In Silicon Cloning and Analysis of a LACS Gene from Glycine Max ( L . )

Long chain acyl-coenzyme A synthetases (LACSs) activate free fatty acids to acyl-CoA thioesters, and play important roles in the biosynthesis and degradation of lipids. In this study, a Glycine max(L.) LACS gene, designated as GmLACS, has been isolated through in silicon cloning. The gene is 2,219 bp with an open reading frame (ORF) of 1,989 bp, which encodes a LACS with 662 amino acid residues, with the isoelectric point of 6.42 and the calculated molecular mass of 65.6 kDa. Sequence analysis showed that GmLACS possessed typical domains of LACSs. Real-time quantitative PCR data analysis suggested that GmLACS was hightly expressed in leaves and young pods.


Introduction
Acyl-coenzyme A synthetases (ACSs) are generally classified by their specificities for fatty acids of varying chain length.The Commission on Enzymes of the International Union of Biochemistry has classified these enzymes as acetyl-CoA synthetase (EC 6.2.1.1),medium-chain ACS (EC 6.2.1.2) and long-chain ACS (EC 6.2.1.3).A unifying feature of all acyl-CoA synthetases is the presence of an "AMP-binding domain signature" (PROSITE PS00455) (Watkins, P.A., 1997, pp.55-83).In all of LACSs, long chain acyl-coenzyme A synthetases (LACSs) play a key role in metabolization of fatty acid (Shrago, E., 2000, pp.290-293).LACS esterifies free fatty acids to acyl-CoAs, a key activation step that is necessary for the utilization of fatty acids by most lipid metabolic enzymes (Bradford, M.M., 1976, pp.248-254).LACS catalyzes the formation of acyl-CoAs by a two-step mechanism.In the first step, the free fatty acid is converted to an acyl-AMP intermediate, called an adenylate, through the pyrophosphorolysis of ATP.The activated carbonyl carbon of the adenylate is then coupled to the thiol group of CoA, releasing AMP and final product, acyl-CoA (Groot, P.H., 1976, pp.75-126).Another special molecular characteristic is that eukaryotic LACSs contain a linker domain with the length about 30 to 40 amino acid residues (Shockey, J.M., 2002(Shockey, J.M., , pp.1710(Shockey, J.M., -1722)).The linker domain does not exist in the other acyl-CoA synthetases.Though the detailed utility is unknown, the linker domain seems to be necessary for eukaryotic LACSs' function (Iijima, H., 1996, pp.186-190).The length of linker domain is also important for the activity of LACSs.Two LACS-like proteins At4g14070 and At3g23790 in Arabidopsis thaliana have high identity with AtLACSs, but they do not encode LACS activity.The abnormal length of their linker domain, about 70 amino acid residues, is a putative reason for their non-LACS function (Shockey, J.M., 2002(Shockey, J.M., , pp.1710(Shockey, J.M., -1722)).
This important class of enzymes affect prominently in several fatty acid-derived metabolic pathways, including phospholipid, triacylglycerol, jasmonate biosynthesis and fatty acid -oxidation.Oil accumulation in oilseeds has significant economic interest for food, feed, cosmetics and detergents etc., and triacylglycerol (TAG) is the main component of plant oil (Shen, B., 2006, pp.377-387).Fatty acyl-CoA thioesters and glycerol 3-phosphate are substrates for acyltransferases to synthesize TAG via Kennedy cycle.During the processes, LACSs play a pivotal role by providing fatty acyl-CoA and link fatty acid de novo synthesis and TAG assembly (Ohlrogge, J.B., 1997, pp.109-113).Another important role LACSs played is in fatty acids transport.This process has been studied in detail in bacteria, yeast (Saccharomyces cerevisiae), and mammalian cells.Escherichia coli contains a single LACS, encoded by FadD gene, which was proved to transport the fatty acids (Black, P.N., 1992, pp.25513-25520).LACS also initiates the process of fatty acid -oxidation.In oilseeds, carbon reserves are stored as triacylglycerol (TAG).With the onset of germination, lipases release free fatty acids from the TAG molecules (Hills, M.J., 1986, pp.671-674;Lin, Y.H., 1986, pp.346-356).LACS activates the free fatty acids to acyl-CoAs that enter the -oxidation pathway in the glyoxysomes of the germinating seedling.
In Arabidopsis thaliana, it has been established that nine LACS genes exist and were named LACS1-9.Nine LACSs could be classified into 3 distinct clades.LACS1 is supposed to be involved in the syntheses of lipids and LACS2 is supposed to be involved in the pathway of cutin synthesis (Schnurr, J., 2004, pp.629-642).LACS6 and LACS7 are localized in peroxisome, and both of them are involved in peroxisomal fatty acid -oxidation (Fulda, M., 2004, pp.394-405).In Ricinus communis, three LACS genes have been cloned.RcLACS2 is likely to be a peroxisomal ACS isoform.RcLACS4 is supposed to be involved in the syntheses of lipids (He, X., 2007, pp.931-938).Capsicum annuum also has been found GaLACS.In this paper, we found a LACS gene from Glycine max through in silicon cloning, designated GmLACS.Sequence analysis indicated that the deduced protein possessed AMP-binding motifs and a linker domain.

