Differential Gene Expression and Characterization of Tissue-specific cDNA Clones in Oil Palm using mRNA Differential Display

The mRNA differential display method was utilized to study the differential expression and regulation of genes in two species of oil palm, the commercially grown variety Elaeis guineensis, var. tenera and the South American species, Elaeis oleifera. We demonstrated the differential expression of genes in the mesocarp and kernel at the week of active oil synthesis (15 week after anthesis) during fruit development as compare to the roots and leaves and the isolation of tissue-specific and species-specific cDNA clones. A total of eight specific cDNA clones were isolated and their specificities were confirmed by Northern hybridization and classified into three groups. Group one contains four clones (KT3, KT4, KT5 and KT6) that are kernel-specific for E. guineensis, tenera and E. oleifera. The second group represents clone FST1, which is mesocarp and kernel-specific for E. guineensis, tenera and E. oleifera. The third group represents clones MLT1, MLT2 and MLO1 that are mesocarp and leaf-specific. Northern analysis showed that their expressions were developmentally regulated. Nucleotide sequencing and homology search in GenBank data revealed that clones KT3 and KT4 encode for the same maturation protein PM3. While clones MLT1 and MLT2 encode for S-ribonuclease binding protein and fibrillin, respectively. The other clones (KT5, KT6, FST1 and MLO1) did not display any significant homology to any known protein.     

Carotenoid hydroxylase from Haematococcus pluvialis: cDNA sequence, regulation and functional complementation.

A cDNA homologous to beta-carotene hydroxylase from Arabidopsis thaliana was isolated from the green alga Haematococcus pluvialis. The predicted amino acid sequence for this enzyme shows homology to the three known plant beta-carotene hydroxylases from Arabidopsis thaliana and from Capsicum annuum (38% identity) and to prokaryote carotenoid hydroxylases (32-34% identities). Heterologous complementation using E. coli strains which were genetically engineered to produce carotenoids indicated that the H. pluvialis beta-carotene hydroxylase was able to catalyse not only the conversion of beta-carotene to zeaxanthin but also the conversion of canthaxanthin to astaxanthin. Furthermore, Northern blot analysis revealed increased beta-carotene hydroxylase mRNA steady state levels after induction of astaxanthin biosynthesis. In accordance with the latter results, it is proposed that the carotenoid hydroxylase characterized in the present publication is involved in the biosynthesis of astaxanthin during cyst cell formation of H. pluvialis.     

Insights from the GC content analysis of 76genome survey sequences (GSS) from Elaeisoleifera

South American oil-palm (Elaeis oleifera) is not cultivated in tropical countries like Malaysia on large scale due to low yield of palm oil derived from its fruit mesocarp. However, its fruit mesocarp oil contains about 68.6 % oleic acid (C_18:1) which is more than double in comparison to commercially cultivated oilpalm, E. guineensis Jacq Tenera (hybrid of Dura (♀) x Pisifera (♂)). It is also known that E. oleifera is a good source of tocotrienols and carotenoids. Therefore, it is of interest to know the genome sequence of E. oleifera. The objective of this study is to generate genome survey sequences (GSS) to get GC content insight in the E. oleifera genome. The nuclear genomic DNA isolated from young leaf‐tissues was digested with EcoRI and NdeI/DraI restriction enzymes; and three genomic DNA libraries were constructed using Lambda ZAP‐II, pGEM®‐T Easy, and pDONR 222™ as cloning vectors. Generated 76 GSSs were analyzed by using Bioinformatics tools. The analysis result indicates that the adenine, cytosine, guanine and thymine content in generated GSSs are 30%, 20%, 20%, and 30% respectively. In conclusion, based on the precise GC content analysis of the randomly isolated 76 GSSs by using Bioinformatics tools we hypothesize that GC content in E. oleifera genome is 40%. The hypothesized 40% GC content in E. oleifera genome is expected to remain close to the GC content based on the whole genome analysis.^ψThe nucleotide sequence data reported in this paper have been submitted to dbGSS division of the international DNA database (GenBank/DDBJ/EMBL) under accession numbers: {"type":"entrez-nucleotide-range","attrs":{"text":"DX575945- DX575972","start_term":"DX575945","end_term":"DX575972","start_term_id":"94323534","end_term_id":"94323561"}}DX575945- DX575972 and {"type":"entrez-nucleotide-range","attrs":{"text":"EI798032-EI798079","start_term":"EI798032","end_term":"EI798079","start_term_id":"146199019","end_term_id":"146199066"}}EI798032-EI798079.

