牡丹PsSPL3基因的克隆和表达特性分析

Cloning and Expression Pattern Analysis of PsSPL3 in Tree Peony

SPL(SQUAMOSA promoter-binding protein-like)转录因子在植物多个生理过程中发挥重要的生理功能,为了研究其在牡丹花芽休眠进程中的作用机理,采用RACE方法,从牡丹花芽中克隆得到了一个SPL基因。该基因全长cDNA 1 057 bp,完整开放阅读框为549 bp,编码182个氨基酸,Blast分析表明,编码的氨基酸序列中具有SBP-box基因家族所特有的SBP-box保守结构域。与拟南芥已知SPLs蛋白构建进化树结果表明,牡丹SPL蛋白与At SPL3聚为一支,该基因被命名为PsSPL3。生物软件预测PsSPL3蛋白分子量为20.349 4 kDa,理论等电点为9.62。同源性分析了牡丹PsSPL3与其他已知植物的SPL3,相似性为47.98%~69.46%,其中与白桦的SPL3蛋白相似性最高,为69.46%。实时定量PCR分析PsSPL3基因在初花期牡丹不同组织中和花芽休眠过程中的表达水平,结果表明,PsSPL3基因在不同组织中表达差异较大,其中在根中转录水平最高,在茎和花瓣中次之;PsSPL3基因在休眠进程中的转录水平呈先上升后下降趋势,其中低温处理14 d时,PsSPL3基因的表达量达到最高。低温7 d结合外源施加GA3的牡丹花芽中PsSPL3基因的表达量显著增加,推测PsSPL3基因的转录受GA3诱导来促进牡丹花芽内休眠解除的进程。

Transcription factor of SPL（ SQUAMOSA promoter-binding protein-like） plays very important role among many physiological processes of plants. In order to study the mechanism of action,a 1 057 bp full-length c DNA of PsSPL3 gene was isolated from Paeonia suffruticosa using RACE method. Bioinformatic analysis showed that the ORF length of PsSPL3 was 549 bp which encodes 182 amino acid residues with relative molecular mass of20. 349 4 k Da and the isoelectric point of 9. 62. Blast analysis showed that PsSPL3 contained a typical SBP-box structure domain. Evolutionary tree analysis revealed that the putative SPL protein of tree peony and At SPL3 were clustered in same group,so the SPL gene of tree peony was named PsSPL3. The homology of amino acid compared with PsSPL3 and other known SPL3 was 47. 98%-69. 46%,and the homology with Bp SPL3 was highest with69. 46%. The expression pattern of PsSPL3 in different tissues at the early stage of flowering was analyzed by quantitative Real-time PCR（ q PCR）,and the results showed that the transcript of PsSPL3 in the root was the highest,and the lower were in stem and petal. The expression pattern of PsSPL3 during dormancy release of tree pony showed that the expression of PsSPL3 gene was first increased and then decreased. When the tree peony flower bud were exposed 14 d chilling,the expression level of PsSPL3 gene was the highest. The expression of PsSPL3 gene in the flower buds after 7 d chilling with exogenous application of GA3 was significantly higher than that in the control,suggested that the expression of PsSPL3 gene was induced by GA3 to promote the process of dormancy release in tree peony flower bud.