梨CBL基因家族全基因组序列的鉴定及非生物胁迫下的表达分析

Identification and Expression Analysis under Abiotic Stresses of the CBL Gene Family in Pear

【目的】最近在植物中发现了一类Ca2+传感蛋白——类钙调磷酸酶B亚基蛋白CBL(calcineurin B-likeproteins),CBL及其靶蛋白CIPK(CBL-interacting protein kinase)构成CBL/CIPK信号网络系统,在植物干旱、盐渍、低温等逆境胁迫应答中起重要作用。鉴定梨基因组中CBL家族基因成员,对其基因进化、结构与表达特征进行分析,为植物CBL基因功能分析与利用提供依据。【方法】通过生物信息学手段,结合梨基因组注释信息,鉴定梨CBL家族成员序列信息;利用MEGA6.0程序进行多序列比对、分类并构建系统进化树;利用per1程序、GSDS工具以及ClustalX软件进行基因结构与保守性分析;通过qRT-PCR技术进行多种非生物胁迫处理下PbCBLs表达分析。【结果】成功鉴定出7个CBL家族成员,基因结构预测表明PbCBL9含5个内含子,其余PbCBLs均含有7—8个内含子;预测的PbCBLs均含4个EF-hand功能域,且相邻EF-hand功能域之间氨基酸数目非常保守;通过系统发育树分析,将7个PbCBLs分为2类;qRT-PCR技术进行不同胁迫处理下杜梨叶片PbCBLs的表达分析,结果表明,在NaCl胁迫下,PbCBL1表达量6h降至最低,24h则明显升高;与之相反,PbCBL2和PbCBL3的表达量在6h达最高,24h最低;PbCBL4和PbCBL8表达量在3h明显增加,随后表达量下降;PbCBL9表现明显的上调趋势,24h达到最大;PbCBL10则在6h表达量最高。10%(w/v)PEG6000胁迫处理下,PbCBL2、PbCBL4和PbCBL8表达量上调,PbCBL1表达量下调;PbCBL3、PbCBL9和PbCBL10表达量均在6h最低,12h和24h较6h明显升高,PbCBL3和PbCBL10于24h表达量达到最高,而PbCBL9在12h表达量最高。4℃低温处理下,PbCBL2、PbCBL4、PbCBL8表达量上调;而PbCBL1和PbCBL3表达量下调;PbCBL9和PbCBL10表达量表现上下波动的变化趋势,均在3h有明显增加,随后显著降低。42℃高温胁迫处理下,PbCBL1、PbCBL3和PbCBL4表达量下调;PbCBL2表达量整体上调,6h达到最高;PbCBL8、PbCBL9和PbCBL10总体呈现相同的变化趋势,在3h表达量达到最高,随后明显下降。ABA处理下,PbCBL3和PbCBL10表达量下调,PbCBL2与PbCBL8表达量上调;PbCBL1在6h表达量最高;PbCBL4和PbCBL9表达量呈现相似的变化趋势,3h明显升高,随后下降,24h时最低。【结论】成功鉴定的7个候选PbCBLs中,2个基因(PbCBL8和PbCBL9)受盐胁迫诱导表达,3个基因(PbCBL2、PbCBL4和PbCBL8)分别受干旱、低温与ABA诱导表达。PbCBL2在高温胁迫下被强烈诱导可能意味着其在高温应答中发挥重要作用。

【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.q RT-PCR was further used to analyse the expression of Pb CBLs under various abiotic stress conditions.【Result】 Seven CBL gene family members were successfully identified.Gene structure prediction indicated that Pb CBL9 consists of only five introns,while others consist of 7 to 8 introns.The predicted Pb CBLs 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 Pb CBLs were classified into two subgroups.Differences in gene expression analysis measured by q RT-PCR in leaves of Pyrus betulaefolia Bunge showed that,in NaCl treatment,the Pb CBL1 gene expression reached its nadir at 6 h,and then increased after 24 h.On the contrary,the expression of Pb CBL2 and Pb CBL3 showed a maximum and minimum at 6 h and 24 h,respectively.Meanwhile,Pb CBL4 and Pb CL8 were significantly increased at 3 h,but then decreased.Pb CBL9 expression was clearly enhanced throughout NaCl treatment and reached a maximal expression at 24 h.Pb CBL10 exhibited a maximal expression level at 6 h.In the stress condition of 10% PEG6000,the expressions of Pb CBL2,Pb CBL4,and Pb CBL8 all increased,while the expression of Pb CBL1 was decreased.Pb CBL3,Pb CBL9,and Pb CBL10 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.Pb CBL3 and Pb CBL10 reached a maximum at 24 h while Pb CBL9 reached a maximum at 12 h.Under cold temperature stress（4℃）,the expressions of Pb CBL2,Pb CBL4,and Pb CBL8 all increased while Pb CBL1 and Pb CBL3 both decreased.Fluctuations in Pb CBL9 and Pb CBL10 gene expression levels were observed,with significant increases around 3 h,followed by a decrease.Under high temperature stress（42℃）,the expressions of Pb CBL1,Pb CBL3,and Pb CBL4 all decreased,while Pb CBL2 was increased,reaching a maximum at 6 h.Pb CBL8,Pb CBL9,and Pb CBL10 had similar trends overall,with the highest levels at 3 h followed by an obvious decline.Under ABA stress conditions,the expressions of Pb CBL3 and Pb CBL10 decreased while the expressions of Pb CBL2 and Pb CBL8 increased.Pb CBL1 reached a maximum at 6 h and the expressions of Pb CBL4 and Pb CBL9 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 Pb CBLs members,two Pb CBLs genes（Pb CBL8 and Pb CBL9） were induced by salt stress,three Pb CBLs genes（Pb CBL2,Pb CBL4 and Pb CBL8） were strongly induced by drought,cold and ABA stress.Pb CBL2 may play an important role in pear under heat stress because of its constitutive high level expression.