cloning and expression analysis of ptfatb gene encoding the acyl

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					Journal of Genetics and Genomics (Formerly Acta Genetica Sinica) March 2007, 34(3): 267 274

Research Article

Cloning and Expression Analysis of PtFATB Gene Encoding the Acyl-acyl Carrier Protein Thioesterase in Populus tomentosa Carr.
Zhou Zhou, Deqiang Zhang, Mengzhu Lu
Key Laboratory of Forest Silviculture of the State Forestry Administration , Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China

Abstract: Acyl-ACP thioesterases (FATs) terminates the fatty acid synthesis and allow the transport of fatty acids out of the plastids, which are the important determinants of cellular metabolism. FATB is a member of FAT enzymes that has been described previously in most of the plants. In silico cloning is a new method that utilizes the bioinformatics on the complete genome and available EST database. In this study, a full-length cDNA clone of PtFATB gene was isolated from Populus tomentosa using this approach. It is 1,450 bp in length and the open reading frame encodes a peptide of 421 amino acids. The predicted amino acid sequence shows significant homology with those from other plant species, which contain typical domains owned by FATB proteins. The transcripts of PtFATB were abundant in leaves, and less in roots detected by using semiquantitative RT-PCR. When the shoots were subjected to the stress treatments (cold, dry, NaCl) and ABA (Abscisic acid), the expression of PtFATB was only slightly reduced under the treatment of low temperature. This suggests that the expression of PtFATB is in a constitutive fashion. This study provides the basis not only for the identification and characterization of this gene but also for the improvement of cold tolerance by controlling the expression of the PtFATB gene in trees in near future. Keywords: Populus tomentosa Carr.; Acyl-acyl carrier protein thioesterase (PtFATB); in silico and molecular cloning; RT-PCR; expression analysis

In plants, the major site for the de novo fatty acid synthesis (FAS) occurs in the plastid [1]. Fatty acids are either utilized in this organelle or transported to supply diverse cytoplasmic biosynthetic pathways and cellular processes. Production of fatty acids for transport depends on the activity of acyl-ACP thioesterases (FATs) that hydrolyze acyl-acyl carrier protein (acyl-ACP) to release free fatty acids and ACP [2]. After transportation, the free fatty acids are reesterified to CoA to form the cytosolic acyl-CoA pool [3]. In mesophyll cells, acyl-CoAs are primarily used for the
Received: 2006 01 19; Accepted: 2006 04 18

biosynthesis of membrane glycerolipids in the endoplasmic reticulum [4]. However, in other cell types the transported fatty acids have different fates. For example, in embryo cells of oilseeds, the major fraction is incorporated into triacylglycerols, whereas in epidermal cells a large fraction is utilized for the synthesis of waxes and cutin[5, 6]. Plant FATs terminate the acyl-acyl carrier protein track of fatty acid biosynthesis and play an essential role in determining the amount and composition of fatty acids entering the storage lipid pool. These

This work was supported by project “Regulation of Composition and Saturation of Fatty Acid in Trees by Genetic Engineering”, Introduction of Foreign Advanced Agricultural Science and Technology into China (No. 2005-4-52). Corresponding author. E-mail: lumz@caf.ac.cn ; Fax: +86-10-6287 2015

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enzymes are important determinants of cellular metabolism. Two classes of FATs, known as FATA and FATB, were described in plants[2]. The FATA class has the highest activity in vitro for 18:1-ACP with low activity for saturated acyl-ACP substrates. In contrast, the FATB class prefers saturated acyl-ACP substrates but shows the activity for oleoyl-ACPs[2,7,8]. The Arabidopsis genome have two FATA genes and one FATB gene[9,10]. Bonaventure[11] have previously described the isolation and analysis of an Arabidopsis mutant disrupted in the FATB gene. In this mutant, the total amounts of saturated fatty acids in various tissues were reduced by 40% to 50% when compared with the wild type. This reduction occurred only in the cytosolic pool of saturated fatty acids, affecting the fatty acid composition of extraplastidial phospholipids, waxes in leaves and stems, and triacylglycerols in seeds. Disruption of the FATB gene reduced the growth of the mutant, resulting in seedlings that are approximately half the size of the wild-type by week 4[11]. On the basis of these results, it was concluded that the reduction in the transportation of saturated fatty acids from plastids affects the cellular processes that are critical for plant growth. In this study, we have reported that the molecular cloning, sequencing, and expression analysis of the PtFATB gene in Populus tomentosa for the first time in poplar, which we believe will provide the information for manipulation of poplar fatty acid by genetic engineering.

