Dataset Information

Jasmonate-Sensitivity-Assisted Screening and Characterization of Nicotine Synthetic Mutants from Activation-Tagged Population of Tobacco (Nicotiana tabacum L.).

ABSTRACT: Nicotine is a secondary metabolite that is important to the defense system and commercial quality of tobacco (Nicotiana tabacum L.). Jasmonate and its derivatives (JAs) are phytohormone regulators of nicotine formation; however, the underlying molecular mechanism of this process remains largely unclear. Owing to the amphitetraploid origin of N. tabacum, research on screening and identification of nicotine-synthetic mutants is relatively scarce. Here, we describe a method based on JA-sensitivity for screening nicotine mutants from an activation-tagged population of tobacco. In this approach, the mutants were first screened for abnormal JA responses in seed germination and root elongation, and then the levels of nicotine synthesis and expression of nicotine synthetic genes in the mutants with altered JA-response were measured to determine the nicotine-synthetic mutants. We successfully obtained five mutants that maintained stable nicotine contents and JA responses for three generations. This method is simple, effective and low-cost, and the finding of transcriptional changes of nicotine synthetic genes in the mutants shows potentials for identifying novel regulators involved in JA-regulated nicotine biosynthesis.

SUBMITTER: Yin G

PROVIDER: S-EPMC5303748 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Jasmonate-Sensitivity-Assisted Screening and Characterization of Nicotine Synthetic Mutants from Activation-Tagged Population of Tobacco (Nicotiana tabacum L.).

Yin Guoying G Wang Wenjing W Niu Haixia H Ding Yongqiang Y Zhang Dingyu D Zhang Jie J Liu Guanshan G Wang Sangen S Zhang Hongbo H

Frontiers in plant science 20170213

Nicotine is a secondary metabolite that is important to the defense system and commercial quality of tobacco (Nicotiana tabacum L.). Jasmonate and its derivatives (JAs) are phytohormone regulators of nicotine formation; however, the underlying molecular mechanism of this process remains largely unclear. Owing to the amphitetraploid origin of N. tabacum, research on screening and identification of nicotine-synthetic mutants is relatively scarce. Here, we describe a method based on J ...[more]

PMID: 28243248

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Project description:BackgroundPollen tube growth is essential for plant reproduction and represents a widely employed model to investigate polarized cell expansion, a process important for plant morphogenesis and development. Cellular and regulatory mechanisms underlying pollen tube elongation are under intense investigation, which stands to greatly benefit from a comprehensive understanding of global gene expression profiles in pollen and pollen tubes. Here, RNA sequencing technology was applied to de novo assemble a Nicotiana tabacum male gametophytic transcriptome and to compare transcriptome profiles at two different stages of gametophyte development: mature pollen grains (MPG) and pollen tubes grown for six hours in vitro (PT6).ResultsDe novo assembly of data obtained by 454 sequencing of a normalized cDNA library representing tobacco pollen and pollen tube mRNA (pooled mRNA isolated from mature pollen grains [MPG] and from pollen tubes grown in vitro for 3 [PT3] or 6 [PT6] hours) resulted in the identification of 78,364 unigenes. Among these unigenes, which mapped to 24,933 entries in the Sol Genomics Network (SGN) N. tabacum unigene database, 24,672 were predicted to represent full length cDNAs. In addition, quantitative analyses of data obtained by Illumina sequencing of two separate non-normalized MPG and PT6 cDNA libraries showed that 8979 unigenes were differentially expressed (differentially expressed unigenes: DEGs) between these two developmental stages at a FDR q-value of <0.0001. Interestingly, whereas most of these DEGs were downregulated in PT6, the minor fraction of DEGs upregulated in PT6 was enriched for GO (gene ontology) functions in pollen tube growth or fertilization.ConclusionsA major output of our study is the development of two different high-quality databases representing the tobacco male gametophytic transcriptome and containing encompassing information about global changes in gene expression after pollen germination. Quantitative analyses of these databases 1) indicated that roughly 30% of all tobacco genes are expressed in the male gametophyte, and 2) support previous observations suggesting a global reduction of transcription after pollen germination. Interestingly, a small number of genes, many of which predicted to function in pollen tube growth or fertilization, were found to be upregulated in elongating pollen tubes despite globally reduced transcription.

Project description:Plant molecular farming (PMF) is an important growing prospective approach in plant biotechnology; it includes production of recombinant pharmaceutical and industrial proteins in large quantities from engineered plants. Elastin is a major protein component of tissues that require elasticity, it helps keep skin smooth as it stretches to allow normal. Elastin is used as a raw material for the cosmetic industry. In this work, we aimed to use plant as a bioreactor for the expression and production of the full human tropoelastin protein. Agrobacterium- mediated transient expression system into Nicotiana tabacum using syringe agroinfiltration was used to provide fast and convenient way to produce recombinant proteins with greater expression overall the plant leaf. This study aimed to establish an efficient and rapid system for transiently expression and production of human recombinant tropoelastin protein in transgenic N. tabacum plants. Modified elastin (ELN) gene was biosynthesized and cloned into pCambia1390 vector to be used into N. tabacum agroinfilteration. Optimization of codon usage for the human tropoelastin gene, without changing the primary structure of the protein was carried out to ensure high expression in tobacco plants. The obtained data proved that the 5(th) day post-infiltration is the optimum interval to obtain the maximum production of our recombinant protein. Southern blot analysis was able to detect 2175 bp fragment length representing the ELN orf (open reding frame). On the other hand, ELN -expression within plant's tissue was visualized by RT-PCR during the period 3-10 days post agroinfiltration. At the protein level, western and ELISA confirmed the expression of recombinant tropoelastin protein. Western blot analysis detected the tropoelastin protein as parent band at ?70 kDa from freshly extracted protein, while two degraded bands of ?55 and ?45 kDa, representing a pattern of tropoelastin were appeared with frozen samples. This study showed that biosynthetic ELN gene was successfully expressed into N. tabacum leaves using agroinfiltration technique.

Dataset Information

Jasmonate-Sensitivity-Assisted Screening and Characterization of Nicotine Synthetic Mutants from Activation-Tagged Population of Tobacco (Nicotiana tabacum L.).

Publications

Jasmonate-Sensitivity-Assisted Screening and Characterization of Nicotine Synthetic Mutants from Activation-Tagged Population of Tobacco (<i>Nicotiana tabacum</i> L.).

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