8(1) 2015 issue
January 2015 issue
Southern Cross Publishing Group©2015
Comparative computational analysis of stigmasterol biosynthetic genes and proteins in plants
Bawankar Raksha, Rajendrarao Priya, Ramamoorthy Siva and Subramanian Babu
School of Bio Sciences and Technology, VIT University, Vellore 632014, India
Plant stigmasterol synthesis involves the partaking of many genes. In the present study, we performed computational comparison of the genes smt2, smo2, ste1, dwf5, dwf1 and c-22 sterol desaturase involved in the phytosterol biosynthetic pathway in different plants. Eleven plants belonging to different botanical families were selected and the gene sequence was retrieved from the KEGG pathway database. Nucleotide sequence homology was observed by Clustal W analysis and phylogeny was studied using MEGA 5 software. Clustal W and MEGA 5 analysis was done for amino acid sequence also. The maximum conserved regions were used in Prosite scan analysis. To determine the domains in protein sequence of the aforesaid genes in eleven plants, Pfam domain database search was performed. Phyre 2 software was used to develop 3D protein models. All the monocots used in the study belong to Poaceae family and hence in our phylogenetic analysis of phytosterol biosynthetic genes showed 90% sequence homology among the plants with same point of origin. The dicot plants chosen for the study belong to different families and hence the genes showed a homology percentile of less than 80.Pfam studies revealed SMT2 protein with a Methyl transf 11 and Sterol MTC domains. SMO2 and STE-1 proteins showed a common FA hydroxylase domain. DWF-5 was found to have domain structure of ERG4/ERG24 family, whereas, DWF-1 showed presence of FAD binding 4 domain. P450 structural domain was represented by sterol c-22 desaturase proteins. The 3D structure prediction revealed structural similarity among all eleven plants for protein involved in the plant stigmasterol biosynthesis.
Pages 1-8 | Full Text PDF | Supplementary Data PDF
Genetic diversity in Indian snapmelon (Cucumis melo var. momordica) accessions revealed by ISSR markers
Anil Kumar Singh, Sanjeev Kumar, Hemant Singh, Ved Prakash Rai, Brahma Deo Singh, Sudhakar Pandey*
Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi- 221 005, India
Division of Crop Improvement, Indian Institute of Sugarcane Research, Lucknow- 226 002 India
School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi- 221 005 India
Division of Crop Improvement, Indian Institute of Vegetable Research, Varanasi- 221 305 India
Present address: College of Agriculture and Research Station, Korea- 497 335, Chattisgarh, India
Snapmelon (Cucumis melo var. momordica) is native to India and many of its accessions have been used as source for disease and insect pest resistance, worldwide. Inter simple sequence repeat (ISSR) markers were used to evaluate intra-specific genetic diversity among twenty-two snapmelon accessions, variable for fruit cracking, peeling patterns, fruit shape, and flesh colour. Of the 32 ISSR markers tested, three produced monomorphic products, nine markers failed to amplify, and rest of the 20 markers produced 127 amplification products, of which 74 (58.38%) were polymorphic. Although the accessions varied greatly in terms of fruit traits, the pair-wise Jaccords similarity coefficient ranged from 0.59 to 0.88, revealing a narrow diversity in the studied samples owing to dominant nature of the ISSR markers. The dendrogram prepared through unweighted pair group method with arithmetic mean (UPGMA) distinguished two main clusters, cluster I consisting of 8 accessions, while cluster II contained 14 accessions. UPGMA clustering was also supported by principal components analysis (PCA). The first three PCs contributed 21.1, 18.9, and 8.7% of the variation, respectively. The first three PCs contributed for 48.7% variation in the studied accessions. This study could provide useful information for Indian snapmelon germplasm management activities, leading to development of a core collection for use in breeding and conservation programs.
Pages 9-16 | Full Text PDF
Genomic sequencing using 454 pyrosequencing and development of an SSR primer database for Lagerstroemia indica L.
