Articles

9(2) 2016 issue
 
March 2016 issue
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Assessment of allelopathic potential of Couroupita guianensis Aubl.
  
Md Sirajul Islam Khan*
, Hisashi Kato-Noguchi

Laboratory of Plant Biochemistry, Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki, Kagawa, 761-0795, Japan

Abstract
Couroupita guianensis Aubl. has been traditionally used for medicinal as well as ornamental purposes, but the allelopathic potential of this species has not yet been reported. Hence, the aim of this study is to explore the allelopathic activity of C. guianensis leaves. We examined the effect of aqueous methanol extracts of the leaves of this species at five different concentrations (1, 3, 10, 30, and 100 mg dry weight (DW) equivalent extract mL-1) on the germination of lettuce (Lactuca sativa L.) and barnyard grass (Echinochloa crus-galli (L.) Beauv.), and on the growth of cress (Lepidium sativum L.), lettuce, alfalfa (Medicago sativa L
.), barnyard grass, timothy (Phleum pretense L.), and Italian ryegrass (Lolium multiflorum Lam.) seedlings. The germination of lettuce and barnyard grass, as well as the growth of all test plants, was inhibited by leaf extracts of C. guianensis at concentrations = 1 mg DW equivalent extract mL-1. The extract obtained from 100 mg C. guianensis leaves completely inhibited germination of lettuce or significantly delayed germination of barnyard grass. The hypocotyl/coleoptile and root growth of all test plants were inhibited by the leaf extracts to less than 10% of control hypocotyl/coleoptile and root growth at the concentration of 100 mg DW equivalent extract mL-1, except for the hypocotyl/coleoptile growth of alfalfa and barnyard grass. The ranges of I50 values of hypocotyl/coleoptile and root growth of all test plants were 1.22-10.24 and 0.76-3.97 mg DW equivalent extract mL-1, respectively. The inhibitory activities increased with increasing extract concentrations. The present results suggest that C. guianensis may have allelopathic potential and thus possesses allelopathic substances. Therefore, the leaves of this species could potentially be used as mulch and/or soil additive to control weeds in sustainable crop production.

Pages 115-120 | Full Text PDF | DOI: 1021475/poj.160902.p7526x
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Main factors affecting the genetic transformation of Chrysanthemum var. Micromargara

Juan Ubaldo Sαnchez-Velαzquez, Guadalupe Lσpez Puc, Ana Luisa Ramos-Dνaz, Julia del Socorro Cano-Sosa, Ingrid Mayanin Rodrνguez Buenfil, Rσmulo Garcνa-Velasco y Alberto Uc Varguez*

Centro de Investigaciσn y Asistencia en Tecnologνa y Diseρo del Estado de Jalisco AC., Unidad Sureste. Parque cientνfico y Tecnolσgico de Yucatαn. Km. 5.5 Carretera Sierra Papacal-Chuburnα Puerto. Cp. 97302, Mexico
Universidad Autσnoma del Estado de Mιxico. Centro Universitario Tenancingo. Exhacienda de Santa Ana Carretera Tenancingo-Villa Guerrero km 1.5 Tenancingo, Mιxico

Abstract
Factors influencing Agrobacterium tumefaciens- mediated genetic transformation of plants have been widely reported: type and concentration of antibiotic, co-culture period, concentration of bacteria, concentration of acetosyringone, and of course, the type and age of the explants, as well as temperature conditions. However, it is not yet understood how these factors interact and how they affect the efficiency of the final transformation. The aim of this work was to evaluate the interaction of the three main factors affecting the transformation of chrysanthemum (Dendranthema grandiflora) var. Micromargara. A 23 factorial design was used with central points, in which three concentrations of A. tumefaciens (1.0, 1.5. and 2.0 of D.O.600
), three concentrations of acetosyringone (50 ΅M, 75 ΅M and 100 ΅M), and three co-culture periods (1, 2 and 3 days) were evaluated. The transformation was verified by GUS staining and by means of RT-PCR amplification and sequencing of fragment of the genes uidA, aph3 II and act-cr. The results show that, among the factors evaluated, only the concentration of A. tumefaciens presented a statistically significant effect (p< 0.05) on transformation efficiency, without interaction with the factors of acetosyringone concentration and co-culture period. It is concluded therefore that an A. tumefaciens concentration of D.O 600: 2.0 is determinant for greater transformation efficiency in chrysanthemum var. Micromargara.