Bioinformatics analysis
Blast in NCBI(http://www.ncbi.nlm.nih.gov/) was used for genes searching in network.DNA and amino acid sequence manipulation was performed with EditSeq program of DNAStar 5.0 package.Domain prediction was done at ExPASy Proteomics Server(http://au.expasy.org/).Sequence alignments were carried out by GeneDoc.Phylogenetic tree was constructed using MEGA4.0 and TreeView was used for exhibition of phylogenetic tree.

Plant materials
Glycine max(L.)cvWillimas was utilized for expression analysis of GmLACS at different reproductive stages.

RNA extraction
RNA samples extracted from different soybean tissues including leaf, root, flower and pod.RNA samples were extracted by plant Trizol reagent (Invitrogen Biotech Co. Ltd., U.S.).All steps were carried out following the instruction of manufacturer.RNA samples were DNase treated with DNA-free(TaKara, Japan) according to the manufacturers directions.RNA concentrations were determined using spectrophotometer (GeneSpecIII, U.S.) at absorbance 260 nm.Aliquots of RNA (free of genomic DNA) were diluted to 50 ng uL 1 in RNase-free water and stored at 70 until use.To verify RNA integrity, 500 ng of total RNA of each sample was examined on a 1% agarose gel following electrophoresis and staining with ethidium bromide.

RT-PCR assay of GmLACS expression profiles
2μg total RNAs were used for the first strand cDNA synthesizing with M-MLV Reverse Transcriptase (Takara, Japan) according to the manufacture's protocol.Real-time reverse transcription-polymerase chain reaction(RT-PCR) was performed with the iCycler using the SYBR Green RT-PCR kit (Takara, Japan) to quantify GmLACS in different tissues.

GmLACS
gene specific primers

In silicon cloning of GmLACS
Arabidopsis LACS1-9 (GeneBank Acc.No. AAM28868-AAM28876) were used as querys to blast Glycine max (L.) database, two Glycine max cDNAs(GeneBank Acc.No. AK245419 and AK245622) with high sequence identity were obtained.From this group, one cDNA(AK245419) which share 75% identity with AtLACS4, 74% identity with AtLACS5 and 73% identity with AtLACS3 was selected.It is a 2,219 bp long fragment.Sequence aligning with GmLACS sequence suggested that the whole fragment contained intact ORF of 1,989 bp, designated as GmLACS.
Translated by DNAStar software, GmLACS was predicted to encode a protein of 662 amino acid residues (Fig. 1), with the theoretical pI of 7.11 and calculated molecular weight of 74.06 KDa.
Multiple sequence alignment of GmLACS and some other LACSs from plants showed that there was some considerable conserved amino acid sequences existed in the form of blocks.Three blocks appeared among these proteins, and three

AMP-binding motifs-I[MCV]TSG[TS][ST]GXPK, GYGXTE and GW[FL]
[HK]TG-orderly located in Block I-III.The conserved tyrosine residue at the position 481 on GmLACS was assumed to be involved in adenylate formation (Fig. 2. A).Multiple sequence alignment of central sequences of GmLACS, AtLACS1, AtLACS2, RcLACS4 and Arabidopsis LACS-like protein At4g14070 demonstrated that GmLACS contained a linker domain of 31 amino acid residues as well as other LACSs (Fig. 2. B), and this length of the linker domain suggested it might encode LACS activity.