Jing Wang, Xiaogang Dai, Yingnan Chen*, Yanling Yang, Xinye Zhang, Shuxian Li, Tongming Yin
The Southern Modern Forestry Collaborative Innovation Centre, Nanjing Forestry University, Nanjing 210037, China
Hubei Forestry Academy, Wuhan 430075, China
Crape myrtles (Lagerstroemia spp.) represent a large group of woody flowing plants. Despite their high ornamental value and popularity, few genomic sequences and marker resources are available for them. Lagerstroemia indica is one of the most widely cultivated crape myrtle species. In this study, we partially sequenced the genome of L. indica using newly updated 454 sequencing technology. Over 1.2 million high-quality reads in a total length of 837.4 Mb were generated. The average read length was 679 bp. Of the reads, 779,744 (63.2%) were assembled into 65,129 contigs covering a physical length of 93.6 Mb and with N50 contig size of 1,648 bp. The contigs were used to recover microsatellites with repeat motifs of 1-6 bp. A total of 33,026 microsatellites were detected. An SSR primer database was established based on the flanking sequences of the detected microsatellites. A PCR survey of subset of these SSR primers revealed that 89.5% amplified successfully,and 66.7% of the loci were polymorphic. The polymorphic information contents of the polymorphic SSRs ranged from 0.08 to 0.79, with an average value of 0.44. This study provided valuable genomic sequences and marker resources for future genetic studies on Lagerstroemia species.
Pages 17-23 | Full Text PDF | Supplementary Data PDF
In silico analysis of simple sequence repeats (SSRs) in chloroplast genomes of Glycine species
Ibrahim Ilker Ozyigit, Ilhan Dogan, Ertugrul Filiz*
Marmara University, Faculty of Science and Arts, Department of Biology, 34722, Goztepe, Istanbul, Turkey
Izmir Institute of Technology, Faculty of Science, Department of Molecular Biology and Genetics, 35430, Urla, Izmir, Turkey
Duzce University, Cilimli Vocational School, Department of Crop and Animal Production, 81750, Duzce, Turkey
Microsatellites, also known as simple sequence repeats, are short (1-6 bp long) repetitive DNA sequences present in chloroplast genomes (cpDNAs). In this work, chloroplast genomes of eight species (Glycine canescens, G. cyrtoloba, G. dolichocarpa, G. falcata, G. max, G. soja, G. stenophita, and G. tomentella) from Glycine genus were screened for cpSSRs by utilisation of MISA perl script with a repeat size of =10 for mono-, 5 for di-, 3 for tri-, tetra-, penta- and hexa-nucleotide, including frequency, distributions, and putative codon repeats of cpSSRs. According to our results, a total of 1273 cpSSRs were identified and among them, 413 (32.4%) were found to be in genic regions and the remaining (67.6%) were all located in intergenic regions, with an average of 1.04 cpSSRs per kb. Trinucleotide repeats (45%) were the most abundant motifs, followed by mononucleotides (36%) and dinucleotides (11.8%) in the plastomes of the Glycine species. In genic regions, trimeric repeats, the most frequent one reached the maximum of 70.7%. Among the other repeats, mono- and tetrameric repeats were represented in proportions of 25.7% and 3.6%, respectively. Interestingly, there were no di-, penta-, and hexameric repeats in coding sequences. The most common motifs found in all plastomes were A/T (97.8%) for mono-, AT/AT (98%) for di-, and AAT/ATT (41.5%) for trinucleotides. Among the chloroplast genes, ycf1 had the highest number of cpSSRs, and G. cyrtoloba and G. falcata species had the maximum number of genes containing cpSSRs. The most frequent putative codon repeats located in coding sequences were found to be glutamic acid (21.2%), followed by serine (15.5%), arginine (8.3%) and phenylalanine (7.8%) in all species. Also, tryptophan, proline, and aspartic acid were not detected in all plastomes.
