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      Parsley ubiquitin promoter displays higher activity than the CaMV 35S promoter and the chrysanthemum actin 2 promoter for productive, constitutive, and durable expression of a transgene in Chrysanthemum morifolium

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          Abstract

          The chrysanthemum ( Chrysanthemum morifolium) is one of the most popular ornamental plants in the world. Genetic transformation is a promising tool for improving traits, editing genomes, and studying plant physiology. Promoters are vital components for efficient transformation, determining the level, location, and timing of transgene expression. The cauliflower mosaic virus (CaMV) 35S promoter is most frequently used in dicotyledonous plants but is less efficient in chrysanthemums than in tobacco or torenia plants. Previously, we used the parsley ubiquitin ( PcUbi) promoter in chrysanthemums for the first time and analyzed its activity in transgenic calli. To expand the variety of constitutive promoters in chrysanthemums, we cloned the upstream region of the actin 2 ( CmACT2) gene and compared its promoter activity with the 35S and PcUbi promoters in several organs, as well as its durability for long-term cultivation. The CmACT2 promoter has higher activity than the 35S promoter in calli but is less durable. The PcUbi promoter has the highest activity not only in calli but also in leaves, ray florets, and disk florets, and retains its activity after long-term cultivation. In conclusion, we have provided useful information and an additional type of promoter available for transgene expression in chrysanthemums.

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          Most cited references27

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          Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation.

          Two genomic clones (lambda Ubi-1 and lambda Ubi-2) encoding the highly conserved 76 amino acid protein ubiquitin have been isolated from maize. Sequence analysis shows that both genes contain seven contiguous direct repeats of the protein coding region in a polyprotein conformation. The deduced amino acid sequence of all 14 repeats is identical and is the same as for other plant ubiquitins. The use of transcript-specific oligonucleotide probes shows that Ubi-1 and Ubi-2 are expressed constitutively at 25 degrees C but are inducible to higher levels at elevated temperatures in maize seedlings. Both genes contain an intron in the 5' untranslated region which is inefficiently processed following a brief, severe heat shock. The transcription start site of Ubi-1 has been determined and a transcriptional fusion of 0.9 kb of the 5' flanking region and the entire 5' untranslated sequence of Ubi-1 with the coding sequence of the gene encoding the reporter molecule chloramphenicol acetyl transferase (CAT) has been constructed (pUBI-CAT). CAT assays of extracts of protoplasts electroporated with this construct show that the ubiquitin gene fragment confers a high level of CAT expression in maize and other monocot protoplasts but not in protoplasts of the dicot tobacco. Expression from the Ubi-1 promoter of pUBI-CAT yields more than a 10-fold higher level of CAT activity in maize protoplasts than expression from the widely used cauliflower mosaic virus 35S promoter of a 35S-CAT construct. Conversely, in tobacco protoplasts CAT activity from transcription of pUBI-CAT is less than one tenth of the level from p35S-CAT.
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            The CRISPR-Cas system for plant genome editing: advances and opportunities.

            Genome editing is an approach in which a specific target DNA sequence of the genome is altered by adding, removing, or replacing DNA bases. Artificially engineered hybrid enzymes, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), and the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) system are being used for genome editing in various organisms including plants. The CRISPR-Cas system has been developed most recently and seems to be more efficient and less time-consuming compared with ZFNs or TALENs. This system employs an RNA-guided nuclease, Cas9, to induce double-strand breaks. The Cas9-mediated breaks are repaired by cellular DNA repair mechanisms and mediate gene/genome modifications. Here, we provide a detailed overview of the CRISPR-Cas system and its adoption in different organisms, especially plants, for various applications. Important considerations and future opportunities for deployment of the CRISPR-Cas system in plants for numerous applications are also discussed. Recent investigations have revealed the implications of the CRISPR-Cas system as a promising tool for targeted genetic modifications in plants. This technology is likely to be more commonly adopted in plant functional genomics studies and crop improvement in the near future.
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              The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression.

              We have isolated and determined DNA sequence for the 5'-flanking regions of three Arabidopsis thaliana polyubiquitin genes, UBQ3, UBQ10, and UBQ11. Comparison to cDNA sequences revealed the presence of an intron in the 5'-untranslated region at the same position immediately upstream of the initiator methionine codon in each of the three genes. An intron at this position is also present in two sunflower and two maize polyubiquitin genes. An intron is also found in the 5'-untranslated regions of several animal polyubiquitin genes, although the exact intron position is not conserved among them, and none are in the same position as those in the higher plant polyubiquitin genes. Chimeric genes containing the 5'-flanking regions of UBQ3, UBQ10, and UBQ11 in front of the coding regions for the reporter enzyme Escherichia coli beta-glucuronidase (GUS) were constructed. When introduced transiently into Arabidopsis leaves via microprojectile bombardment, all resulted in readily detectable levels of GUS activity that were quantitatively similar. The introns of UBQ3 and UBQ10 in the corresponding promoter fragments were removed by replacement with flanking cDNA sequences and chimeric genes constructed. These constructs resulted in 2.5- to 3-fold lower levels of marker enzyme activity after transient introduction into Arabidopsis leaves. The UBQ10 promoter without the 5' intron placed upstream of firefly luciferase (LUX) resulted in an average of 3-fold lower LUX activity than from an equivalent construct with the UBQ10 intron. A UBQ3 promoter cassette was constructed for the constitutive expression of open reading frames in dicot plants and it produced readily detectable levels of GUS activity in transient assays.
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                Author and article information

                Journal
                Breed Sci
                Breed. Sci
                Breeding Science
                Japanese Society of Breeding
                1344-7610
                1347-3735
                September 2019
                23 July 2019
                : 69
                : 3
                : 536-544
                Affiliations
                Institute of Vegetable and Floriculture Science, National Agriculture and Food Research Organization (NARO) , Fujimoto 2-1, Tsukuba, Ibaraki 305-0852, Japan
                Author notes
                [* ]Corresponding author (e-mail: kattu@ 123456affrc.go.jp )

                Communicated by Hiroshi Ezura

                Article
                69_19036
                10.1270/jsbbs.19036
                6776152
                31598089
                881908bf-573c-4047-8957-585afcb29a50
                Copyright © 2019 by JAPANESE SOCIETY OF BREEDING

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 11 March 2019
                : 04 June 2019
                Categories
                Note

                Animal agriculture
                chrysanthemum morifolium,gene expression,promoter activity,parsley
                Animal agriculture
                chrysanthemum morifolium, gene expression, promoter activity, parsley

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