title: Identification and computational annotation of genes differentially expressed in pulp development of Cocos nucifera L. by suppression subtractive hybridization.
abstract: BACKGROUND
  Coconut (Cocos nucifera L.) is one of the world's most versatile, economically important tropical crops. Little is known about the physiological and molecular basis of coconut pulp (endosperm) development and only a few coconut genes and gene product sequences are available in public databases. This study identified genes that were differentially expressed during development of coconut pulp and functionally annotated these identified genes using bioinformatics analysis.
  
  
  RESULTS
  Pulp from three different coconut developmental stages was collected. Four suppression subtractive hybridization (SSH) libraries were constructed (forward and reverse libraries A and B between stages 1 and 2, and C and D between stages 2 and 3), and identified sequences were computationally annotated using Blast2GO software. A total of 1272 clones were obtained for analysis from four SSH libraries with 63% showing similarity to known proteins. Pairwise comparing of stage-specific gene ontology ids from libraries B-D, A-C, B-C and A-D showed that 32 genes were continuously upregulated and seven downregulated; 28 were transiently upregulated and 23 downregulated. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis showed that 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT), phospholipase D, acetyl-CoA carboxylase carboxyltransferase beta subunit, 3-hydroxyisobutyryl-CoA hydrolase-like and pyruvate dehydrogenase E1 β subunit were associated with fatty acid biosynthesis or metabolism. Triose phosphate isomerase, cellulose synthase and glucan 1,3-β-glucosidase were related to carbohydrate metabolism, and phosphoenolpyruvate carboxylase was related to both fatty acid and carbohydrate metabolism. Of 737 unigenes, 103 encoded enzymes were involved in fatty acid and carbohydrate biosynthesis and metabolism, and a number of transcription factors and other interesting genes with stage-specific expression were confirmed by real-time PCR, with validation of the SSH results as high as 66.6%. Based on determination of coconut endosperm fatty acids content by gas chromatography-mass spectrometry, a number of candidate genes in fatty acid anabolism were selected for further study.
  
  
  CONCLUSIONS
  Functional annotation of genes differentially expressed in coconut pulp development helped determine the molecular basis of coconut endosperm development. The SSH method identified genes related to fatty acids, carbohydrate and secondary metabolites. The results will be important for understanding gene functions and regulatory networks in coconut fruit.
authors.forename: Yuanxue
authors.initials: Y
authors.lastname: Liang
authors.forename: Yijun
authors.initials: Y
authors.lastname: Yuan
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Mao
authors.forename: Yusheng
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Department of Biotechnology, College of Materials and Chemical Engineering, Hainan University, Haikou 570228, Hainan, PR China. liddfym@hotmail.com.
authors.forename: Dongdong
authors.initials: D
authors.lastname: Li
mesh_terms: D003059:Cocos / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D056625:Endosperm / Q000254:growth & development / Q000378:metabolism*; D005227:Fatty Acids / Q000096:biosynthesis; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D008697:Methane / Q000378:metabolism; D058977:Molecular Sequence Annotation; D060888:Real-Time Polymerase Chain Reaction; D013213:Starch / Q000378:metabolism; D066251:Subtractive Hybridization Techniques; D013395:Sucrose / Q000378:metabolism
chemical_list: D005227:Fatty Acids; D013395:Sucrose; D013213:Starch; D008697:Methane
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