Molecular evolution analysis
A neighbor-joining phylogenetic tree was generated to exhibit the distances among GmLACS and other plant LACSs(Fig.3).Bootstrap analysis was performed for the reliability of phylogenetic tree.The phylogenetic tree demonstrated that the LACSs derived from a common ancestor and diverged into two separate clades.AtLACS3, AtLACS4, AtLACS5, AtLACS6, AtLACS7 and AtLACS8 were in the first clade; RcLACS4, GmLACS, CaLACS, AhLACS, AtLACS1, AtLACS2 and AtLACS9 were in the second clade.The phylogenetic tree revealed that there was remarkable species specificity among LACSs.The sequence homology among those proteins was probably coincident with their function or subcellular location.A phylogenetic tree was drawn based on the deduced amino acid sequence and other LACSs (Fig. 3).In the phylogenetic tree, it showed that GmLACS had a higher homology with the second clade, especially with the RcLACS4 in the second clade.

Analysis of GmLACS expression profile
To investigate the GmLACS expression profile in different tissues, total RNA from root, leaf, flower and pod tissues were used as templates to detect the transcription of GmLACS by real-time quantitative PCR.The actin gene was used as the internal control to ensure that the amount of RNA used is equal.The results from real-time PCR assay indicated that GmLACS genes were expressed in all tissues tested, but the patterns is quite different, stronger expression exhibited in leaves and young pods (Fig. 4).The most distinctive result was the high accumulation of GmLACS in young pod.
Based on the high sequence similarity of GmLACS to RcLACS4 which was supposed to be related with the syntheses of lipids, GmLACS was likely to be involved in the syntheses of lipids (Fig. 3).

Discussions
The long chain acyl-coenzyme A synthetases (LACSs), such as AtLACS1, AtLACS2, AtLACS6 and AtLACS7, play essential roles in the biosynthesis and degradation of lipids in model plant Arabidopsis thaliana (Shockey, J.M., 2002(Shockey, J.M., , pp.1710(Shockey, J.M., -1722;;Fulda, M., 2004, pp.394-405;Schnurr, J., 2004, pp.629-642).But their functions in Glycine max(L.) are not reported.In this study, we successfully identified a novel gene, named as GmLACS, which might be involved in lipids metabilasim in soybean.We obtained a complete cDNA of GmLACS from Glycine max(L.)using in silico cloning.Sequence analysis indicated that GmLACS belonged to AMP-binding super-family and contained a linker domain of 31 amino acid residues as well as other LACSs, it is suggested GmLACS presumably encoded LACS activity.GmLACS was highly homologous to RcLACS4, an gene involved in the syntheses of lipids, in the second clade in phylogenic tree.(He, X., 2007, pp.931-938).Real-time quantitative PCR analysis showed that the GmLACS was strongly expressed in leaves and young pods.This indicated that GmLACS may be involved in the syntheses of lipids in soybean seed development like AtLACS1 or AtLACS2 play roles in cutin biosynthesis.Based on the sequence similarity of GmLACS to RcLACS4, GmLACS is likely to be involved in the syntheses of lipids (Fig. 3).To indentify its functional characterization, more evidences need to get.

Figure 1 .
Figure 1.The nucleotide sequence and deduced amino acid sequences of GmLACS First line: nucleotide sequence.The initiation codon (ATG) is underlined.The stop codon (TGA) is asterisked.Second line: deduced amino acid sequence.

Figure 4 .
Figure 4. Expression of GmLACS genes in the soybean plant Aliquots of total RNA were analyzed for expression of the gene in different tissues by real-time quantitative PCR using GmLACS gene-specific primers.Values were normalized to actin and represent mean ± SE (p < 0.05).