石榴MAPK家族基因鉴定及其响应冷胁迫的表达分析

Identification of MAPK family genes and analysis of their expression patterns in response to cold stress in pomegranate

【目的】评价不同石榴种质资源籽粒硬度及抗寒性,筛选可能参与调控石榴抗寒性的MAPK家族基因。【方法】以31份石榴种质资源为试材,进行抗寒性及籽粒硬度评价;基于全基因组筛选石榴MAPK家族基因,对其进行进化、基因结构和蛋白理化性质分析,同时利用实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)分析冷胁迫对石榴MAPK家族基因表达模式的影响。【结果】31个石榴品种籽粒硬度及半致死温度测定结果表明,峄城粉红牡丹、淮北六棱甜和鲁白榴2号等硬籽石榴抗寒性较强,华紫、以3和玛丽斯等软籽石榴抗寒性较弱。基于石榴全基因组鉴定出17个MAPK家族基因,广泛分布于8条染色体上;MAPK家族所有成员主要分为3个亚类,其中,A和B亚类成员主要包含PKc_MAPKK_plant_like和PTZ00024结构域,C亚类主要包含PLN00034结构域,所有成员均具有S_TKc结构域;各成员氨基酸残基数量分布在314~860 aa,外显子数目1~18个,蛋白分子质量为34910.05~97965.26 u,等电点4.94~9.35;PgMKK2、PgMPK6、PgMPK9、PgMPK16和PgMPK13在峄城粉红牡丹响应冷胁迫过程中表现为显著上调,PgMKK8、PgMPK1-1和PgMKK4在玛丽斯响应冷胁迫过程中表现为显著上调;PgMKK2、PgMPK6、PgMPK9、PgMPK16和PgMPK13在峄城粉红牡丹响应冷胁迫过程中的表达量显著高于玛丽斯,PgMKK8和PgMPK1-1在玛丽斯响应冷胁迫过程中的表达量显著性高于峄城粉红牡丹;PgMKK3在峄城粉红牡丹不同时间均未检测到表达,在玛丽斯中表现为先升高后降低的趋势;PgMPK12-2在玛丽斯不同时间均未检测到表达,在峄城粉红牡丹中表现为逐渐升高的趋势。【结论】石榴MAPK家族基因响应冷胁迫信号,其中,PgMKK2、PgMPK6、PgMPK12-2和PgMPK9可能参与正调控石榴的抗寒性。

【Objective】Cold stress is one of the most important factors limiting the progress of pomegranate production.The objectives of this study were to compare the cold hardiness among 31 pomegranate cultivars with wide distribution of seed hardness,and analyze the expression pattern of Mitogenactivated protein kinase(MAPK)family genes in response to cold stress.The results can pave the way for shedding light to the function of Mitogen-activated protein kinase cascades in pomegranate cod tolerance.【Methods】Plant materials were cultivated in the National Horticulture Germplasm Resources Center of China(NHGRC),with conventional cultivation management.Juvenile branches of 31 pomegranate cultivars were sampled in mid-January,the relative electrolyte conductivity(REC)was measured,and the semi-lethal temperature(LT50)was calculated for the evolution of cold hardiness.Seed hardness was detected by the TA-XT texture apparatus.MAPK family genes of Arabidopsis were used as queries to search in the whole pomegranate genome database,and reference genome sequence of‘Tunisia’was obtained from NCBI database.Potential members of MAPK family were identified.Phylogeny relationship,gene structure and protein physicochemical properties were analyzed.Moreover,we performed real-time quantitative PCR(qRT-PCR)to analyze the expression pattern of 17 MAPK family genes in response to cold stress.【Results】The seed hardness and cold tolerance were evaluated.A total of 17 MAPK family genes were identified,which widely distributed on different chromosomes.All the members of the MAPK family could be mainly divided into three sub-classes,among which the members of subclass A and B mainly included PKc_MAPKK_plant_like and PTZ00024 Domain,subclass C mainly contained the PLN00034 domain,and all members contained a S_TKc domain.The results of analysis of physicochemical properties of proteins showed that the number of amino acid residues of each member distributed from 314 to 860 aa,the number of exons was from 1 to 18,the molecular weight of the protein was from 34910.05 to 97965.26 u,and the isoelectric point was from 4.94 to 9.35.Specific primer was designed for each PgMAPKs and PgMAPKKs,and their expression patterns were detected.The results showed that 12 out of 17 members were activated after low-temperature treatment.Yichengfenhongmudan that showed strong cold tolerance,and Malisi with weak cold tolerance,were both selected to compare the expression pattern of MAPKs.PgMKK2,PgMPK6,PgMPK9,PgMPK16 and PgMPK13 were all rapidly activated after low-temperature treatment in Yichengfenhongmudan.PgMKK8,PgMPK1-1 and PgMKK4 were rapidly activated in Malisi.PgMKK2,PgMPK6,PgMPK9,PgMPK16 and PgMPK13 showed significantly higher expression level in Yichengfenhongmudan than those in Malisi after low-temperature treatment.The expression of PgMPK3,PgMPK12-1,PgMPK20,PgMPK18 and PgMKK6 was not affected by low-temperature treatment.PgMKK8,PgMPK1-1 and PgMKK4 were up-regulated in Malisi than Yichengfenhongmudan.The expression of PgMPK12-2 was not activated after low-temperature treatment in Malisi,but showed gradual increase in Yichengfenhongmudan.The expression level of PgMKK3 increased at first and then decreased during low-temperature treatment in Malisi,whereas it was not detected in Yichengfenhongmudan.【Conclusion】Pomegranate MAPK family genes responded to cold stress signals.PgMKK2,PgMPK6,PgMPK12-2 and PgMPK9 might positively regulate cold tolerance.