关键词: 梨, CBL家族基因, 全基因组鉴定, 系统进化, 胁迫响应, 基因表达

Abstract: 【Objective】 Calcineurin B-like proteins (CBL) is a kind of Ca²⁺ sensor protein recently found in plants. CBL /CIPK (CBL-interacting protein kinase) signal network system plays an important role in plant stress response such as salinity, drought and chilling. The evolution and structure of Calcineurin B-like proteins (CBLs) in pear were identified and their tissue expression was analyzed for providing a basis for the functions of plant CBL genes and their application. 【Method】 The annotation information of the pear genome was analysed using bioinformatics tools to identify the sequence information of CBL family members in pear. The MEGA 6.0 software was used for multiple sequence alignment, classification, and construction of the phylogenetic tree; per1-based program, GSDS and Clustal X software were adopted to analyze gene structure and conservative. qRT-PCR was further used to analyse the expression of PbCBLs under various abiotic stress conditions. 【Result】 Seven CBL gene family members were successfully identified. Gene structure prediction indicated that PbCBL9 consists of only five introns, while others consist of 7 to 8 introns. The predicted PbCBLs contain four EF-hand domains and a highly conservative number of amino acids between every two adjacent EF-hand domains. According to the phylogenetic analysis, the seven PbCBLs were classified into two subgroups. Differences in gene expression analysis measured by qRT-PCR in leaves of Pyrus betulaefolia Bunge showed that, in NaCl treatment, the PbCBL1 gene expression reached its nadir at 6 h, and then increased after 24 h. On the contrary, the expression of PbCBL2 and PbCBL3 showed a maximum and minimum at 6 h and 24 h, respectively. Meanwhile, PbCBL4 and PbCL8 were significantly increased at 3 h, but then decreased. PbCBL9 expression was clearly enhanced throughout NaCl treatment and reached a maximal expression at 24 h. PbCBL10 exhibited a maximal expression level at 6 h. In the stress condition of 10% PEG6000, the expressions of PbCBL2, PbCBL4, and PbCBL8 all increased, while the expression of PbCBL1 was decreased. PbCBL3, PbCBL9, and PbCBL10 had a minimum gene expression at 6 h, but then increased significantly at 12 h and 24 h as compared with that at 6 h. PbCBL3 and PbCBL10 reached a maximum at 24 h while PbCBL9 reached a maximum at 12 h. Under cold temperature stress (4℃), the expressions of PbCBL2, PbCBL4, and PbCBL8 all increased while PbCBL1 and PbCBL3 both decreased. Fluctuations in PbCBL9 and PbCBL10 gene expression levels were observed, with significant increases around 3 h, followed by a decrease. Under high temperature stress (42℃), the expressions of PbCBL1, PbCBL3, and PbCBL4 all decreased, while PbCBL2 was increased, reaching a maximum at 6 h. PbCBL8, PbCBL9, and PbCBL10 had similar trends overall, with the highest levels at 3 h followed by an obvious decline. Under ABA stress conditions, the expressions of PbCBL3 and PbCBL10 decreased while the expressions of PbCBL2 and PbCBL8 increased. PbCBL1 reached a maximum at 6 h and the expressions of PbCBL4 and PbCBL9 significantly increased at 3 h followed by a decrease, reaching its lowest at 24 h. 【Conclusion】 The pear CBL genome family consists of seven members and among all the seven PbCBLs members, two PbCBLs genes (PbCBL8 and PbCBL9) were induced by salt stress, three PbCBLs genes (PbCBL2, PbCBL4 and PbCBL8) were strongly induced by drought, cold and ABA stress. PbCBL2 may play an important role in pear under heat stress because of its constitutive high level expression.

Key words: pear, CBL gene family, genome-wide identification, phylogeny analysis, stress responses, gene expression