Identification and Characterization of PTE-2, a Stowaway-like MITE Activated in Transgenic Chinese Cabbage Lines.
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ABSTRACT:
SUBMITTER: Jeon YJ
PROVIDER: S-EPMC9319602 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundMiniature inverted-repeat transposable elements (MITEs) are expected to play important roles in evolution of genes and genome in plants, especially in the highly duplicated plant genomes. Various MITE families and their roles in plants have been characterized. However, there have been fewer studies of MITE families and their potential roles in evolution of the recently triplicated Brassica genome.ResultsWe identified a new MITE family, BRAMI-1, belonging to the Stowaway super-family in the Brassica genome. In silico mapping revealed that 697 members are dispersed throughout the euchromatic regions of the B. rapa pseudo-chromosomes. Among them, 548 members (78.6%) are located in gene-rich regions, less than 3 kb from genes. In addition, we identified 516 and 15 members in the 470 Mb and 15 Mb genomic shotgun sequences currently available for B. oleracea and B. napus, respectively. The resulting estimated copy numbers for the entire genomes were 1440, 1464 and 2490 in B. rapa, B. oleracea and B. napus, respectively. Concurrently, only 70 members of the related Arabidopsis ATTIRTA-1 MITE family were identified in the Arabidopsis genome. Phylogenetic analysis revealed that BRAMI-1 elements proliferated in the Brassica genus after divergence from the Arabidopsis lineage. MITE insertion polymorphism (MIP) was inspected for 50 BRAMI-1 members, revealing high levels of insertion polymorphism between and within species of Brassica that clarify BRAMI-1 activation periods up to the present. Comparative analysis of the 71 genes harbouring the BRAMI-1 elements with their non-insertion paralogs (NIPs) showed that the BRAMI-1 insertions mainly reside in non-coding sequences and that the expression levels of genes with the elements differ from those of their NIPs.ConclusionA Stowaway family MITE, named as BRAMI-1, was gradually amplified and remained present in over than 1400 copies in each of three Brassica species. Overall, 78% of the members were identified in gene-rich regions, and it is assumed that they may contribute to the evolution of duplicated genes in the highly duplicated Brassica genome. The resulting MIPs can serve as a good source of DNA markers for Brassica crops because the insertions are highly dispersed in the gene-rich euchromatin region and are polymorphic between or within species.
| S-EPMC3626606 | biostudies-literature
Project description:Miniature inverted repeat transposable elements (MITEs) lack protein coding capacity and often share very limited sequence similarity with potential autonomous elements. Their capability of efficient transposition and dramatic amplification led to the proposition that MITEs are an untapped rich source of materials for transposable element (TE) based genetic tools. To test the concept of using MITE sequence in gene transfer, a rice Stowaway MITE previously shown to excise efficiently in yeast was engineered to carry cargo genes (neo and gfp) for delivery into the budding yeast genome. Efficient excision of the cargo gene cassettes was observed even though the excision frequency generally decreases with the increase of the cargo sizes. Excised elements insert into new genomic loci efficiently, with about 65% of the obtained insertion sites located in genes. Elements at the primary insertion sites can be remobilized, frequently resulting in copy number increase of the element. Surprisingly, the orientation of a cargo gene (neo) on a construct bearing dual reporter genes (gfp and neo) was found to have a dramatic effect on transposition frequency. These results demonstrated the concept that MITE sequences can be useful in engineering genetic tools to deliver cargo genes into eukaryotic genomes.
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Project description:Ammonium transporters (AMTs), which include AMT1 and AMT2 subfamilies, have been identified and partially characterized in many plants. In this study, two AMT2-type genes from Brassica campestris, namely BcAMT2 and BcAMT2like, were identified and characterized. BcAMT2 and BcAMT2like are 2666 bp and 2952 bp, encode proteins of 490 and 489 amino acids, respectively, and contain five exons and four introns. Transient expression of these proteins labelled with green fluorescence protein in onion epidermal cells indicated that both are located on the plasma membrane. When expressing BcAMT2 or BcAMT2like, the mutant yeast strain 31019b could grow on medium containing 2 mM ammonium as the only nitrogen source when expressing BcAMT2 or BcAMT2like, indicating that both are functional AMT genes. Quantitative PCR results showed that BcAMT2 and BcAMT2like were expressed in all tissues, but they displayed different expression patterns in the reproductive stages. BcAMT2s transcript levels in leaves were positively correlated with ammonium concentration and external pH. Moreover, the expression BcAMT2s responded to diurnal change. Furthermore, the uncharged form of ammonium, i.e., ammonia, might also be transported by BcAMT2s. These results provide new insights into the molecular mechanisms underlying ammonium absorption and transportation by the AMT2 subfamily in B. campestris.
