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(E)-?-farnesene gene reduces Lipaphis erysimi colonization in transgenic Brassica juncea lines.

ABSTRACT: Aphids are the major concern that significantly reduces the yield of crops. (E)-?-farnesene (E?f) is the principal component of the alarm pheromone of many aphids. The results of current research support the direct defense response of (E)-?-farnesene (E?f) against aphid Lipaphis erysimi (L.) Kaltenbach in Brassica juncea. E?f gene was isolated from Mentha arvensis and transformed into B. juncea, showed direct repellent against aphid colonization. The seasonal mean population (SMP) recorded under field condition showed significantly higher aphid colonization in wild type in comparison to most of the transgenic lines, and shows positive correlation with the repellency of transgenic plant expressing (E)-?-farnesene. The current research investigation provides direct evidence for aphid control in B. juncea using E?f, a non-toxic mode of action.

SUBMITTER: Verma SS

PROVIDER: S-EPMC4623259 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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(E)-β-farnesene gene reduces Lipaphis erysimi colonization in transgenic Brassica juncea lines.

Verma Shiv Shankar SS Sinha Rakesh Kumar RK Jajoo Anajna A

Plant signaling & behavior 20150101 7

Aphids are the major concern that significantly reduces the yield of crops. (E)-β-farnesene (Eβf) is the principal component of the alarm pheromone of many aphids. The results of current research support the direct defense response of (E)-β-farnesene (Eβf) against aphid Lipaphis erysimi (L.) Kaltenbach in Brassica juncea. Eβf gene was isolated from Mentha arvensis and transformed into B. juncea, showed direct repellent against aphid colonization. The seasonal mean population (SMP) recorded under ...[more]

PMID: 26251882

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Lipaphis erysimi

Project description:Lipaphis erysimi Raw sequence reads

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Functional characterization of Rorippa indica defensin and its efficacy against Lipaphis erysimi.

Project description:Rorippa indica, a wild crucifer, has been previously reported as the first identified plant in the germplasm of Brassicaceae known to be tolerant towards the mustard aphid Lipaphis erysimi Kaltenbach. We herein report the full-length cloning, expression, purification and characterization of a novel R. indica defensin (RiD) and its efficacy against L. erysimi. Structural analysis through homology modeling of RiD showed longer ?-helix and 3rd ?-sheet as compared to Brassica juncea defensin (BjD). Recombinant RiD and BjD was purified for studying its efficacy against L. erysimi. In the artificial diet based insect bioassay, the LC50 value of RiD against L. erysimi was found to be 9.099 ± 0.621 µg/mL which is far lower than that of BjD (43.51 ± 0.526 µg/mL). This indicates the possibility of RiD having different interacting partner and having better efficacy against L. erysimi over BjD. In the transient localization studies, RiD signal peptide directed the RiD: yellow fluorescent protein (YFP) fusion protein to the apoplastic regions which indicates that it might play a very important role in inhibiting nutrient uptake by aphids which follow mainly extracellular route to pierce through the cells. Hence, the present study has a significant implication for the future pest management program of B. juncea through the development of aphid tolerant transgenic plants.

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Development of transgenic Brassica juncea lines for reduced seed sinapine content by perturbing phenylpropanoid pathway genes.

Project description:Sinapine is a major anti-nutritive compound that accumulates in the seeds of Brassica species. When ingested, sinapine imparts gritty flavuor in meat and milk of animals and fishy odor to eggs of brown egg layers, thereby compromising the potential use of the valuable protein rich seed meal. Sinapine content in Brassica juncea germplasm ranges from 6.7 to 15.1 mg/g of dry seed weight (DSW) which is significantly higher than the prescribed permissible level of 3.0 mg/g of DSW. Due to limited natural genetic variability, conventional plant breeding approach for reducing the sinapine content has largely been unsuccessful. Hence, transgenic approach for gene silencing was adopted by targeting two genes-SGT and SCT, encoding enzymes UDP- glucose: sinapate glucosyltransferase and sinapoylglucose: choline sinapoyltransferase, respectively, involved in the final two steps of sinapine biosynthetic pathway. These two genes were isolated from B. juncea and eight silencing constructs were developed using three different RNA silencing approaches viz. antisense RNA, RNAi and artificial microRNA. Transgenics in B. juncea were developed following Agrobacterium-mediated transformation. From a total of 1232 independent T0 transgenic events obtained using eight silencing constructs, 25 homozygous lines showing single gene inheritance were identified in the T2 generation. Reduction of seed sinapine content in these lines ranged from 15.8% to 67.2%; the line with maximum reduction had sinapine content of 3.79 mg/g of DSW. The study also revealed that RNAi method was more efficient than the other two methods used in this study.

