龙眼中性转化酶基因(DlNI)的克隆及分析

Cloning and Sequence Analysis of Neutral Invertase Gene (DlNI) from Longan Fruit

【目的】中性转化酶是果实蔗糖代谢关键酶之一,克隆中性转化酶基因对于揭示龙眼果实糖代谢具有重要的意义。【方法】以‘石硖’龙眼试材,采用RT-PCR结合RACE技术成功克隆了3个龙眼中性转化酶基因全长cDNA序列。【结果】3个龙眼中性转化酶基因全长cDNA序列分别命名为DlNI-1、DlNI-2和DlNI-3,NCBI登录号为KP769773、KP769774和KP769775,其中DlNI-1全长2090 bp,编码589个氨基酸,比对结果发现与木薯(82%)的中性转化酶基因的氨基酸序列同源性最高;DlNI-2全长2558 bp,编码709个氨基酸,比对结果发现与克莱门柚(80%)的中性转化酶基因的氨基酸序列同源性最高;DlNI-3全长2444 bp,编码805个氨基酸,比对结果发现与克莱门柚(80%)的中性转化酶基因的氨基酸序列同源性最高。3个龙眼中性转化酶基因的氨基酸序列都具有中性转化酶的12个高度保守的结构域,并且都具有2个催化残基,推测其蛋白都属于α类,且都定位于细胞器中。3个基因在不同组织中表达量具有较大差异,其中DlNI-1和DlNI-2在叶片中都具有较高的表达量,而DlNI-3在果皮组织中具有最高的表达量。【结论】成功克隆了3个龙眼中性转化酶基因,为后期深入研究中性转化酶基因结构和功能奠定基础。

【Objective】Neutral invertase was a key enzyme of fruit sucrose metabolism,and cloning neutral invertase gene sequences had a significance for sugar metabolism. 【Method】Taken Dimocarpus longan‘Shixia＇cultivar as tested materials,the whole lengths of its three neutral invertase gene cDNA were cloned and sequenced by RT-PCR and RACE. 【Result】Three neutral invertase gene cDNAs named as DlNI-1,DlNI-2 and DlNI-3,the NCBI numbers were KP769773,KP769774 and KP769775,of which the DlNI-1 gene consisted of 2090 bp encoding a polypeptide of 589 amino acids,which had the highest homology with the Manihot esculenta crantz（ 82 %） through the NCBI blastx; The DlNI-2 gene consists of 2558 bp encoding a polypeptide of 709 amino acids,which had the highest homology with the Citrus clementina（ 80 %） through the NCBI blastx; The DlNI-3 gene consists of 2444 bp encoding a polypeptide of 805 amino acids,which had the highest homology with the Citrus clementine（ 80 %） through the NCBI blastx. Amino acids of three longan neutral invertase gene sequence had 12 highly conservative domain structure and two catalytic residues,it was concluded that the three neutral invertase protein belonged toα class and located in organelles. The expression level of three neutral invertase genes had bigger difference in different tissues,the DlNI-1 and DlNI-2 had the highest expression level in leaves,but the DlNI-3 had the highest expression in pericarp. 【Conclusion】Three longan neutral invertase gene were cloned,which would provide references for the investigation of the relation between structure and function of neutral invertase gene.