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Project description:Transgenic plants are usually produced through tissue culture, which is an essential step in Agrobacterium-mediated plant transformation. However, genomic variations, termed somaclonal variations, have been detected in transgenic plants cultured in vitro. The occurrence of these variations should be as low as possible to secure the stability of transgenic crops. Determining the cause and mechanism of somaclonal variations in tissue culture-derived plants will help reduce the rate of variation and promote the stable expression of genes in transgenic plants. In order to determine the genetic variability in transgenic Chinese cabbage plants, we performed whole-genome resequencing and compared the sequencing data with the 'CT001' reference genome. The variation candidates that were expected to consistently occur in the transgenic lines were selected and validated. The single nucleotide polymorphism (SNP) and insertion and deletion (InDel) candidates were identified using the resequencing data and validated by reverse transcription (RT)-PCR analysis. The deduced amino acid sequences were used to determine whether the variations caused changes in the resulting polypeptide, and the annotations of the mutated genes were analyzed to predict the possible effects of the SNPs on gene function. In conclusion, we selected and validated the genetic variations identified in transgenic Chinese cabbage plants. Their genomes were expected to be affected by the process of Agrobacterium-mediated transformation. The findings of our study will provide a genetic basis for transgenic plant research.
| S-EPMC8143544 | biostudies-literature
Project description:Mitochondria are the energy sources of plant cells and are involved in regulating cell development. Ubiquinol-cytochrome c reductase iron-sulfur protein, which is necessary for mitochondrial respiration, is a subunit of mitochondrial electron transport chain multimeric enzyme complexes. To better understand the biological function of the ubiquinol-cytochrome c reductase iron-sulfur protein, the full-length cDNA of BcRISP1 was cloned; it was found to contain 810 base pairs and encode 269 amino acids. Unusually, high expression of the BcRISP1 gene in the archesporial cell stages was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of cytoplasmic male sterile lines and maintainer lines. The seed set was affected by the overexpression of BcRISP1, and shorter siliques with lower seed sets were observed in 35S::BcRISP1 Arabidopsis plants. These characteristics may have resulted from the reduced formation of pollen and impaired pollen tube growth. qRT-PCR results revealed that in 35S::BcRISP1 plants, the expression levels of the mitochondrial respiratory chain-related genes, COX10 and RIP1, were enhanced, whereas the expression levels of QCR7 and SDH2-1 were reduced. This result implies that overexpression of BcRISP1 in transgenic Arabidopsis plants may disrupt the mitochondrial electron transport chain by affecting the expression of mitochondrial respiratory chain-related genes and therefore, reducing the seed set.
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Project description:The S1fa transcription factor is part of a small family involved in plant growth and development and abiotic stress tolerance. However, the roles of the S1fa genes in abiotic stress tolerance in Chinese cabbage are still unclear. In this study, four S1fa genes in the Chinese cabbage genome were identified and characterized for abiotic stress tolerance. Tissue-specific expression analysis suggested that three of these four S1fa genes were expressed in all tissues of Chinese cabbage, while Bra006994 was only expressed in the silique. Under Hg and Cd stresses, the S1fa genes were significantly expressed but were downregulated under NaCl stresses. The Bra034084 and Bra029784 overexpressing yeast cells exhibited high sensitivity to NaCl stresses, which led to slower growth compared with the wild type yeast cells (EV) under 1 M NaCl stress. In addition, the growth curve of the Bra034084 and Bra029784 overexpressing cells shows that the optical density was reduced significantly under salt stresses. The activities of the antioxidant enzymes, SOD, POD and CAT, were decreased, and the MDA, H2O2 and O2- contents were increased under salt stresses. The expression levels of cell wall biosynthesis genes Ccw14p, Cha1p, Cwp2p, Sed1p, Rlm1p, Rom2p, Mkk1p, Hsp12p, Mkk2p, Sdp1p and YLR194c were significantly enhanced, while Bck1p, and Ptc1p were downregulated under salt stresses. These results suggest that the Bra034084 and Bra029784 genes regulate cell wall biosynthesis and the defense regulatory system under salt stresses. These findings provide a fundamental basis for the further investigation of crop genetic modification to improve crop production and abiotic stress tolerance in Chinese cabbage.