tion kit (RNeasy Plant Mini Kit, Qiagen, Stanford, CA) according to the manufacturer’s instructions. The cDNA was synthesized according to the manual enclosed in the OligotexTM mRNA Mini Kit (Clonetech, Mountain View, CA). 1. 3 Analysis of FATB sequence in P. tomentosa based on ESTs On the basis of complete poplar genome and available EST database[12], FATB gene in P. tomentosa was cloned using in silicon cloning method. The cDNA sequence of Arabidopsis thaliana FATB (GenBank accession number: NM_100724) was used as a quarry to search the corresponding FATB cDNA fragment in poplar expressed sequence tags (ESTs) database by tBJASTn program. The poplar EST database (http://dendrome.ucdavis.edu/Gen_res.htm) includes PopulusDB, AspenDB, and PoplarDB EST database. The EST with high score was selected and used as a probe to search the poplar EST database in National Centre for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/blast/) using the BLASTn program. The matched segments with high-score, especially in identity to the 3′ or 5′ end of the obtained ESTs, were combined and the homologous sequences were extended to the 3′ and 5′ direction until the theoretical full-length coding region was obtained. 1. 4 Isolation of PtFATB cDNA using the RTPCR method

1 Materials and Methods
1. 1 Plant materials

Cambium was collected from a 4-year-old tree of Populus tomentosa Carr., which was grown in Renqiu County of Hebei Province. The plant samples were immediately frozen in liquid nitrogen and stored at −70 1. 2 for later use. RNA isolation and cDNA synthesis Total RNA was extracted using the RNA extrac-

Primers were designed on the basis of the putative nucleotide sequence of poplar FATB, the primers sequences are: Fr 5′-GTTGAGCTGCTCTGCCTCTC-3′, Re 5′-GGAGACTAACGCCATTCCAGC-3′. In a 50 µL volume of PCR reaction, 2 µL cDNA was used as template along with 15 pmol of each primer. The PCR program was used as follows: predenaturing at 94 for 4 min, then denaturing at 94 for 30 s, extension at 72 for 30 s, for 1.5 annealing at 59

min for 35 cycles, followed by 7 min of extension at

Zhou Zhou et al.: Cloning and Expression Analysis of PtFATB Gene Encoding the Acyl-acyl Carrier Protein Thioesterase

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. PCR products were separated on a 1.0% aga-

rose gel. The band of interest was excised from the gel and purified using the Wizard PCR prep DNA purification kit (Promega, Shanghai, China). The amount of PCR product was quantified by gel electrophoresis by comparing with DNA low mass standards (Tiangen, Beijing, China) and ligated into the plasmid pGEM-T Easy Vector Systems (Promega, madison, USA). 3 µL of purified PCR products and 0.5 µL of pGEM-T Easy Vector were used in ligation reaction. The plasmid was transformed into Escherichia coli DH5α. Clones containing the PCR products were selected by blue/white screening, and the constructed plasmid was named as pGEM-TPtFATB, which was further purified by the Wizard Plus Plasmid DNA purification kit (Sbsbio,Beijing, China) and identified by the digestion with EcoR . The PCR product was sequenced from the plasmid in both directions using the T7 and SP6 primers at the Invitrogen Biotechnology Company (Beijing, China). 1.5 Analysis of PtFATB sequence

used for the analysis of PtFATB expression, which were about 10 cm high with 12 leaves. The tissue culture plantlets were germinated and grown in Murashige & Skoog basal medium supplemented with 6-BA 1 mg/L + NAA 0.2 mg/L in a growth chamber for 30 days, the growth conditions were: temperature, 25 ; light:dark, 16:8 h. The shoots were under four different treatments and the leaves were collected at 0, 10, 30, 60 min and 2, 6, 12, and 24 h after treatment. The treatments were achieved by adding 20% PEG8000 for drought, chilling at 4 in a growth chamber for coldness, adding 250 mmol/L NaCl for salt, and adding 100 µmol/L ABA for ABA treatment.

