Keywords

327.1 Introduction

Fatty acyl–acyl carrier protein thioesterases (FAT) was initially identified in the Escherichia coliand has been found to be widespread in eukaryotes, bacteria, and archea and to be involved in a range of cellular processes including fatty acid biosynthesis [1]. Two different classes of FATs have been described in plants, based upon their amino acid sequence and substrate specificity, namely FATA and FATB [2]. The FATA thioesterases have the highest in vitro activity for 18:1-ACP substrates and exhibit a much lower activity for saturated acyl-ACP substrates [3]. Conversely, FATBs prefer saturated acyl group substrates, but also have activity for unsaturated acyl-ACPs [4]. In the current study, a putative acyl–acyl carrier protein thioesterase cDNA (CpF ATB) was cloned by screening a cDNA library of wintersweet (Chimonanthus praecox). The Bioinformatics Toolkit is a platform that integrates a great variety of tools for CpFATB sequence analysis to predict the possible functions, inquiry this protein properties.

327.2 Methods

327.2.1 CpFATB Cloning and Analysis

A wintersweet cDNA library was constructed using a SMART™ cDNA Library Construction Kit from wintersweet corolla material (Sambrook et al. 1989). The coding region of CpFATB was amplified by PCR from the wintersweet cDNA library using the following primers

sense primer: 5′-GCTCTAGAACCATGGCCGCTACT,

anti-sense primer: 5′-CCGCTCGAGTCTTTCATTCATTC ATCACAA.

Thirty thermal cycles were carried out, each consisting of 45 s at 95 °C, 1 min at 54 °C, and 90 s at 72 °C in an automatic thermal cycler. PCR products were separated on a 1 % agarose gel and visualized under UV light.

327.2.2 Bioinformatics Analysis

Physicochemical properties and molecular features of CpFATB were predicted by bioinformatic approaches including physical and chemical properties analysis, hydrophobicity analysis, domain analysis, phylogenetic tree analysis and subcellular localization analysis. Homology searche using the program BLAST (Basic Local Alignment Search Tool) from NCBI (National Center for Biotechnology Information, Washington, D.C.). BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families. Protein Secondary Structure Prediction using SOPMA from http://npsa-pbil.ibcp.fr/. The Hydrophobicity profile predicted of FAT amino acid sequence by DNAMAN and MEGA4x1 software. SignalP server predicts the presence and location of signal peptide cleavage sites in amino acid sequences. PSORT software predicts protein subcellular location.

327.3 Results

327.3.1 Cloning and Characterization of CpFATB cDNA Sequence

A coding region of wintersweet cDNA (CpFATB) was cloned via screening a wintersweet cDNA library. The CpFAT cDNA is 1107 bp form a single open reading frame, predicting a 369 amino acid polypeptide (Fig. 327.1). Sequence homology was investigated using NCBI-BLAST provided by the National Center for Biotechnology Information. Sequence analysis showed that the gene had typical characteristics encoding FAT protein family, the gene was named as CpFATB (Fig. 327.2).

Fig. 327.1
figure 1

The ORF nucleotide sequence and putative amino acid sequence of CpFATB

Full size image
Fig. 327.2
figure 2

Aligment of the CpFATB gene amino acid sequence in GenBank

Full size image

327.3.2 CpFAT Conservative Regional and Phylogenetic Tree

Use of NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) this gene encoding amino acids are conservative regional analysis, CpFAT belongs the plant acyl–acyl carrier protein (ACP) thioesterases (TEs). Acyl-ACP thioesterase,This family consists of various acyl–acyl carrier protein (ACP) thioesterases (TE) these terminate fatty acyl group extension via hydrolysing an acyl group on a fatty acid (Fig. 327.3).

Fig. 327.3
figure 3

Analysis of conserved domain

Full size image

A simple phylogenetic tree to show the relatedness of CpFATB to other FATs also indicates the protein from wintersweet belongs to FATB class (Fig. 327.4). These results suggest that CpFATB may have similar function with B class members in plant.

Fig. 327.4
figure 4

Phylogenetic tree analysis of Fat

Full size image

327.3.3 Fundamental Properties of Protein

Use http://www.expasy.ch/tools/ Primary structure analysis, A coding region of wintersweet cDNA was cloned via screening a wintersweet cDNA library. The CpFATB cDNA is 1110 bp in length with an open reading frame (ORF) of 1107 bp encoding a protein of 369 amino acids. Molecular weight and isoelectric point of the protein is 41.72 kD and 7.72, respectively. Formula is C1843H2934N520O548S18. Extinction coefficients are in units of M−1 cm−1, at 280 nm measured in water. The N-terminal of the sequence considered is M (Met). The estimated half-life is 30 h (mammalian reticulocytes, in vitro), >20 h (yeast, in vivo), >10 h (Escherichia coli, in vivo). The instability index (II) is computed to be 38.27. This classifies the protein as stable. Aliphatic index is 86.29.

327.3.4 Protein Secondary Structure Prediction and Analysis of Hydrophobicity

Protein secondary structure prediction using SOPMA. The gene containing 40.33 % Alpha helix (Hh), 4.63 % Beta bridge (Bb) 14.99 % Extended strand (Ee), 40.05 % Random coil (Ee) (Fig. 327.5).

Fig. 327.5
figure 5

Secondary structure prediction of CpFATB

Full size image

The software of DNAman are applied to analyze to these FATs in clustering (Fig. 327.6). The hydrophobic protein of the gene expression of the maximum value of 2.91, is located in 205 bp, the minimum value is −3.60, located in 211, 18 bp as the hydrophobic region.

Fig. 327.6
figure 6

Analysis of Hydrophobicity

Full size image

327.3.5 Chloroplast Transit Peptides and Protein Localization Sites

Use http://www.cbs.dtu.dk/services/, The ChloroP server predicts the presence of chloroplast transit peptides (cTP) in protein sequences and the location of potential cTP cleavage sites. The predicted length of the presequence is 49 bp.

Name

Length

Score

cTP

CS-score

cTP-length

Sequence

369

0.543

Y

2.303

49

PSORT—Prediction of Protein Localization Sites. The possibility exists in the chloroplast stroma reached 50.3 %

Final Results:

chloroplast stroma—Certainty = 0.503 (Affirmative) < succ>

mitochondrial matrix space—Certainty = 0.432 (Affirmative) < succ > chloroplast thylakoid membrane—Certainty = 0.255 (Affirmative) < succ > microbody (peroxisome)—Certainty = 0.245 (Affirmative).

327.4 Conclusion

A full-length complementary deox-yribunucleic acid (cDNA) of a fatty acyl–acyl carrier protein thioesterase (CpFATB) was isolated from a Chimonanthus praecox (wintersweet) cDNA library. Wintersweet is a hardy shrub native to Chinese montane regions and is known to be tolerant to many biotic and abiotic stresses including cold, drought, and avariety of plant pathogens [5]. Bioinformatic analysis also demonstrated that CpFATB contains the hotdog fold/domain [6]. The cDNA is 1, 110 nucleotides in length, of which 1, 107 bp form a single open-reading frame, predicting a 369 amino acid polypeptide. Molecular weight and isoelectric point of the protein is 41.72 kD and 7.72, respectively. Bioinformatics analysis shows that a signal peptide with 49 amino acid residues and Protein Localization Sites exists in the chloroplast stroma.This classifies the protein as stable. This gene may be of use in the production of drought-tolerant crops pecies and, as such, is worthy of further characterization and investigation.