Pages 24-29 | Full Text PDF
A novel plant code optimization phosphomannose isomerase (pPMI) and its application in rice (Oryza sativa L.) transformation as selective marker
Chunhong QIU, Hao LI, Fengshun SONG, Yongbo DUAN, Yicheng SUN, Yachun YANG, Ruiying Qin, Li LI, Pengcheng WEI*, Jianbo YANG*
College of Life Sciences, Anhui University, Hefei, China
Key Laboratory of Rice Genetics Breeding of Anhui Province, Rice Research Institute, Anhui Academy of Agricultural Sciences, Hefei, China
College of Life Sciences, Huaibei Normal University, Huaibei, China
The phosphomannose isomerase (PMI) gene has been developed as selective marker gene for plant transformation. This positive selection system does not use toxic compounds such as antibiotics or herbicides. In this study, we used PMI effectively and safely. The original nucleotide sequence of PMI was codon optimized as rice (Oryza sativa L.) synonymous codon usage bias. The nucleotides in the PMI were optimized according to the preferred codons in rice. This plant code optimized phosphomannose isomerase (pPMI) has higher GC content (61.31%), especially GC content at the third position in a codon (83.42%). The vectors harboring pPMI and PMI were transferred into rice by Agrobacterium-mediated transformation. There were higher transformation frequency (54.5%) and single copy rate (44.5%) using pPMI as selective marker than PMI. This work showed that pPMI could be a suitable substitution for PMI in production of transgenic crops. Moreover, pPMI could be used in gene editing technology for higher transformation frequency and safety.
Pages 30-36 | Full Text PDF | Supplementary Data PDF
A simple method for transient transformation of pumpkin (Cucurbita maxima) seedlings
Francisco Arturo Ramνrez-Ortega, Beatriz Xoconostle-Cαzares, Roberto Toscano-Morales, Roberto Ruiz-Medrano*
Departamento de Biotecnologνa y Bioingenierνa, CINVESTAV-IPN, Col. San Pedro Zacatenco 07360, Mιxico D.F., Mιxico
Currently there are no efficient stable transformation methods for pumpkin. In this work we report an efficient genetic transient transformation method of whole pumpkin (Cucurbita maxima cv. Big Max, a dicot) plants in the cotyledonary stage via direct inoculation of Agrobacterium tumefaciens and A. rhizogenes. A vector harboring a uidA-GFP translational fusion and the Cauliflower mosaic virus (CaMV) 35S promoter was employed. Transformation with A. rhizogenes resulted in the generation of root tumor phenotype within the lower portion of the stem, two months after inoculation. On the other hand, A. tumefaciens induces callus tissue also in the lower section of the stem one month after inoculation. Sections from these tissues were confirmed to harbor the transgene through histochemical GUS analysis and quantitative (q) PCR and RT-qPCR. Transgene RNA was also detected in phloem sap exudates, suggesting a via for delivery of foreign-expressed proteins in pumpkin. Plants expressing the maize knotted1 (KN1) genes showed distinct phenotype, consisting in leaf deformations with its known role in determining leaf shape. Transformation efficiency, based on transgene presence and appearance of a phenotype, ranged between 17.6 and 56%. The method described in this work can be used to transiently transform adult plants, and may be applicable to other species recalcitrant to Agrobacterium-mediated stable transformation; also, novel traits may be introduced to pumpkin and other plants using this technique.
Pages 37-46 | Full Text PDF
Proteome changes of Pteridium aquilinum during postharvest storage
Jinsong Huang, Li Jiang, Lin-hui Jiang, Chen Huan, Zhifang Yu*
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China
Department of Materials and Chemical Engineering, Chizhou College, Chizhou, 247000, China
Postharvest quality deterioration directly limited the shelf life and edibility of the stem of P. aquilinum. Proteome changes in response to quality worsening of the vegetable were investigated using 2-DE and differential proteins were identified by MALDI-TOF/TOF. The results demonstrated seventeen proteins were identified and these proteins were classified into five functional groups based on the protein functions and relevant literatures. Among them, six proteins were related to carbohydrate metabolism, three proteins were connected with to energy and amino acid metabolism, three proteins were involved in cell structure formation, three proteins were associated with stress response and defense and two proteins were engaged in protein synthesis. The biological mechanisms of quality deterioration was shows as follow: (1) The decline of antioxidant capacity would be responsible for accumulation of ROS, and excessive ROS leads to cell oxidation and senescence (2) P. aquilinum senescence begins and protein content declines when the vegetable is harvested and stored. (3) Cell structure integrity could be destroyed during postharvest storage.