Abbreviations

gDNA - Nuclear genomic DNA, GSSs - Genome survey sequences K12, SAOP - South American oil‐palm Db1     

观赏凤梨烯醇酶基因的克隆及分析

烯醇酶催化着糖酵解途径中的唯一一步脱水反应,与植物的抗逆性有着密切的联系.基于前期从擎天凤梨全长cDNA文库中获得的烯醇酶基因的EST单克隆,进行引物步移测序,获得其全长cDNA序列.序列长1 703bp,编码445个氨基酸残基,命名为GoEnolase1(GenBank登录号JN896863),蛋白质理论分子质量为47.9kD,等电点为5.7,包含1个TIM磷酸结合超家族保守区,二级结构主要由α螺旋(44.49%)、随机卷曲(33.71%)、延伸链(13.71%)和β转角(8.09%)组成.系统进化树分析表明,GoEnolase1蛋白与水稻、玉米、油棕等的烯醇酶蛋白聚为一类,它们的亲缘关系最近.     

Screening for lipase activity in the oil palm

The oil palm mesocarp contains an endogenous lipase which is strongly activated at low temperature. Lipase activity is thus very conveniently assayed by prior exposure of the fruits to low temperature. More than 100 oil palm samples from the germplasm collection of the Palm Oil Research Institute of Malaysia (now known as the Malaysian Palm Oil Board) were screened for non-esterified fatty acid activity using both the low-temperature activation assay and a radioactivity assay. The results showed good correlation between assay procedures. The different samples had a very wide range of lipase activity. Elaeis oleifera samples had significantly lower lipase activity compared with E. guineensis (var. tenera) samples. Even within E. guineensis (var. tenera), there was a wide range of activity. The results confirmed that lipase activity is genotype-dependent. Selection for lipase genotypes is thus possible and this will have obvious commercial value.     

Use of Trichoderma harzianum cultured on ground mesocarp fibre of oil-palm as seed treatment to control Pythium aphanidermatum,a causal agent of damping-off of Chinese kale seedling

Trichoderma harzianum, isolate T 01-22, was cultured on either sorghum grains, ground mesocarp fibre of oil-palm or oil-palm shell, both amended with urea fertilizer (100:1, w/w). T. harzianum cultured on ground mesocarp fibre was then used to coat seeds of Chinese kale (Brassica alboglabra Bailey) to control damping-off of seedlings caused by Pythium aphanidermatum. Biomass of T. harzianum cultured on ground mesocarp fibre of oil-palm was more effective than Captan and Benomyl, but less effective than Metalaxyl, in controlling damping-off of Chinese kale seedlings. Viability of T. harzianum growing on sorghum grains was reduced significantly during 7 months storage, followed by that of T. harzianum cultured on ground mesocarp fibre and oil-palm shell, both amended with urea fertilizer (46-0-0) at 100:1 (w/w).     

cDNA cloning and expression of bovine aspartyl (asparaginyl) beta-hydroxylase.

Aspartyl (asparaginyl) beta-hydroxylase which specifically hydroxylates 1 Asp or Asn residue in certain epidermal growth factor-like domains of a number of proteins, has been previously purified to apparent homogeneity from detergent-solubilized bovine liver microsomes (Wang, Q., VanDusen, W. J., Petroski, C. J., Garsky, V. M., Stern, A. M., and Friedman, P. A. (1991) J. Biol. Chem. 266, 14004-14010). Three oligonucleotides, corresponding to three amino acid sequences of the purified hydroxylase, were used to screen bovine cDNA libraries. Several overlapping positive cDNA clones containing a full length open reading frame of 754 amino acids encoding a 85-kDa protein were isolated, and a cDNA, containing the full length open reading frame, was constructed from two of these clones. The resulting clone was then transcribed and translated in vitro to produce recombinant protein which possessed Asp beta-hydroxylase activity. These results constitute proof that the protein purified from bovine liver is an Asp beta-hydroxylase. Comparisons of deduced amino acid sequences of two other alpha-ketoglutarate-dependent dioxygenases, prolyl-4-hydroxylase and lysyl hydroxylase, with that of Asp beta-hydroxylase showed no significant homologies. Indeed, Asp beta-hydroxylase appears to be unique as no striking homology was found with known protein sequences. Furthermore, structural predictions derived from the deduced amino acid sequence are in accord with earlier Stokes' radius and sedimentation coefficient determinations of the enzyme, suggesting that the enzyme contains a relatively compact carboxyl-terminal catalytic domain and an extended amino terminus. This amino-terminal region has a potential transmembrane type II signal-anchor domain that could direct the catalytic domain into the lumen of the endoplasmic reticulum.     

Characterization of the chloroplast genome sequence of oil palm (Elaeis guineensis Jacq.)

