腾冲红花油茶花蜜蛋白1,6-二磷酸果糖醛缩酶的鉴定、克隆与分析

为鉴定出花蜜中某些影响花蜜化学组成的功能蛋白,并探讨其在花蜜分泌及组分调控过程中的功能,以腾冲红花油茶为试验材料,利用MALDI-TOF/MS质谱分析方法初步鉴定出腾冲红花油茶花蜜中含有1,6-二磷酸果糖醛缩酶(FBP2),然后根据质谱得到的部分肽段序列设计特异性引物,采用SMART RACE方法克隆腾冲红花油茶花蜜FBP2全长cDNA,命名为CRFBPase (GenBank登录号:MG764086)。序列分析表明,该cDNA全长1 077 bp,编码358个氨基酸,分子质量为38 428.13 Da,等电点为7.56。经同源比对分析发现CRFBPase基因序列与普通油茶的FBP2基因序列同源性高达98.8%,同时酶活检测结果也进一步证实该花蜜中含有FBP2,且该酶的活性会随着花蜜分泌累积时间的延长而增加。实时荧光定量PCR结果显示,CRFBPase基因在开花后第5天的蜜腺和花部组织中均有一定量的表达且蜜腺中表达量最高,开花后第5天(泌蜜高峰期)蜜腺显著高于花蕾期。本研究结果为进一步揭示FBP2在植物花蜜形成、分泌及糖组分调控过程中的作用机制提供了一定的理论依据。

Identification and Cloning of Fructose -1,6-bisphosphate Aldolase From Floral Nectar of Camellia reticulata

In order to identify proteins from Camellia reticulata nectar which may be responsible for regulating the composition of the nectar and discuss their related functions in nectar secretion, MALDI-TOF/MS analysis was applied to identify. Fructose -1, 6-Bisphosphate Aldolase (FBP2) was identified. Based on the amino acid sequence identified by MALDI-TOF/MS, a specific primer was designed and the full length cDNA of FBP2 gene was amplified using SMART RACE. This gene was named as CRFBPase (GenBank accession No. MG764086). The sequence analysis revealed that cDNA of CRFBPase was 1 077 bp in length, encoding 358 amino acid residues with an estimated molecular mass of 38 428.13 Da and a calculated isoelectric point(pI)of 7.56. CRFBPase gene shared a similarity of 98.8% with the Camellia oleifera clone (JX914588). The FBP2 activity in Camellia reticulata nectar increased along with the nectar accumulation. The results of quantitative real-time PCR showed that CRFBPase was expressed in leaf, nectary, petal, stamen, and pistil, and the expression level in nectary was the highest in nectary. Moreover, the mRNA expression level of CRFBPase gene was significantly higher in nectary tissue on the fifth day after anthesis than that in nectary tissue on the bud stage. The results of this study provide theoretical foundation for the research on the regulation mechanisms of FBP2 protein in the nectar generation, secretion and components regulation.