Genomics and Applied Biology, 2010, Vol. 1 - Sophia Publishing ...

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Genomics and Applied Biology, 2010, Vol.1 No.2http://gab.sophiapublisher.comFigure 4 Neighbor-joining tree based on the deduced aminoacid sequences of HD homologsNote: Sequences were shown by their accession numbers (locustags) and strain names; Bootstrap values from neighbor-joininganalyses were listed to the left of each node, with values morethan 50 were shownFigure 7 Expression analysis of AhKR, AhHD and AhENRgenes in four different tissuesFigure 6 Neighbor-joining tree based on the deduced aminoacid sequences of ENR homologsNote: Sequences were shown by their accession numbers (locustags) and strain names; Bootstrap values from neighbor-joininganalyses were listed to the left of each node, with values morethan 50 were shown2 DiscussionPeanut is an important crop internationally for bothfood and oil production (Luo et al., 2005). One of themajor factors influencing peanut oil quality is thecomposition of fatty acid. So validating themechanism of the fatty acid synthesis and metabolismis the central goal to increase peanut quality. Fattyacid biosynthesis in higher plants is carried out bytype II FAS. In the present study, we isolated AhKR,AhHD and AhENR orthologues in peanut seedling.Sequence comparisons revealed that the primarystructure of plant plastid type II FAS enzymes (KR,HD, ENR) was strictly conserved, especially thecatalytic residues, which suggested that these enzymesmay have similar functions in higher plants as those inE. coli. Real-time RT-PCR analysis revealed that thethree genes were expressed dominantly in leaf amongfour tissues tested, and expressed at the lowest level instem. AhKR and AhENR genes shared similarexpression behaviors over the developmental stagescompared to that of AhHD gene. These results indicated- 17 -
Genomics and Applied Biology, 2010, Vol.1 No.2http://gab.sophiapublisher.comreal-time RT-PCR analysis. In addition, the immaturepeanut seeds from 25 to 60 days after pegging (DAP)were also collected for expression analysis.3.2 Nucleic acid manipulationTotal RNA was extracted from samples using theRNeasy Mini Kit (Qiagen) according to the manufacturer’sinstructions. The RNA samples were used forreal-time RT-PCR after RQ1 RNase-free DNaseI(Promega, Wisconsin, USA) treatment to removegenomic DNA. The first-strand cDNA was synthesizedwith RT-PCR kit (Promega, Wisconsin, USA) using500 ng of total RNA according to the manufacturer’sinstructions. Controls received water instead ofreverse transcriptase to assess any contamination fromgenomic DNA as described by Zhou et al. (2007).Figure 8 Expression analysis of AhKR, AhHD and AhENRgenes in seed at different developmental stagesthat the three genes may have different biochemicalfunctions during vegetative growth and seeddevelopment. To improve peanut oil quality shouldcoordinate the expression of the three enzymesinvolved in the fatty acid synthesis pathway. Thiswork may provide a basis for elucidating themolecular mechanism of fatty acid synthesis andprovide candidate genes for modifying oil quality viatransgenic plants.3 Materials and methods3.1 Plant materialsPeanut seeds (Arachis hypogaea L. cultivar Huayu19)were sown in sand and soil mixture (1:1), grown in agrowth chamber under a 16~8 h light-dark cycle at26°C and 22°C, respectively. Three kinds of12-day-old tissues including root, stem and leaf werecollected as experimental materials for quantitative3.3 Full-length cDNA sequence isolationPCR was performed with the LA PCR system (Takara)using 2.5 μL of 10×PCR buffer with MgCl 2 , 1 μL of10 μM each primer, 4.0 μL of 10 mM dNTPs, 1 μLcDNA samples and 0.5 μL LA Taq DNApolymerase, and 15 μL double distilled water. ThePCR products were run on 1% agarose gel andpurified with Gel Extraction Kit (Takara) according tothe manufacturer’s protocol. The purified productswere then cloned into the pMD18-T Easy vector(Takara) and sequenced (Shangon, Shanghai) (Table 2).3.4 Multiple sequence alignment and phylogeneticanalysisPhysicochemical properties of the deduced proteinwere predicted by Protparam (http://www.expasy.ch/tools/protparam.html). The putative subcellular localizationsof the candidate proteins were estimated byTargetP (http://www. cbs.dtu.dk/services/TargetP/)and Predotar (http://urgi.versailles.inra.fr/ predotar/predotar.html). The potential N-terminal presequencecleavage site was predicted by ChloroP (http://www.cbs.dtu.dk/services/ChloroP/). Amino acid sequenceswere aligned using ClustalX program with theimplanted BioEdit (Thompson et al., 1994). Theneighbor-joining (NJ) method in MEGA4 (Tamura etal., 2007) was used to construct the phylogenetic tree.Bootstrap with 1000 replicates was used to establishthe confidence limit of the tree branches. Defaultprogram parameters were used.- 18 -
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