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2. 1

Results
Isolation of a cDNA clone of PtFATB

A 1.5 kb cDNA fragment of PtFATB was amplified using RT-PCR method with the designed genespecific primers, and ligated into plasmid pGEM-T. The constructed pGEM-T-PtFATB plasmid was confirmed through digestion with EcoR (Fig. 1). The

DNA sequence, coding sequence and its’ putative amino acid sequence were analyzed by DNAstar, Primer Premier 5.00 and Match code biosoftwares (http://www.bioon.com/soft/class1/index.html/). The multisequence alignment was carried out using the CLUSTAL X program (Toby Gibson, Heidelberg, Germany). Phylogenic tree of FATBs was constructed for P. tomentosa and other plants using DNAman software (Lynnon Biosoft, USA). 1.6 Expression analysis of PtFATB using RTPCR

The expression pattern of the PtFATB gene was studied by semi-quantitative RT-PCR (semi-QRTPCR) in different tissues and in leaves under various stress conditions, in which actin gene was used as a control. The actin fragment was amplified by primers: 5′-GGGTAGACCAAGACACACTGG-3′ (forward) and 5′-GGATGGCATGTGGAAGGGCAT-3′ (reverse). The shoots from the same clone of P. tomentosa were

Fig. 1 PtFATB amplified products and recombinant plasmid pGEM-T-PtFATB DNA 1: DL2000 DNA Marker; 2: PtFATB PCR products; 3: recombinant pGEM-T-PtFATB plasmid DNA digested with EcoR ; 4: recombinant pGEM-T-PtFATB plasmid DNA; 5: 1 kb DNA Ladder.

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cDNA clone of PtFATB (GenBank number DQ321500) was 1,450 bp in length, containing 1,263 bp open reading frame (ORF), which encoded a protein of 421 amino acids with 144 bp 5′ untranslated region (5′ UTR) and 43 bp 3′ UTR (Fig. 2). 2. 2 Sequence analysis of PtFATB gene

When PtFATB was analyzed in NCBI using tblastn program, the hit genes with high scores were FATB family genes from other 5 plant species:

Arabidopsis thaliana (NM_100724), Garcinia mangostana (U92878), Gossypium hirsutum (AF034266), Helianthus annuus (AJ242915), and Oryza sativa (XM_493749). This indicates that PtFATB belongs to the FATB family. CLUATALX was used to align the amino acid sequences of PtFATB with that from other plant species. The alignment of the six species’ FATB sequences showed that the amino acid residues are highly conserved with 250 in identical and 110 in high similarity (Fig. 3).

Fig. 2 The nucleotide acid sequence and deduced amino acids sequence for PtFATB Stop codon is indicated by asterisk(*); Filled box indicates the EcoR site.

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Fig. 3 Alignment of the deduced amino acids sequence of P. tomentosa’s PtFATB and the FATB sequences of other plants The accession number for Arabidopsis thaliana: NM_100724; Garcinia mangostana: U92878; Gossypium hirsutum: AF034266; Helianthus annuus: AJ242915; Oryza sativa: XM_493749. “*” is used to indicate the identical amino acids, the “‫”׃‬or “·” means that the amino acids are similar, “ ”means the gap.

Phylogenetic tree was constructed using DNAman software (Fig. 4). which showed that the homology between PtFATB and FATBs from other five plant species: G. mangostana, A. thaliana, G. hirsutum, H. annuus, O. sativa were 82%, 76%, 74%, 68%,

and 64%, respectively. PtFATB and G. mangostana FATB were clustered together while PtFATB and O. sativa FATB were more distant in the phylogenic tree. This is in the agreement with their taxonomic positions.

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Fig. 4 Phylogenetic tree of FATBs from different plants The accession number of the sequences is as the same as Fig. 3.

2. 3

PtFATB transcripts in different tissues and in leaves under various stress conditions

ment; but slightly reduced after treatment with low temperature for 12 h (Fig. 5).

