芥菜开花抑制因子SVP表达分析及其与FLC互作的调节位点鉴定  

Expression Analysis of Flowering Regulation Factor SVP and Identification of Mediation Sites of SVP/FLC in Brassica juncea

作　　者：李朝闯 马关鹏 杨修勤[1] 王志敏[1] 宋明[1] 汤青林[1] 

机构地区：[1]西南大学园艺园林学院,南方山地园艺学教育部重点实验室,重庆市蔬菜学重点实验室,重庆400715

出　　处：《园艺学报》2016年第8期1513-1524,共12页Acta Horticulturae Sinica

基　　金：国家重点基础研究发展计划(‘973’)项目(2012CB113900);国家自然科学基金项目(31000908);中央高校基本科研业务费专项(XDJK2012B020)

摘　　要：为阐明芥菜开花抑制因子SVP基因的表达特性及其与FLC蛋白互作的调节机制,从‘青叶芥’中克隆了SVP基因。定量PCR分析表明:低温春化途径和长日照光周期途径中SVP在叶片和茎尖均有表达。营养生长初期表达量较低(茎尖和叶片中平均相对表达量分别为0.56和0.35),生殖生长早期则显著增加(春化途径的茎尖和叶片分别为0.60和1.27,光周期途径的茎尖和叶片分别为0.49和1.42)。茎尖中SVP对低温春化的反应比光周期敏感;而叶片中SVP对光周期的反应比低温敏感。酵母双杂交和β–半乳糖苷酶活性测定显示:SVP蛋白I域突变体SVP^(E90L)以及K域突变体SVP^(K104C)和SVPH106I均会削弱SVP/FLC_2蛋白的互作,但不会导致相互作用消失。SVP蛋白K域突变体SVP^(R137L)能完全破坏SVP/FLC_2的互作,但SVP^(R137L)仍然能与芥菜FLC_1、FLC_3、FLC_4和FLC_5相互作用,说明SVP/FLC_2的蛋白互作受到SVP第137位氨基酸的特异性调控。序列比对发现:芥菜FLC_4和FLC_5氨基酸序列完全相同,它们与FLC_3仅有1个变异位点;FLC_2与FLC_1、FLC_3、FLC_(4-5)之间分别有28、19、18个变异位点;FLC_2与FLC_1、FLC_3、FLC_4或FLC_5均不相同的位点有11个。推测FLC_2与FLC家族其他成员之间的变异位点很可能对SVP^(R137L)/FLC_2特异性调控有贡献。SHORT VEGETATIVE PHASE（SVP） was a key regulatory factor in flowering-time control of Brassica juncea.In order to clarify the expression characteristics of SVP gene and the mediation sites of protein interactions between SVP with FLC in flowering pathways,we cloned SVP gene in＇ Qingyejie＇ germplasm of Brassica juncea.Expression analysis of qRT-PCR revealed that SVP gene expressed in leaves as well as shoot apexes in the flowering pathways of vernalization and long-day photoperiod.SVP gene expressed at very low level（0.56 in stem tip and 0.35 in leaf blade） at early stage of vegetative phase.However,it significantly increased at early stage of the reproductive phase（The relative expression values in the vernalization pathway are 0.60 in stem tip and 1.27 in leaf blade,respectively.However,the values in the photoperiod pathway are 0.49 in stem tip and 1.42 in leaf blade）.In stem tips,the expression of SVP was more sensitive to vernalization than that of long-day photoperiod.Contrarily,the sensitivity of SVP gene expression in leaves was exactly reversed.Yeast two-hybrid experiments and β-galactosidase activity assays showed that FLC_2 retained protein interactions with Ⅰ-domain mutant of SVP^（E90L） as well as K-domain mutants of SVP^（K104C） and SVP^（H1061）.However,the interacting strength of SVP/FLC_2 was weakened by each of the three mutation sites above mentioned.Another K-domain mutant of SVP failed to interact with FLC_2 but still interacted with FLC_1,FLC_3,FLC_4 and FLC_5.It suggested that the interaction of SVP/FLC_2 was specifically regulated by the 137 th amino acid site of SVP.Sequence comparison showed that two different genes of FLC_4 and FLC_5 in B.juncea encoded the same FLC protein,which had only one variable amino acid site compared with FLC_3.However,there are respectively28,19 and 18 variable amino acid sites in FLC_1,FLC_3 and FLC_（4-5） compared with FLC_2.Additionally,FLC_2 had 11 unique amino acid sites which were different from any of other FLC family members

关 键 词：芥菜 SVP 酵母双杂交 开花调控 

分 类 号：S637.1[农业科学—蔬菜学]