Pages 121-125 | Full Text PDF | DOI: 10.21475/poj.160902.p7542x
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An efficient method for isolating large quantity and high quality RNA from oleaginous microalgae for transcriptome sequencing

Anongpat Suttangkakul, Piyada Juntawong, Anchalee Sirikhachornkit, Chonlada Yaisumlee, Kanidtha Jariyachawalid, Kunn Kangwansaichol, Somsak Apisitwanich
, and Supachai Vuttipongchaikij*

Special Research Unit in Microalgal Molecular Genetics and Functional Genomics (MMGFG), Department of Genetics, Faculty of Science, Kasetsart University, 50 Ngarm Wong Wan road, Chatuchak, Bangkok 10900, Thailand
Center of Advanced studies for Tropical Natural Resources, Kasetsart University, Ngam Wong Wan, Chatuchak, Bangkok 10900, Thailand
PTT Research and Technology Institute, PTT Co., LTD., 71 Moo 2 Pahol Yothin road Wang Noi, Ayuthaya 13170, Thailand

Abstract
Transcriptome analysis requires a large quantity of high-quality DNase-treated RNA for poly(A)+ mRNA isolation and sequencing. This could be problematic in many oleaginous microalgal species that harbor strong cell walls and accumulate high lipid content. Using Scenedesmus obliquus
, a microalga with high oil content and potential as a source of algal biofuel, we assessed the efficiency of four RNA isolation methods: direct extraction using TriPure, mechanical breakage using either freeze-thawed with bead beating or grinding in liquid nitrogen followed by TriPure, and grinding in liquid nitrogen before using Qiagen RNeasy Plant Mini Kit. Liquid nitrogen grinding with TriPure method gave the best RNA yields at 15.15 ΅g mg-1 cell dry weight and ~148.9 ΅g total RNA from 100 ml culture of S. obliquus. Despite lower yields, RNA isolation of oil accumulating cells (~22% w/w lipid content) provided ~68.1 ΅g total RNA with the yield of 1.70 ΅g mg-1 cell dry weight. Transcriptome sequencing and de novo assembly with the average contig length of 824 bp reflected high quality of RNA obtained using this method. The RNA isolation protocol was tested on six other oleaginous microalgae including Chlamydomonas reinhardtii, S. acuminatus, Chorella vulgaris, Chlorococcum humicola, Tetradesmus cumbricus and Coelastrum sp. and yielded 0.86 - 5.42 ΅g mg-1 cell dry weight. For large scale RNA isolation from microalgae, grinding with liquid nitrogen before TriPure provided the best yield and quality. This finding helps simplify RNA isolation for upcoming transcriptome analyses in microalgae.

Pages 126-135 | Full Text PDF | DOI: 10.21475/poj.160902.p7617x
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Genome wide analysis of heat shock transcription factor (HSF) family in chickpea and its comparison with Arabidopsis

Syed Adeel Zafar, Muzammil Hussain, Mubashar Raza, Hafiz Ghulam Muhu-Din Ahmed, Iqrar Ahmad Rana, Bushra Sadia, Rana Muhammad Atif*

Institute of crop science, Chinese Academy of agricultural sciences, Beijing, China
Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan
Center of Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad, Pakistan
US-Pakistan centre for Advanced Studies in Agriculture and Food Security, University of Agriculture, Faisalabad, Pakistan

