Stable transformation ofphaC2 gene in tobacco chloroplast genome

2006 ◽  
Vol 3 (3) ◽  
pp. 201-207
Author(s):  
Wang Yu-Hua ◽  
Wu Zhong-Yi ◽  
Zhang Xiu-Hai ◽  
Wang Yong-Qin ◽  
Huang Cong-Lin ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Chloroplast Genome ◽  
Marker Gene ◽  
Chloroplast Transformation ◽  
Single Copy ◽  
Stable Transformation ◽  
Pha Synthase ◽  
Type Ii ◽  
Tobacco Chloroplast ◽  
Chloroplast Genomes

AbstractMedium-chain-length polyhydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The key enzyme for mcl-PHA biosynthesis is type II PHA synthase. The genephaC2 encoding type II PHA synthase was placed under the control ofpsbA-pro andpsbA-ter of rice (Oryza sativa) to construct aphaC2 cassette, which was ligated with the screening marker geneaadAcassette (prrn–aadA–TpsbA-ter). These recombined fragments were cloned between the plastidrbcLandaccDgenes for targeting to the large single copy region of the chloroplast genome. A chloroplast transformation vector, pTC2, was constructed and introduced into the tobacco (Nicotiana tobacum) chloroplast genome by particle bombardment. PCR and Southern blot analysis confirmed stable integration ofphaC2 into the chloroplast genomes of T0and T1transgenic plants, and T1transgenic plants exhibited homoplasmy. The expression ofphaC2 at transcription level was detected by reverse transcriptase–polymerase chain reaction (RT-PCR). Recombinant transgenes in the tobacco chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. To our knowledge, this is the first report on the stable transformation ofphaC2 encoding type II PHA synthase in tobacco via chloroplast genetic engineering.

scholarly journals A Circular Chloroplast Genome of Fagus sylvatica Reveals High Conservation between Two Individuals from Germany and One Individual from Poland and an Alternate Direction of the Small Single-Copy Region

Forests ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 180
Author(s):  
Bagdevi Mishra ◽  
Bartosz Ulaszewski ◽  
Sebastian Ploch ◽  
Jaroslaw Burczyk ◽  
Marco Thines
Keyword(s):  
Chloroplast Genome ◽  
Fagus Sylvatica ◽  
Pollen Dispersal ◽  
European Beech ◽  
Single Copy ◽  
Migration History ◽  
Chloroplast Genomes ◽  
Long Read ◽  
The Individual ◽  
Small Single Copy

Chloroplasts are difficult to assemble because of the presence of large inverted repeats. At the same time, correct assemblies are important, as chloroplast loci are frequently used for biogeography and population genetics studies. In an attempt to elucidate the orientation of the single-copy regions and to find suitable loci for chloroplast single nucleotide polymorphism (SNP)-based studies, circular chloroplast sequences for the ultra-centenary reference individual of European Beech (Fagus sylvatica), Bhaga, and an additional Polish individual (named Jamy) was obtained based on hybrid assemblies. The chloroplast genome of Bhaga was 158,458 bp, and that of Jamy was 158,462 bp long. Using long-read mapping on the configuration inferred in this study and the one suggested in a previous study, we found an inverted orientation of the small single-copy region. The chloroplast genome of Bhaga and of the individual from Poland both have only two mismatches as well as three and two indels as compared to the previously published genome, respectively. The low divergence suggests low seed dispersal but high pollen dispersal. However, once chloroplast genomes become available from Pleistocene refugia, where a high degree of variation has been reported, they might prove useful for tracing the migration history of Fagus sylvatica in the Holocene.

scholarly journals A systematic comparison of eight new plastome sequences from Ipomoea L

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6563
Author(s):  
Jianying Sun ◽  
Xiaofeng Dong ◽  
Qinghe Cao ◽  
Tao Xu ◽  
Mingku Zhu ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Next Generation Sequencing ◽  
Chloroplast Genome ◽  
Dna Sequences ◽  
Repetitive Sequences ◽  
Single Copy ◽  
Next Generation ◽  
Variable Regions ◽  
Chloroplast Genomes ◽  
Generation Sequencing