| S-EPMC9495863 | biostudies-literature
Project description:Vernalization is a phenomenon in which plants must undergo a period of continuous low temperatures to change from the vegetative growth stage to the reproductive growth stage. Chinese cabbage is a heading vegetable, and flowering time is an essential developmental trait. Premature vernalization leads to premature bolting, which causes a loss of product value and yield. While research into vernalization has provided a wealth of information, a complete understanding of the molecular mechanism for controlling vernalization requirements has not yet been elucidated. In this study, using high-throughput RNA sequencing, we analyzed the plumule-vernalization response of mRNA and long noncoding RNA in the bolting-resistant Chinese cabbage double haploid (DH) line 'Ju Hongxin' (JHX). A total of 3382 lncRNAs were identified, of which 1553 differentially expressed (DE) lncRNAs were characterized as plumule-vernalization responses. The ceRNA network revealed that 280 ceRNA pairs participated in the plumule-vernalization reaction of Chinese cabbage. Through identifying DE lncRNAs in Chinese cabbage and analyzing anti-, cis-, and trans-functional analysis, some candidate lncRNAs related to vernalization promoting flowering of Chinese cabbage and their regulated mRNA genes were found. Moreover, the expression of several critical lncRNAs and their targets was verified using qRT-PCR. Furthermore, we identified the candidate plumule-vernalization-related long noncoding RNAs that regulate BrFLCs in Chinese cabbage, which was interesting and different from previous studies and was a new discovery. Our findings expand the knowledge of lncRNAs in the vernalization of Chinese cabbage, and the identified lncRNAs provide rich resources for future comparative and functional studies.
| S-EPMC10043383 | biostudies-literature
Project description:We analyze the differentially expressed genes (DEGs) in RNA-seq data.
2019-10-25 | GSE139375 | GEO
Project description:Brassica downy mildew, a severe disease caused by Hyaloperonospora brassicae, can cause enormous economic losses in Chinese cabbage (Brassica rapa L. ssp. pekinensis) production. Although some research has been reported recently concerning the underlying resistance to this disease, no studies have identified or characterized long noncoding RNAs involved in this defense response. In this study, using high-throughput RNA sequencing, we analyzed the disease-responding mRNAs and long noncoding RNAs in two resistant lines (T12-19 and 12-85) and one susceptible line (91-112). Clustering and Gene Ontology analysis of differentially expressed genes (DEGs) showed that more DEGs were involved in the defense response in the two resistant lines than in the susceptible line. Different expression patterns and proposed functions of differentially expressed long noncoding RNAs among T12-19, 12-85, and 91-112 indicated that each has a distinct disease response mechanism. There were significantly more cis- and trans-functional long noncoding RNAs in the resistant lines than in the susceptible line, and the genes regulated by these RNAs mostly participated in the disease defense response. Furthermore, we identified a candidate resistance-related long noncoding RNA, MSTRG.19915, which is a long noncoding natural antisense transcript of a MAPK gene, BrMAPK15. Via an agroinfiltration-mediated transient overexpression system and virus-induced gene silencing technology, BrMAPK15 was indicated to have a greater ability to defend against pathogens. MSTRG.19915-silenced seedlings showed enhanced resistance to downy mildew, probably because of the upregulated expression of BrMAPK15. This research identified and characterized long noncoding RNAs involved in resistance to downy mildew, laying a foundation for future in-depth studies of disease resistance mechanisms in Chinese cabbage.
| S-EPMC7917106 | biostudies-literature
Project description:Branching is an important agronomic trait determining plant architecture and yield; however, the molecular mechanisms underlying branching in the stalk vegetable, flowering Chinese cabbage, remain unclear. The present study identified two tandem genes responsible for primary rosette branching in flowering Chinese cabbage by GradedPool-Seq (GPS) combined with Kompetitive Allele Specific PCR (KASP) genotyping. A 900 kb candidate region was mapped in the 28.0-28.9 Mb interval of chromosome A07 through whole-genome sequencing of three graded-pool samples from the F2 population derived by crossing the branching and non-branching lines. KASP genotyping narrowed the candidate region to 24.6 kb. Two tandem genes, BraA07g041560.3C and BraA07g041570.3C, homologous to AT1G78440 encoding GA2ox1 oxidase, were identified as the candidate genes. The BraA07g041560.3C sequence was identical between the branching and non-branching lines, but BraA07g041570.3C had a synonymous single nucleotide polymorphic (SNP) mutation in the first exon (290th bp, A to G). In addition, an ERE cis-regulatory element was absent in the promoter of BraA07g041560.3C, and an MYB cis-regulatory element in the promoter of BraA07g041570.3C in the branching line. Gibberellic acid (GA3) treatment decreased the primary rosette branch number in the branching line, indicating the significant role of GA in regulating branching in flowering Chinese cabbage. These results provide valuable information for revealing the regulatory mechanisms of branching and contributing to the breeding programs of developing high-yielding species in flowering Chinese cabbage.
| S-EPMC9806259 | biostudies-literature