橡胶树糖代谢调控基因HbF2KP的克隆及表达

Molecular cloning and expression analysis of HbF2KP from Hevea brasiliensis

根据拟南芥、蓖麻及杨树果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶基因(F2KP)的核苷酸序列,使用橡胶树胶乳转录组数据库拼接到1条1 228 bp的核苷酸序列,通过两次RACE-PCR获得了橡胶树长2 629 bp的F2KP基因cDNA序列,命名为HbF2KP。ORF Finder软件分析结果显示,该序列含有211 bp的5'端非编码区,173 bp的3'端非编码区,以及长度为2 244 bp的开放阅读框(ORF)。氨基酸同源性分析发现,该蛋白含有保守的激酶和酯酶结构域,属于具有双功能的果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶。生物信息学分析显示,HbF2KP蛋白无导肽酶切位点,不具有跨膜结构域且属于亲水蛋白,推测该蛋白可能在细胞质中发挥作用。实时荧光定量PCR分析表明,HbF2KP基因在光合组织及非光合组织中均有表达。对非光合库组织胶乳(产胶细胞的细胞质)中的表达模式进行分析,结果显示该基因表达受割胶、伤害处理及死皮胁迫调控,推测其在橡胶树胶乳糖代谢中发挥重要的调控作用,参与橡胶烃的生物合成调控及死皮胁迫应答。此外,HbF2KP表达受部分植物激素或激素类似物如乙烯利(ET)、植物生长素(2,4-D)、脱落酸(ABA)、水杨酸(SA)及赤霉素(GA)的调控,但调控作用不明显。

The cDNA sequence of HbF2KP was isolated and analyzed in Hevea brasiliensis for the first time. HbF2KP contained a 2 244 bp long ORF that predicted a protein of 747 amino acids. Bioinformatics analyses were performed on HbF2KP protein. HbF2KP had highly conserved kinase and phosphatase domains of fructose-6-phosphate,2-kinase /fructose-2,6-bisphosphatase. Due to the characteristics of high hydrophilicity,no signal peptide and transmembrane domains,it was inferred that HbF2KP function mainly in the cytoplasm. Real-time PCR analysis revealed that the expression of HbF2KP was constitutive in both photosynthetic and non-photosynthetic tissues. HbF2KP expressions were affected in the latex by tapping,wounding and tapping panel dryness,which suggests that HbF2KP play an important role in the regulation of sugar metabolism,especially in rubber biosynthesis and stress response to tapping panel dryness. In addition,the expression of HbF2KP was induced by some hormones,such as ET,2,4-D,ABA,SA and GA.