桂花OfSPLs基因克隆及其在不同温度下花芽分化时期的表达分析  

OfSPL Genes Cloning and Its Expression Analysis During Flower Bud Differentiation at Different Temperature in Osmanthus fragrans

作　　者：吴鸿飞 周敏舒 朱守阔 杨丽媛 赵宏波[1,2,3] 董彬[1,2,3] WU Hong-Fei;ZHOU Min-Shu;ZHU Shou-Kuo;YANG Li-Yuan;ZHAO Hong-Bo;DONG Bin(School of Landscape Architecture,Zhejiang A&F University,Hangzhou 311300,China;Zhejiang Provincial Key Laboratory of Germplasm Innovation and Utilization for Garden Plants,Zhejiang Agriculture&Forestry University,Hangzhou 311300,China;Key Laboratory of National Forestry and Grassland Administration on Germplasm Innovation and Utilization for Southern Garden Plants,Zhejiang Agriculture&Forestry University,Hangzhou 311300,China)

机构地区：[1]浙江农林大学风景园林与建筑学院,杭州311300 [2]浙江农林大学浙江省园林植物种质创新与利用重点实验室,杭州311300 [3]浙江农林大学南方园林植物种质创新与利用国家林业和草原局重点实验室,杭州311300

出　　处：《农业生物技术学报》2020年第8期1390-1399,共10页Journal of Agricultural Biotechnology

基　　金：浙江省自然科学基金(LQ19C160012);浙江省教育厅一般项目(Y201839931);国家级大学生创新创业训练计划项目(201910341027);浙江农林大学科研发展基金(2017FR016)。

摘　　要：环境温度变化能显著影响桂花(Osmanthus fragrans)的花芽分化和开放,为了明确SPL(SQUAMOSA promoter-binding protein-like)基因和miR156(microRNA156)响应环境温度变化对桂花花芽分化的调控,本研究以桂花品种'堰虹桂'(O.fragrans‘Yanhonggui’)为材料,克隆得到了10个OfSPL基因(OfSPL1A:MT756841;OfSPL1B:MT756842;OfSPL5:MT756843;OfSPL6:MT756844;OfSPL7:MT756845;OfSPL8:MT756849;OfSPL10:MT756846;OfSPL11:MT756847;OfSPL12:MT756848;OfSPL13:MT756850),运用qRT-PCR对OfSPLs和miR156在不同花芽分化阶段的表达特异性进行分析.通过序列分析发现克隆得到OfSPLs cDNA序列长为1004~3824 bp,编码232~1006个氨基酸,OfSPLs均含有保守的SBP结构域;系统进化分析表明桂花OfSPLs基因与木犀科油橄榄(Olea europaea)OlSPLs基因具有较近的亲缘关系.另外,通过生物信息学分析发现,10个OfSPL基因均定位于细胞核内;miR156通过抑制翻译或降解来调节SPL基因表达发挥作用,对OfSPL基因的miRNA结合位点分析发现,OfSPL6、OfSPL10、OfSPL11、OfSPL12具有与miR156作用的位点.在桂花花芽分化的不同时期,与常温25℃相比,只有OfSPL1A、OfSPL5、OfSPL6、OfSPL10和OfSPL13在环境低温19℃下表达量显著增加,另外也发现miR156-1和miR156-2在19℃的表达显著低于25℃,说明环境低温可能抑制了miR156的表达,进而促进了OfSPL6和OfSPL10的上调使花芽分化进程加快;OfSPL1A、OfSPL5和OfSPL13也同时参与了环境低温下桂花的生长发育,但是并不受到miR156的调控.本研究可为进一步探讨环境温度调控桂花花芽分化和花期提供参考依据.Ambient temperature is significantly affected flower bud development and flower opening of sweet osmanthus(Osmanthus fragrans).To further understand SQUAMOSA promoter-binding protein-like(SPL)genes and miR156(microRNA156)on how to respond to ambient temperature in regulating flower bud development of sweet osmanthus,the variety of Osmanthus fragrans‘Yanhonggui’were selected,and 10 OfSPLs(OfSPL1A:MT756841;OfSPL1B:MT756842;OfSPL5:MT756843;OfSPL6:MT756844;OfSPL7:MT756845;OfSPL8:MT756849;OfSPL10:MT756846;OfSPL11:MT756847;OfSPL12:MT756848;OfSPL13:MT756850)were cloned by PCR.Meanwhile,the expression of miR156 and OfSPLs were identified by real-time quantitative PCR(qRT-PCR)to further analyze their temporal and spatial expression patterns.The results of OfSPL sequences analysis revealed that the cloned OfSPLs cDNA length between 1004~3824 bp,the sizes of OfSPL proteins ranged from 232 amino acid(aa)to 1006 aa,and all of OfSPLs included a conserved SBP domain.Furthermore,all of OfSPL genes showed high homology with OlSPL genes from the species of Olea europaea by amino acid sequence alignment and evolutionary analysis.To investigate the intracellular localization of the proteins coded by the OfSPL genes,the bioinformatics method was used for predicting.The predicted results showed that all of the OfSPL proteins located in the nucleus.miR156 plays its role through regulating SPL gene expression by translational repression or degradation of mRNA in a sequence-specific manner.Therefore,the binding site of SPL genes was further analyzed.The expression of OfPL6,OfPL10,OfPL11,and OfSPL12 might be affected by miR156 through targeting their binding sites.Under different temperature treatments,the expression of OfSPL1A,OfSPL5,OfSPL6,OfSPL10,and OfSPL13 significantly increased at 19℃comparing with the control 25℃.In contrast,the expression of miR156-1 and miR156-2 under 19℃treatment were lower than the control 25℃.The results indicated that the expression of miR156-1 and miR156-2 may be inhibited by low ambient tempera

关 键 词：桂花 环境温度 SPL基因 花芽分化 表达分析 

分 类 号：S722.3[农业科学—林木遗传育种]