Abstract
Plants cope with thermo-stress by increased expression of heat shock genes. These genes encode various heat shock proteins (HSPs) which rapidly accumulate and protect plants following hasty heat stress. Heat shock transcription factors (HSFs) primarily regulate expression of HSP genes by deciphering conserved binding motifs in promoter region. We retrieved HSF genes of Arabidopsis and chickpea from the online data bases and analyzed their structure and properties using bioinformatics tools. Here, we reported 20 non-redundant genes encoding HSF domain containing proteins in chickpea. Comparative phylogenetic analysis of HSF genes with Arabidopsis revealed four major groups with several paralogous and orthologous genes. Gene localization studies showed that HSF genes are unevenly distributed across all of the eight chromosomes. Segmental duplications were principally involved in HSF gene family expansion during evolution. HSF genes predominantly contain a single intron. However, quite a few genes also retain two introns, which suggest gain of intron during the evolutionary process. Combined conserved-domain analysis of Arabidopsis and chickpea HSF proteins revealed presence of 19 most common domains. Comparison of conserved domains with phylogenetic tree has shown that some domains were present in a clade-specific manner. The presence of multiple conserved domains in HSF proteins suggested that the respective genes originate from duplication events. Our in-silico work may prove helpful in understanding the evolutionary pathways of HSFs in chickpea.

Pages 136-141 | Full Text PDF | DOI: 10.21475/poj.160902.p7644x
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Cloning and characterization of a putative gene encoding serine protease inhibitor (251Hbpi) with antifungal activity against Trichophyton rubrum from Hevea brasiliensis leaves

Dutsadee Chinnapun*, Sarawoot Palipoch, Hatairat Hongphruk

School of Medicine, Walailak University, Nakhon Si Thammarat 80161, Thailand
Center for Scientific and Technological Equipments, Walailak University, Nakhon Si Thammarat  80161, Thailand

Abstract
A novel serine protease inhibitor gene was isolated from Hevea brasiliensis leaves, a RRIT251 cultivar and designated RRIT251 H. brasiliensis protease inhibitor (251Hbpi). Reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were used to isolate 251Hbpi.  A full-length cDNA of 251Hbpi encoded a 70 amino acid protein. 251HbPI is a member of the potato inhibitor I (PI-I) family of serine protease inhibitors. The amino acid residues at the active site of 251HbPI were predicted as Met46-Glu47. Multiple alignments of the homologous PI-I family revealed one motif WPELVG of 251HbPI conserved across the family. 251Hbpi was cloned into expression vector pFLAG-ATS and expressed in Escherichia coli strain BL21. Molecular weight of the recombinant 251HbPI (r251HbPI) was approximately 11 kDa. Protease inhibition analysis revealed that r251HbPI inhibited the activity of chymotrypsin and subtilisin A but did not trypsin protease. Moreover, purified r251HbPI protein inhibited Trichophyton rubrum with a minimum inhibitory concentration of 0.7 mg/ml and a minimum fungicidal concentration of 1.4 mg/ml. The specific T. rubrum protease targets of r251HbPI were analyzed by co-immunoprecipitation. r251HbPI interacted with approximate 27 and 61 kDa T. rubrum proteins, suggesting a role in the inhibition of T. rubrum growth. These results suggest that 251HbPI could be a candidate for the development of a novel drug to treat T. rubrum infection.

Pages 142-148 | Full Text PDF |
DOI: 10.21475/poj.160902.p7796x
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Overexpression of potato transcription factor (StWRKY1) conferred resistance to Phytophthora infestans and improved tolerance to water stress

Raheel Shahzad*, Putri Widyanti Harlina, Xie Cong-hua, Mohamed Ewas, Elsayed Nishawy, Pan Zhenyuan, Moatzbellah Mohamed Foly

Department of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
Department of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan 430070, China
Department of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
Department of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