The expression pattern of the PtFATB gene was detected by semi-quantitative RT-PCR in different tissues and leaves under various stress conditions, using actin gene as the inner expressing control (Fig. 5). The transcripts of PtFATB were abundant in leaves, but less in roots. The expression level of PtFATB was not affected by drought, saline stress, and ABA treat-

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Discussion

In this study, bioinformatics approach was used in rapid cloning of the PtFATB gene. This cloning method has certain advantages for cloning genes. Firstly, the approach is time saving, only 2 weeks is required to clone a poplar cDNA clone due to that

Fig. 5

Expression of PtFATB in different tissues, and the leaves under cold (4 ), drought (PEG8000), Salt (NaCl) and ABA

treatments

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most work on the identification of the target gene was carried out using bioinformatics based on the available poplar ESTs database. Secondly, it is not very difficult to go through the experimental procedure only including RNA isolation, cDNA synthesis, and PCR amplification, i.e., a routine molecular manipulation. However, in practice, the sequence of the target genes should not be too long and the genetic distances to the genes hit from database is not too far, which is favorable to get the putative full gene sequence thus easy the primer designing for RT-PCR. The expression pattern of PtFATB was quite similar to that of AtFATB from Arabidopsis thaliana[11]. Transcriptional regulation of the PtFATB gene was not associated with the acclimation of stress condition. This indicates that it is essential to express the gene of plant to keep normal growth and development. Therefore, the possibility that altering the composition and saturation of fatty acids by regulation of Acyl-ACP thioesterases activity is still open to discussion, in light of the improvement of the cold tolerance of plants. Nevertheless, this study provides the basis not only for cloning and characterization of this gene, but also for the further alteration of poplar fatty acid through genetic engineering of the gene. References
1 Ohlrogge JB, Kuhn DN, Stumpf PK. Subcellular localization of acyl-carrier protein in leaf protoplasts of Spinacia oleracea. Proc Natl Acad Sci USA, 1979, 76(3): 1194–1198. Voelker TA, Jones A, Cranmer AM, Davies HM, Knutzon DS.

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Broad-range and binary-range acyl-acyl-carrier protein thioesterases suggest an alternative mechanism for medium-chain production in seeds. Plant Physiol, 1997, 114(2): 669–677. 3 Pollard MR, Ohlrogge JB. Testing models of fatty acid transfer and lipid synthesis in spinach leaf using in vivo oxygen-18 labeling. Plant Physiol, 1999, 121(4): 1217–1226. 4 Browse J, Somerville C. Glycerolipid synthesis: biochemistry and regulation. Annu Rev Plant Physiol Plant Mol Biol, 1991, 42: 467–506. 5 Post-Beittenmiller D. Biochemistry and molecular biology of wax production in plants. Annu Rev Plant Physiol Plant Mol Biol, 1996, 47: 405–430. 6 Kolattukudy PE. Polyesters in higher plants. Adv Biochem Eng Biotechnol, 2001, 71: 1–49. 7 Doermann P, Voelker T, Ohlrogge JB. Cloning and expression in Escherichia coli of a novel thioesterase from Arabidopsis thaliana specific for long chain acyl-acyl carrier proteins. Arch Biochem Biophys, 1995, 316(1): 612–618. 8 Salas JJ, Ohlrogge JB. Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases. Arch Biochem Biophys, 2002, 403(1): 25–34. 9 Mekhedov S, de Ilarduya OM, Ohlrogge J. Toward a functional catalog of the plant genome. A survey of genes for lipid biosynthesis. Plant Physiol, 2000(2), 122: 389–402. 10 Beisson F, Koo AJ, Ruuska S, Schwender J, Pollard M, Thelen JJ, Paddock T, Salas JJ, Savage L, Milcamps A, Mhaske VB, Cho Y, Ohlrogge JB. Arabidopsis genes involved in acyl lipid metabolism. A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a web-based database. Plant Physiol, 2003, 132(2): 681–697. 11 Bonaventure G, Salas JJ, Pollard MR, Ohlrogge JB. Disruption of the FATB gene in Arabidopsis demonstrates an essential role of saturated fatty acids in plant growth. Plant Cell, 2003, 15(4): 1020–1033. 12 Zhu ZF, Sun CQ, Fu YC, Qian XY, Yang JS, Wang XK. Isolation and analysis of a novel MYC gene from rice.Acta Genetica Sinica , 2005, 32 (4): 393–398.

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-

(PtFATB)

100091 FATB EST PtFATB 1,450 bp 421 NaCl (1978 ) ABA PtFATB PCR E-mail: zhouzhou_caf@yahoo.com.cn ATG RT-PCR 144 bp 5′ PtFATB 24 h PtFATB RT-PCR FATB TGA 40 bp cDNA 3′ cDNA (FATB)

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