A New Adenylate Kinase Gene From Sweetpotato: Molecular Cloning, Chatacterization And Plant Expression Vector Construction

Sweetpotato (Ipomoea batatas (L.) Lam) is one of the most important crops in the world, as well as the starch-producing crop. The development of biotechnology and genetic engineering makes it possible to obtain high-strach-content new cultivars of sweetpotato, the precondition of which is researching the metabolic pathway of starch.Adenylate kinase (EC. 2.7.4.3, ADK) catalyzes the interconversion of ATP, AMP and ADP, which is the key enzyme regulating the balance of the three molecules (ATP, AMP and ADP) between the the nucleic acid pool and the starch pool. To investigate the role of ADK in the starch biosynthesis and provide a candidate gene for metabolic engineering of sweetpotato starch pathway, a new full-length cDNA encoding adenylate kinase, designated as IbADK (GenBank Accession Number: EF562533 ), was cloned with RACE method from " YUSU303 " , a high-starch-content cultivar of sweetpotato. And then, bioinformatic methods were employed to analyze the cloned gene; and the tissue expression profile was produced by semi-quantity RT-PCR. Finally, the gene was subcloned into the plant expression vector.A 592-bp core fragment of IbADK was amplified with the two primers designed according to the conservative sequences of the reported plant ADK genes. Based on the sequence of the core fragment, the nested gene-specific primers were designed and used to generate the 3' and 5'-end. Using RACE method, a 548-bp (3'-end) fragment and a 314-bp fragment (5'-end) were obtained respectively. The three fragments including the core, 3'-end and 5'-end fragment were assymbled by ContigExpress (Vector NTI Suite 6.0) and the full-length cDNA of IbADK was electronically generated. Then, a pair of gene-specific primers were used to successfully amplify the physical full-length sequnece of IbADK. The full-length cDNA of IbADK was 1314 bp in length with an 855-bp open reading frame (ORF), which encoded a 284-amino-acid polypeptide (designated as IbADK) with the calculated molecular mass of 30.86 kDa and the theoretic p1 of 6.46. BLAST search results showed that the sequence of IbADK was 83% similar to that of potato. The subcellular prediction found that IbADK had a 22-amino-acid transit peptide at N-terminus, which directed IbADK to the plastids. And this result was consistent with the fact that starch was biosynthesized in plastids.The phylogenetic analysis using ADKs of plant origin revealed that IbADK and potato ADK could be devided into the same group that was consistent with the BALST research results. The secondary structure analysis revealed that IbADK was composed of 26.06%α-helica, 23.59% extended strands and 50.35% random coils. Further, the homology-based structural modelling generated the putative 3-D structure of IbADK in which the AMP-binding and ATP-AMP (Ap5A) binding sites were found.Semi-quantitative one-step RT-PCR was carried out to reveal the tissue expression profile of IbADK. The results indicated that IbADK expression could be detected in the tubers and tender leaves of sweetpoato at the highest levels, followed by mature leaves and veins; but the expression of IbADK was not found in petioles. The previous studies found that starch was synthesized in two kinds of plastids: one was choloroplast in leaves and the other was amyloid in tubers. The highest expression of IbADK in tender leaves and tubers could facilitate the metabolism of ATP, AMP and ADP in the two kinds of metabolism-activing tissues, and further regulate the metablism of starch and that of nucleic acids. In the present study, the cloning arid chatacterization will be helpful to understand the function of IbADK at the level of molecular genetics and to unveil the molecular michanism of starch biosynthesis.The previous studies indicated that the down-regulation of ADK could put the adenylate pools forward to the starch pools. In the present, IbADK was introduced into a plant expression vector, pHB, in the reverse direction to obtain pHB-aIbADK that could express antisense IbADK; and at the same time, IbADK was constructed into pHB in the forward direction to get pHB-IbADK that could express sense IbADK used as control in the future genetic transfomation. Finally, pHB-aIbADK and pHB-IbADK were introduced into Agrobacterium tumerfaciens strain LBA4404 and disarmed Agrobacterium tumerfaciens strain C58C1 (haboring pRiA4) respectively to generate relative engineered bacteria. The construction of pHB-aIbADK was fundamental work for genetic modification of the starch metabolism through the strategy of regulating the balance of the starch pools and the nucleic acid pools at the molecular level by genetic transformation of sweetpotato.