Pages 47-54 | Full Text PDF
Comparative phylogenetic analysis of phenylpropanoid metabolism genes of legume plants
Neslihan Turgut-Kara*, Φzgόr Ηakir
Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, 34118, Vezneciler, Istanbul, Turkey
Plant phenylpropanoids contribute various physiological functions in accordance with environmental influences; therefore, most of secondary metabolites are synthesized through phenylpropanoid pathway. In this study, National Center for Biotechnology Information (NCBI) was searched to collect protein sequences that encode legume phenylpropanoid metabolism enzymes homologues. A total of 95 phenylpropanoid metabolism enzymes sequences from several legume species were phyletically analysed to light the way for the evolution characteristics of legume-specific homologues. One of the main emphases of this study was the elucidation of the conserved sequences of phenylpropanoid enzymes, and designing a set of quantitative PCR primers to standardize gene expression experiment for different legumes at once. As a result of the analyses, conserved sequences of phenylpropanoid enzymes were determined, and the sets of real-time PCR primers were generated for 5 main phenylpropanoid genes, phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). This study will also assist in furthering our understanding of the evolutionary relation of phenylpropanoids between legume species.
Pages 55-61 | Full Text PDF
Identification of novel miRNA-target pairs in rice (Oryza sativa) by a reversed approach
Chaogang Shao, Guofu Lu, Lan Yu, Qianer Wu, Ming Chen*, Yijun Meng*
College of Life Sciences, Huzhou University, Huzhou 313000, P.R. China
Department of Bioinformatics, College of Life Sciences, Zhejiang University, Hangzhou 310058, P. R. China
College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, P.R. China
MicroRNAs (miRNAs) are crucial regulators of gene expression in plants. Here, we performed a comprehensive computational search for novel miRNA--target pairs in rice by a reversed approach. First, the potential cleavage sites in all the 67,392 cDNAs of rice were searched by degradome sequencing datasets. Then, the bait sequences, which may be acted as the miRNAs binding sites in the targets, were generated by adding 10 nt sequences both upstream and downstream of the cleavage sites. Finally, the potential miRNA candidates were extracted by BLAST of these bait sequences against the AGO1-associated sRNA sequencing data and were further validated considering the structure and expression characteristics of miRNA. As a result, 144 previously validated miRNA--target pairs and 7 novel miRNA candidates together with 15 mRNA targets were identified. According to the literature, some of these 15 corresponding targets play very important role in the stress signaling pathways in rice. Other interesting findings were the non-canonical slicing sites and higher accumulation isomiRs were identified in some of the previously validated miRNA--target pairs.
Pages 62-68 | Full Text PDF | Supplementary Data PDF | Supplementary Data Excel
Potential anti-inflammatory and anti-oxidative properties of Thai colored-rice extracts
Thitinan Kitisin, Nisakorn Saewan, Natthanej Luplertlop*
Department of Anatomy, Faculty of Science, Mahidol University, Ratchathewi, Bangkok, Thailand
School of Cosmetic Science, Mae Fah Luang University, Muang, Chiang Rai, Thailand
Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Ratchathewi, Bangkok, Thailand
In Thailand, there has been growing interest in the use of colored rice extracts as a new source of anti-oxidative and anti-inflammatory effects. This study investigates the effects of different colored rice extracts in terms of their biological content, anti-oxidative activity, and their ability to reduce pro-inflammatory cytokines and matrix metalloproteinase (MMP) expression. Various colored rice from different rice cultivating areas in Thailand were used to obtain ethanolic extracts. The biological compounds in colored-rice extracts were determined by Folin-Ciocalteu colorimetric and pH-differential methods. To determine the anti-oxidative properties of colored-rice extract, DPPH radical scavenging, ferrous reducing power, and lipid peroxidation assays were used. The cytotoxicity of colored rice extracts was determined by MTT assay on a human promyelocytic leukemia (HL-60) cell line in vitro. The inhibition of pro-inflammatory cytokines (IL-6, TNF-a, NF-?B) and MMP expression in LPS-induced HL-60 cells was determined by ELISA assay. Moreover, MMP activity was determined by gelatinolytic zymography. The results found that red (Mun Poo, MP) rice exhibited high anti-oxidative activity and reduced pro-inflammatory cytokines and MMP-2 expression in LPS-induced HL-60 cells. This study provides new insights into the potential use of Thai colored rice extracts, especially red rice, as a source of anti-oxidants and anti-inflammation.
Pages 69-77 | Full Text PDF