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A Novel Insecticidal Peptide SLP1 Produced by Streptomyces laindensis H008 against Lipaphis erysimi.

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Expression of an endochitinase gene from Trichoderma virens confers enhanced tolerance to Alternaria blight in transgenic Brassica juncea (L.) czern and coss lines.

Project description:An endochitinase gene 'ech42' from the biocontrol fungus 'Trichoderma virens' was introduced to Brassica juncea (L). Czern and Coss via Agrobaterium tumefaciens mediated genetic transformation method. Integration and expression of the 'ech42' gene in transgenic lines were confirmed by PCR, RT-PCR and Southern hybridization. Transgenic lines (T1) showed expected 3:1 Mendelian segregation ratio when segregation analysis for inheritance of transgene 'hpt' was carried out. Fluorimetric analysis of transgenic lines (T0 and T1) showed 7 fold higher endochitinase activity than the non-transformed plant. Fluorimetric zymogram showed presence of endochitinase (42 kDa) in crude protein extract of transgenic lines. In detached leaf bioassay with fungi Alternaria brassicae and Alternaria brassicicola, transgenic lines (T0 and T1) showed delayed onset of lesions as well as 30-73 % reduction in infected leaf area compared to non-transformed plant.

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Comprehensive evaluation of candidate reference genes for qRT-PCR studies of gene expression in mustard aphid, Lipaphis erysimi (Kalt).

Project description:Mustard aphid, also known as turnip aphid (Lipaphis erysimi) is a major insect pest of rapeseed-mustard group of crops. Tremendous economic significance has led to substantial basic research involving gene-expression studies in this insect species. In qRT-PCR analysis of gene-expression, normalization of data against RNA variation by using appropriate reference gene is fundamental. However, appropriate reference genes are not known in case of L. erysimi. We evaluated 11 candidate reference genes for their expression stability in 21 samples of L. erysimi subjected to various regimes of experimental treatments. Unlike other studies, we validated true effects of the treatments on the samples either by gene-expression study of an associated marker gene or by biochemical tests. In the validated samples, expression stability of the reference genes was analysed by employing four different statistical softwares geNorm, NormFinder, BestKeeper and deltaCt. Drawing consensus on the results from different softwares, we recommend three best reference genes 16S, RPS18 and RPL13 for normalization of qRT-PCR data in L. erysimi. This study provides for the first time a comprehensive list of suitable reference genes for mustard aphid and demonstrates the advantage of using more than one reference gene in combination for certain experimental conditions.

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Identification of genes involved in wild crucifer Rorippa indica resistance response on mustard aphid Lipaphis erysimi challenge.

Project description:Mustard aphid, Lipaphis erysimi (L.) Kaltenbach is a perpetual annual threat in the cultivation of rapeseed- mustard (Brassica spp.) crop in tropical and sub-tropical climate. Cultivated Brassica germplasm has failed so far to provide any source of resistance. Wild germplasm is a potential source of resistance against many threatening herbivores. On wild germplasm screening, we noted that the wild crucifer Rorippa indica (L.) Hiern confers resistance against L. erysimi. In the present study L. erysimi challenged transcriptome of R. indica was compared to un-infested R. indica sample to get a molecular insight about the aphid resistance mechanism and identify the candidate defense response genes. Cloning, sequencing and in silico sequence analysis of complimentary DNA amplified fragment length polymorphism identified 116 differentially expressed transcript derived fragments revealed thirty candidates which are from different functional categories including redox regulation, signalling, photosynthesis, structure, metabolism, defense response as well as a few of unknown function. Twenty four identifications were then studied by quantitative real time RT PCR analysis at 6, 12, 24 and 48 hour time point post infestation to understand the early-to-late defense response through their relative gene expression profiles. Seventeen fragments showed significant up or down regulation at p<0.05 level. The response was influenced by different phytohormonal signalling pathways simultaneously. The candidate defense response expressed sequence tags specifically for the resistance genes identified in this study have implication in building desired mustard aphid resistance in susceptible rapeseed-mustard plants in future. This is the first molecular report on crucifer defense response against mustard aphid L. erysimi.

