龙眼SPS基因克隆及其表达分析

Cloning and expression of SPS gene in longan

【目的】克隆龙眼蔗糖磷酸合成酶基因(DlSPS)并分析其表达特性,为深入探究SPS基因家族成员在植物生长发育过程中的调控机制提供理论依据。【方法】以石硖龙眼为材料,利用RT-PCR和RACE技术克隆DlSPS基因,对其进行生物信息学分析,并用实时荧光定量PCR检测龙眼不同组织[根、茎、叶、幼果、果皮、花、熟果(果肉和果皮)]的相对表达量;测定分析果实发育过程中蔗糖含量、SPS活性及DlSPS基因表达的相关性。【结果】克隆获得的DlSPS基因(GenBank登录号为KP769779)cDNA全长3504 bp,编码1057个氨基酸残基,其编码蛋白分子量118.38 kD,理论等电点6.09,脂溶指数85.53,不稳定系数43.69,亲/疏水性平均值-0.412,为不稳定的亲水蛋白。DlSPS蛋白与温州蜜桔(BAA23213.1)SPS蛋白同源性最高,聚在同一小分支上,表明二者亲缘关系较近。DlSPS蛋白二级结构中,α-螺旋占35.0%,延伸链占13.8%,无规则卷曲占51.2%,与其三级结构预测结果相符。DlSPS基因在不同组织中均有表达,但表达量存在明显差异,其中在叶中的表达量显著高于其他组织(P<0.05,下同),其次是在幼果和花中的表达量,在根、果肉、果皮和茎中的表达量较低。龙眼果实发育过程中,蔗糖含量和SPS活性均呈先上升后下降的趋势,但蔗糖含量在谢花后101 d达最大值,而SPS活性在谢花后94 d活性达最大值,说明SPS活性与蔗糖积累具有一定的相关性。DlSPS基因的表达量随着果实发育呈略微下降—大幅上升—略微下降的变化趋势,在谢花后101 d表达量达到最大值,此时蔗糖含量也达最大值。【结论】SPS在龙眼不同组织生长发育过程中对蔗糖积累发挥重要作用,尤其是对叶片和果实中蔗糖积累发挥关键作用。

【Objective】To explore the expression characteristics of sucrose phosphate synthase gene(DlSPS)of longan,which provided a theoretical basis for further exploring the regulatory mechanism of SPS gene family members duringlongan growth and development.【Method】Using Shixia longan as experimental material,DlSPS gene was cloned byRT-PCR combined with RACE,and bioinformatics analysis was carried out. The relative expression of different longan tissues[root,stem,leaf,young fruit,pericarp,flower and ripe fruit(pulp and pericarp)]was detected by real-time fluorescencequantitative PCR. The correlation among sucrose content,SPS activity and DlSPS gene expression during fruit developmentwas measured and analyzed.【Result】The full length cDNA of DlSPS gene(GenBank number:KP769779)was3504 bp,encoding 1057 amino acids residues,protein molecular weight 118.38 kD,theoretical isoelectric point 6.09,lipophilicindex 85.53,instability coefficient 43.69,hydrophilic/hydrophobic average -0.412,which was an unstable hydrophilicprotein. The amino acid sequence of DlSPS protein had the highest homology with SPS protein of Citrus unshiu(BAA23213.1),and they were clustered on the same small branch,indicating that they were closely related to each other.In the secondary structure of DlSPS protein,α-helix accounted for 35.0%,extension chain accounted for 13.8%,and irregularcurl accounted for 51.2%,which was consistent with the predicted results of the tertiary structure. DlSPS gene wasexpressed in different tissues,but the expression level was different,in which the expression in leaves was significantlyhigher than that in other tissues(P<0.05,the same below),followed by the expression in young fruit and flower,andlower in root,pulp,pericarp and stem. During longan fruit development,sucrose content and SPS activity increased at first and then decreased,but sucrose content reached the maximum at 101 d after flowering,while SPS activity reachedthe maximum at 94 d after flowering,indicating that there was a certain correlation between SPS activity and sucrose accumulation.The expression of DlSPS gene decreased slightly,increased sharply then decreased slightly with the developmentof fruit,and reached the highest value at 101 d after flowering,and the sucrose content also reached the maximumvalue.【Conclusion】SPS plays a key role in sucrose accumulation during longan fruit development,especially in leavesand fruits.