Oil palm (Elaeis guineensis Jacq.) is an economically important crop, which is grown for oil production. To better understand the molecular basis of oil palm chloroplasts, we characterized the complete chloroplast (cp) genome sequence obtained from 454 pyrosequencing. The oil palm cp genome is 156,973 bp in length consisting of a large single-copy region of?85,192 bp flanked on each side by inverted repeats of 27,071 bp with a small single-copy region of 17,639 bp joining the?repeats. The genome contains 112 unique genes: 79 protein-coding genes, 4 ribosomal RNA genes and 29 tRNA genes. By aligning the cp?genome sequence with oil palm cDNA sequences, we observed 18 non-silent and 10 silent RNA editing events among 19 cp protein-coding genes. Creation of an initiation codon by RNA editing in rpl2 has been reported in several monocots and was also found in the oil palm cp genome. Fifty common chloroplast protein-coding genes from 33 plant taxa were used to construct ML and MP?phylogenetic trees. Their topologies are similar and strongly support for the position of E. guineensis as the sister of closely related species Phoenix dactylifera in Arecaceae (palm families) of monocot subtrees.     

The Kazusa cDNA project for identification of unknown human transcripts

The Kazusa cDNA project is unique by its focus on sequencing large human cDNAs (>4 kb). We describe an overview of the human cDNA sequence data accumulated during the first phase of the project on over 2000 cDNAs and its integration with the genome sequence. In the second phase of the project, which aims at bridging the human genome and proteome using the output of the first phase, we are very carefully evaluating our cDNA clones and, when necessary, experimentally revising them.     

Assessment of nuclear,mitochondrial and chloroplast RFLP markers in oil palm (Elaeis guineensis Jacq.)

A variety of DNA probes was used to screen a diverse set of oil palm accessions in order to identify markers with a utility in genotype discrimination. This survey included samples of the commercial oil palm native to Africa (Elaeis guineensis Jacq.), the closely-related South American species [E.oleifera (HBK) Cortes] and inter-specific hybrids of the two. Of 106 major chloroplast bands none showed differences between E. guineensis and E. Oleifera. Mitochondrial and ribosomal probes were more informative inter-specifically (the former allowing identification of the maternal inheritance of mitochondria) and may be useful in hybrid breeding programmes; however, they were unable to identify polymorphism within E. guineensis. In contrast, low-copy nuclear genomic clones were able to identify intra-specific variation, though in most cases they revealed a relatively small number of allelic variants. One DNA probe showed a much larger number of band variants, revealing ten patterns amongst 13 E. guineensis accessions, and should prove useful in genetic fingerprinting and evaluation of oil-palm germplasm collections.     

Comprehensive definition of genome features in Spirodela polyrhiza by high‐depth physical mapping and short‐read DNA sequencing strategies

Spirodela polyrhiza is a fast‐growing aquatic monocot with highly reduced morphology, genome size and number of protein‐coding genes. Considering these biological features of Spirodela and its basal position in the monocot lineage, understanding its genome architecture could shed light on plant adaptation and genome evolution. Like many draft genomes, however, the 158‐Mb Spirodela genome sequence has not been resolved to chromosomes, and important genome characteristics have not been defined. Here we deployed rapid genome‐wide physical maps combined with high‐coverage short‐read sequencing to resolve the 20 chromosomes of Spirodela and to empirically delineate its genome features. Our data revealed a dramatic reduction in the number of the rDNA repeat units in Spirodela to fewer than 100, which is even fewer than that reported for yeast. Consistent with its unique phylogenetic position, small RNA sequencing revealed 29 Spirodela‐specific microRNA, with only two being shared with Elaeis guineensis (oil palm) and Musa balbisiana (banana). Combining DNA methylation data and small RNA sequencing enabled the accurate prediction of 20.5% long terminal repeats (LTRs) that doubled the previous estimate, and revealed a high Solo:Intact LTR ratio of 8.2. Interestingly, we found that Spirodela has the lowest global DNA methylation levels (9%) of any plant species tested. Taken together our results reveal a genome that has undergone reduction, likely through eliminating non‐essential protein coding genes, rDNA and LTRs. In addition to delineating the genome features of this unique plant, the methodologies described and large‐scale genome resources from this work will enable future evolutionary and functional studies of this basal monocot family.     

NRDRiso酶cDNA的序列测定及生物信息学分析

通过鉴定分析人肝组织中辅酶II依赖性视黄醇脱氢酶不同剪接体全长cDNA核苷酸序列与氨基酸序列的结构特征,为今后进一步研究体内维甲酸的代谢情况奠定基础。根据人、小鼠NRDR编码区的一致性序列,设计一对引物,应用RTPCR方法从人肝组织中得到一条377bp的新的cDNA片段。采用RACE法得到了NRDR新亚型cDNA,并以生物信息学软件分析其生物学特征。 测序得知该cDNA长为1003bp,以NADP-dependent retinol dehydrogenase/reductase short isoform（NRDRiso）登录GenBank。其读码框为525bp,拟编码174个氨基酸的蛋白。