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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。
In order to elucidate the expression characteristics of flowering inhibitor SVP gene of Brassica juncea and its regulatory mechanism of interaction with FLC protein, SVP gene was cloned from ’ Quantitative PCR analysis showed that SVP was expressed in leaves and shoot tips in the pathways of vernalization and long-day photoperiod. The expression level was lower at the beginning of vegetative growth (the average relative expression in shoot tip and leaf was 0.56 and 0.35 respectively), and significantly increased in early reproductive stage (stem tips and leaves of vernalization pathway were 0.60 and 1.27, respectively. The tips and leaves were 0.49 and 1.42, respectively). SVP in shoot tips was more sensitive to photoperiod than that in photoperiod; however, SVP response to photoperiod in leaves was more sensitive than hypothermia. Yeast two-hybrid and β-galactosidase activity assays showed that SVP ~ (E90L) and K domain mutants SVP ~ (K104C) and SVPH106I of SVP I protein both attenuated the interaction of SVP / FLC_2 protein but not Will lead to the disappearance of interaction. SVP ~ (R137L) could completely disrupt the interaction of SVP / FLC_2, but SVP ~ (R137L) still interacted with FLC_1, FLC_3, FLC_4 and FLC_5, indicating that protein interaction of SVP / FLC_2 was affected Specific regulation of the 137th amino acid of SVP. Sequence alignment showed that the amino acid sequences of FLC_4 and FLC_5 in mustard were identical, which had only one mutation site with FLC_3. There were 28, 19 and 18 variation sites between FLC_2 and FLC_1, FLC_3 and FLC_ (4-5) ; There were 11 sites in FLC_2 that were not the same as FLC_1, FLC_3, FLC_4 or FLC_5. It is speculated that the variation between FLC_2 and other members of the FLC family may contribute to the specific regulation of SVP ~ (R137L) / FLC_2.