi. (A) The Pak-choi leaf phenotype (2.5 cm scale) after BcHHP5-silencing test; (B) relative expression of BcHHP5; (C) ion leakage of Pak-choi leaves; (D) relative expression of BcPDS; (E) ion leakage of Pak-choi leaves.