Abstract
Potato (Solanum tuberosum) late blight caused by oomycete Phytophthora infestans (Pi) is highly destructive to potato yield and cost huge losses each year. Regulation of a network of transcription machinery, controlled by transcription factors (TF’s), is required to overcome the susceptibility. WRKY TF’s are known to regulate transcription machinery upon biotic and abiotic stresses in different crop plants. We cloned and characterized a WRKY gene, StWRKY1, from potato cDNA synthesized from Pi infested leaves. StWRKY1 protein localized typically in the nucleus. Overexpression (OE) of StWRKY1 positively regulates Pi resistance as well as drought tolerance in transgenic potato. The elevated resistance in OE lines was co-related with higher accumulation of pathogenesis-related (PR) genes as compared to untransformed control plants. Interestingly, increased susceptibility of co-suppression (CSP) plants was associated with down regulation of PR genes expression. Moreover, transgenic lines overexpressing StWRKY1 showed tolerance in terms of less rate of water loss, during dehydration assay. Importantly, expression of StWRKY1 was upregulated upon treatment with plant hormones, suggesting its involvement in basal signal transduction pathway. Overall, our findings provided evidence that StWRKY1 positively regulate biotic and abiotic stress resistance thereby modulating plant basal defense networks, thus play a significant role for crop improvement.

Pages 149-158 | Full Text PDF |
DOI: 10.21475/poj.160902.p7649x
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In silico identification of candidate microRNAs and their targets in potato somatic hybrid Solanum tuberosum (+) S. pinnatisectum for late blight resistance

Ritu Singh, Jagesh Kumar Tiwari*, Shashi Rawat, Vinay Sharma, Bir Pal Singh

ICAR-Central Potato Research Institute, Shimla, Himachal Pradesh - 171 001, India
Department of Bioscience and Biotechnology, Banasthali University, Rajasthan - 304022, India

Abstract
We employed computational tools to identify candidate miRNAs from 153 putative known genes obtained from a previous microarray experiment by Singh et al. (2016) for late blight resistance. In this study, we identified eight miRNAs families (miR395, 821, 1030, 1510, 2673, 3979, 5021 and 5213) to the nine potato genes. Mature miRNAs located at different positions of pre-miRNAs are varied from 19 to 21 nucleotides in length. Of the total 343 published miRNAs in potato (miRBase), all identified miRNAs family are new to potato crop except miR395. In particular, these novel miRNAs were identified for pathogen-induced late blight resistance mechanism in interspecific potato somatic hybrid. Majority of the predicted target genes (Table 2) of these miRNAs are involved in different biological functions, including disease resistance proteins (NBS-LRR domains) and transcription factors families. This study offers an insight to identify potential candidate miRNAs and their targets to dissect late blight resistance mechanism in potato somatic hybrid Solanum tuberosum (+) S. pinnatisectum
.

Pages 159-164 | Full Text PDF | DOI: 10.21475/poj.160902.p7734x
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Isolation, cloning and bioinformatics analysis of ί-amyrin 11-oxidase coding sequence from licorice

Zahra Shirazi, Ali Aalami*, Masoud Tohidfar, Mohammad Mehdi Sohani

Department of Plant Biotechnology, Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran 
Department of Biotechnology, Shahid Beheshti University, Tehran, Iran

Abstract
Licorice roots and stolons have several chemical compounds. Triterpene saponins such as glycyrrhizin and glycyrrhetinic acid and flavonoids like liquiritin, isoliquiritigenin and glabron are main compounds detected in liquorice root. The plant’s major constituent is a glycyrrhizin. The
ί-amyrin 11-oxidase catalyzes the sequential two-step oxidation of ί-amyrin in C-11 to produce 11-oxo-ί-amyrin, a possible biosynthetic intermediate between ί-amyrin and glycyrrhizin. In this study, the total RNA was extracted from licorice roots and cDNA synthesis, then PCR products were cloned into pTZ57R/T vector. Sequencing confirmed piece length of 1482 bp that encodes a protein of 493 amino acid residues. The results of alignment showed 99% similarity to ί- amyrin sequence of Glycyrrhiza uralensis. Subcellular studies using Softberry and Psort software showed that the activity of this protein is in endoplasmic reticulum. Moreover the protein has a signal peptide and is targeted to the secretory pathway. The results of phylogenetic tree determined most similar amino acid sequence to the CYP88D subfamily of cytochrome P450. These findings can be used for nucleotide or protein manipulation and transformation.

Pages 165-171 | Full Text PDF | DOI: 10.21475/poj.160902.p7778x
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Plant Omics | March 2016