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Mitochondrial genome and transcriptome analysis of five alloplasmic male-sterile lines in Brassica juncea.

Project description:BackgroundAlloplasmic lines, in which the nuclear genome is combined with wild cytoplasm, are often characterized by cytoplasmic male sterility (CMS), regardless of whether it was derived from sexual or somatic hybridization with wild relatives. In this study, we sequenced and analyzed the mitochondrial genomes of five such alloplasmic lines in Brassica juncea.ResultsThe assembled and annotated mitochondrial genomes of the five alloplasmic lines were found to have virtually identical gene contents. They preserved most of the ancestral mitochondrial segments, and the same candidate male sterility gene (orf108) was found harbored in mitotype-specific sequences. We also detected promiscuous sequences of chloroplast origin that were conserved among plants of the Brassicaceae, and found the RNA editing profiles to vary across the five mitochondrial genomes.ConclusionsOn the basis of our characterization of the genetic nature of five alloplasmic mitochondrial genomes, we speculated that the putative candidate male sterility gene orf108 may not be responsible for the CMS observed in Brassica oxyrrhina and Diplotaxis catholica. Furthermore, we propose the potential coincidence of CMS in alloplasmic lines. Our findings lay the foundation for further elucidation of male sterility gene.

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A proteomic analysis of seeds from Bt-transgenic Brassica napus and hybrids with wild B. juncea.

Project description:Transgene insertions might have unintended side effects on the transgenic host, both crop and hybrids with wild relatives that harbor transgenes. We employed proteomic approaches to assess protein abundance changes in seeds from Bt-transgenic oilseed rape (Brassica napus) and its hybrids with wild mustard (B. juncea). A total of 24, 15 and 34 protein spots matching to 23, 13 and 31 unique genes were identified that changed at least 1.5 fold (p < 0.05, Student's t-test) in abundance between transgenic (tBN) and non-transgenic (BN) oilseed rape, between hybrids of B. juncea (BJ) × tBN (BJtBN) and BJ × BN (BJBN) and between BJBN and BJ, respectively. Eight proteins had higher abundance in tBN than in BN. None of these proteins was toxic or nutritionally harmful to human health, which is not surprising since the seeds are not known to produce toxic proteins. Protein spots varying in abundance between BJtBN and BJBN seeds were the same or homologous to those in the respective parents. None of the differentially-accumulated proteins between BJtBN and BJBN were identical to those between tBN and BN. Results indicated that unintended effects resulted from transgene flow fell within the range of natural variability of hybridization and those found in the native host proteomes.

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<i>Brassica juncea</i> Lines with Substituted Chimeric GFP-CENH3 Give Haploid and Aneuploid Progenies on Crossing with Other Lines.

Project description:Haploids and doubled haploids are invaluable for basic genetic studies and in crop improvement. A novel method of haploid induction through genetic engineering of the Centromere Histone Protein gene, CENH3, has been demonstrated in Arabidopsis. The present study was undertaken to develop haploid inducer (HI) lines of Brassica juncea based on the principles elaborated in Arabidopsis. B. juncea was found to carry three copies of CENH3 which generated five different transcripts, of which three transcripts resulted from alternative splicing. Unlike Arabidopsis thaliana where native CENH3 gene was knocked out for constructing HI lines, we used RNAi approach to knockdown the native CENH3 genes. Further, to rescue CENH3 silenced cells, a GFP-CENH3-tailswap construct having N terminal GFP fused to H3.3 tail sequences and synthetic CENH3 histone fold domain sequences was devised. A total 38 transgenic B. juncea plants were regenerated following co-transformation with both silencing and rescue cassettes and transgenics carrying either or both the constructs were obtained. Transgenic status was confirmed through PCR, Southern and qRT-PCR analyses. Co-transformed lines were crossed to untransformed B. juncea or a line expressing only GFP-tailswap. FACS and cytological analyses of progenies revealed partial or complete elimination of B. juncea chromosomes thereby giving rise to aneuploids and haploid. This is the first report in a polyploid crop demonstrating that CENH3 engineering could be used to develop HI lines.

| S-EPMC5216839 | biostudies-literature

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