甘蔗△^1-吡咯啉-5-羧酸合成酶基因（P5CS）的克隆及其表达分析  

Molecular Cloning and Expression of the P5CS Gene from Sugarcane

作　　者：张积森[1,2] 陈由强[1,2] 李伟[2] 阙友雄[2] 叶冰莹[1] 陈如凯[2] 张木清[2] 

机构地区：[1]福建师范大学生命科学学院,福州350108 [2]农业部甘蔗遗传改良重点开放实验室,福州350002

出　　处：《热带作物学报》2009年第9期1337-1344,共8页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金(No.30370901);国家"863"计划"高蓄能能源甘蔗新品种的创制与应用"(No.2007AA100701);国家农业公益性行业科研专项(No.nyhyzx07-019);福建省自然科学基金(No.2009J05058)

摘　　要：△1-吡咯琳-5-羧酸合成酶是植物脯氨酸合成过程的关键酶。应用同源性克隆结合RACE技术获得甘蔗P5CS基因cDNA全长序列2714bp,其中5′端非编码区为226bp,3′端非编码区340bp,poly(A)上游有1个聚腺苷酸五碱基AATAAA;开放读码框为2148bp编码715个氨基酸,含双功能域;其编码的蛋白分子量为77.7ku,等电点为6.47。序列比对分析结果表明,P5CS基因的4个主要功能区(domain)中亮氨酸功能区(Leucinedomain)相对不保守,其它功能区均较为保守。对17个物种的P5CS基因进行聚类分析,结果与公认的生物学分类及进化关系吻合。对甘蔗P5CS在根、茎、叶的表达进行实时PCR分析,结果表明,该基因在茎、叶表达量相近,但在根的表达量极低。Most plants synthesize and accumulate proline under osmotic stress. △^1-pyrroline-5-carboxylate synthetase （P＇SCS） plays a key role for proline biosynthesis. The PSCS was obtained from sugarcane Saccharum officinarum L. through homology-based cloning and rapid amplification of cDNA ends（RACE）. The full-length cDNA of P＇SCS termed as Sc-P5CS from sugarcane was 2 714 bp, containing a 2 148 bp open reading frame（ORF） that encoded a 715 amino acids protein, with a 226 bp UTR at the 5＇ end and a 340 bp UTR at the 3＇ end, and included 2 poly（A） signals at the 2 571st position. The molecular weight of Sc-PSCS was 77.7 ku, with a theoretical isoelectric point of 6.47. The alignment of the sequences showed that the P＇SCS genes from different species were not conserved in the leucine domain, but were highly conserved in the other functional domains such as the ATP binding site, Glu-5-kinase domain, NAD（P） H binding site and GSA-DH domain. The phylogenetic tree of the P5CS from 17 species was consistent with putative evolutionary relationships within these 17 species. The expression of the Sc-P5CS in roots, stalks and leaves of sugarcane seedling was analysed by Real-time PCR. The result indicated that the expression of the Sc-P5CS was very low in roots but normal in leaves and stems. This study provides theoretical basis of the molecular mechanism for Sc-P5CS regulation in sugarcane.

关 键 词：甘蔗 脯氨酸 △1-吡咯啉-5-羧酸合成酶 荧光实时PCR 

分 类 号：S566.1[农业科学—作物学] Q943.2[生物学—植物学]