甘蔗ATP柠檬酸裂解酶基因的克隆与表达分析

ATP柠檬酸裂解酶(ACL)为细胞质中乙酰辅酶A合成途径的关键调控酶，在生物体正常生长发育中扮演着重要角色。本研究通过Race和电子克隆技术获得编码甘蔗ACL蛋白2个亚基的基因SoACLA-1和SoACLB-1，其编码框长度分别为1 272 bp和1 827 bp，编码423个和608个氨基酸，推测的氨基酸序列与其他物种具有高度相似性，都优先与禾本科植物聚于同一进化分支。2个基因在ATP-grasp功能域、柠檬酸结合位点、组氨酸磷酸化位点和ATP结合、CoA结合、磷酸化区域等，序列高度保守。实时荧光定量PCR结果表明，2个基因均受外源ABA、水分胁迫、水分胁迫加ABA处理诱导表达，且叶中表达量显著高于根系，其中又以水分胁迫处理下的表达量最高，2个基因表现出协同表达模式。SoACLA-1和SoACLB-1的表达与ABA、ROS含量具有相关性，说明它们可能参与了ABA调控的植物对逆境胁迫反应的代谢过程。

Cloning and Expression Analysis of ATP-Citrate Lyase Genes from Sugarcane

Acetyl-CoA plays an important role in the cytosol of plant cells for the synthesis of a diverse set of phytochemicals, and the cytosolic acetyl-CoA is from the reaction of citrate and CoA catalyzed by ATP-citrate lyase (ACL) coupled with the hydrolysis of ATP. In this research, two genes encoding two distinct subunits of ACL were identified from sugarcane (Saccharum officinarum) by RACE and insilico cloning technology, named SoACLA-1 and SoACLB-1, which contained 1 272 and 1 828 bp open reading frames, and encoded 423 and 608 amino acids, respectively. Sequence analysis indicated that both amino acids sequences showed high homology with ACLs from other species and were close clustered with ACLs from gramineous plants in the phylogenetic tree constructed by MEGA5.0 software using a Neighborhood-Joining bootstrap method. Both sequences showed high conservation in the ATP-grasp domain, citrate binding site, active binding site of histidine phosphorylated by ATP, potential ATP-binding, phosphorylated site and CoA-binding site when compared with ACLs from other species. Quantitative real-time PCR results indicated that SoACLA-1 and SoACLB-1 were both induced by the treatment of control+ABA, water stress, water stress +ABA, and their expression levels were higher in leaves than in roots, with the highest express level in water stress treatment, SoACLA-1 and SoACLB-1 showed a synergetic expression pattern. The expression of SoACLA-1 and SoACLB-1 was close correlated with the ABA and ROS contents, indicating that ACL maybe involved in stress responsive metabolic process triggered by ABA in plants.