Background Ipomoea is the largest genus in the family Convolvulaceae. The species in this genus have been widely used in many fields, such as agriculture, nutrition, and medicine. With the development of next-generation sequencing, more than 50 chloroplast genomes of Ipomoea species have been sequenced. However, the repeats and divergence regions in Ipomoea have not been well investigated. In the present study, we sequenced and assembled eight chloroplast genomes from sweet potato’s close wild relatives. By combining these with 32 published chloroplast genomes, we conducted a detailed comparative analysis of a broad range of Ipomoea species. Methods Eight chloroplast genomes were assembled using short DNA sequences generated by next-generation sequencing technology. By combining these chloroplast genomes with 32 other published Ipomoea chloroplast genomes downloaded from GenBank and the Oxford Research Archive, we conducted a comparative analysis of the repeat sequences and divergence regions across the Ipomoea genus. In addition, separate analyses of the Batatas group and Quamoclit group were also performed. Results The eight newly sequenced chloroplast genomes ranged from 161,225 to 161,721 bp in length and displayed the typical circular quadripartite structure, consisting of a pair of inverted repeat (IR) regions (30,798–30,910 bp each) separated by a large single copy (LSC) region (87,575–88,004 bp) and a small single copy (SSC) region (12,018–12,051 bp). The average guanine-cytosine (GC) content was approximately 40.5% in the IR region, 36.1% in the LSC region, 32.2% in the SSC regions, and 37.5% in complete sequence for all the generated plastomes. The eight chloroplast genome sequences from this study included 80 protein-coding genes, four rRNAs (rrn23, rrn16, rrn5, and rrn4.5), and 37 tRNAs. The boundaries of single copy regions and IR regions were highly conserved in the eight chloroplast genomes. In Ipomoea, 57–89 pairs of repetitive sequences and 39–64 simple sequence repeats were found. By conducting a sliding window analysis, we found six relatively high variable regions (ndhA intron, ndhH-ndhF, ndhF-rpl32, rpl32-trnL, rps16-trnQ, and ndhF) in the Ipomoea genus, eight (trnG, rpl32-trnL, ndhA intron, ndhF-rpl32, ndhH-ndhF, ccsA-ndhD, trnG-trnR, and pasA-ycf3) in the Batatas group, and eight (ndhA intron, petN-psbM, rpl32-trnL, trnG-trnR, trnK-rps16, ndhC-trnV, rps16-trnQ, and trnG) in the Quamoclit group. Our maximum-likelihood tree based on whole chloroplast genomes confirmed the phylogenetic topology reported in previous studies. Conclusions The chloroplast genome sequence and structure were highly conserved in the eight newly-sequenced Ipomoea species. Our comparative analysis included a broad range of Ipomoea chloroplast genomes, providing valuable information for Ipomoea species identification and enhancing the understanding of Ipomoea genetic resources.

scholarly journals The complete chloroplast genome of Saxifraga sinomontana (Saxifragaceae) and comparative analysis with other Saxifragaceae species

2019 ◽  
Vol 42 (4) ◽  
pp. 601-611 ◽  
Author(s):  
Yan Li ◽  
Liukun Jia ◽  
Zhihua Wang ◽  
Rui Xing ◽  
Xiaofeng Chi ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Chloroplast Genome ◽  
Phylogenetic Relationships ◽  
De Novo ◽  
Single Copy ◽  
Bootstrap Support ◽  
Protein Coding ◽  
Complete Chloroplast Genome ◽  
Protein Coding Genes ◽  
Chloroplast Genomes

Abstract Saxifraga sinomontana J.-T. Pan & Gornall belongs to Saxifraga sect. Ciliatae subsect. Hirculoideae, a lineage containing ca. 110 species whose phylogenetic relationships are largely unresolved due to recent rapid radiations. Analyses of complete chloroplast genomes have the potential to significantly improve the resolution of phylogenetic relationships in this young plant lineage. The complete chloroplast genome of S. sinomontana was de novo sequenced, assembled and then compared with that of other six Saxifragaceae species. The S. sinomontana chloroplast genome is 147,240 bp in length with a typical quadripartite structure, including a large single-copy region of 79,310 bp and a small single-copy region of 16,874 bp separated by a pair of inverted repeats (IRs) of 25,528 bp each. The chloroplast genome contains 113 unique genes, including 79 protein-coding genes, four rRNAs and 30 tRNAs, with 18 duplicates in the IRs. The gene content and organization are similar to other Saxifragaceae chloroplast genomes. Sixty-one simple sequence repeats were identified in the S. sinomontana chloroplast genome, mostly represented by mononucleotide repeats of polyadenine or polythymine. Comparative analysis revealed 12 highly divergent regions in the intergenic spacers, as well as coding genes of matK, ndhK, accD, cemA, rpoA, rps19, ndhF, ccsA, ndhD and ycf1. Phylogenetic reconstruction of seven Saxifragaceae species based on 66 protein-coding genes received high bootstrap support values for nearly all identified nodes, suggesting a promising opportunity to resolve infrasectional relationships of the most species-rich section Ciliatae of Saxifraga.

scholarly journals Complete Chloroplast Genome of Paphiopedilum delenatii and Phylogenetic Relationships among Orchidaceae

Plants ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 61 ◽  
Author(s):  
Huyen-Trang Vu ◽  
Ngan Tran ◽  
Thanh-Diem Nguyen ◽  
Quoc-Luan Vu ◽  
My-Huyen Bui ◽  
...  
Keyword(s):  
Chloroplast Genome ◽  
Inverted Repeat ◽  
Gc Content ◽  
Single Copy ◽  
Rrna Genes ◽  
Trna Genes ◽  
Complete Chloroplast Genome ◽  
Critically Endangered Species ◽  
Plastid Genomes ◽  
Chloroplast Genomes

Paphiopedilum delenatii is a native orchid of Vietnam with highly attractive floral traits. Unfortunately, it is now listed as a critically endangered species with a few hundred individuals remaining in nature. In this study, we performed next-generation sequencing of P. delenatii and assembled its complete chloroplast genome. The whole chloroplast genome of P. delenatii was 160,955 bp in size, 35.6% of which was GC content, and exhibited typical quadripartite structure of plastid genomes with four distinct regions, including the large and small single-copy regions and a pair of inverted repeat regions. There were, in total, 130 genes annotated in the genome: 77 coding genes, 39 tRNA genes, 8 rRNA genes, and 6 pseudogenes. The loss of ndh genes and variation in inverted repeat (IR) boundaries as well as data of simple sequence repeats (SSRs) and divergent hotspots provided useful information for identification applications and phylogenetic studies of Paphiopedilum species. Whole chloroplast genomes could be used as an effective super barcode for species identification or for developing other identification markers, which subsequently serves the conservation of Paphiopedilum species.

scholarly journals Long-reads reveal that the chloroplast genome exists in two distinct versions in most plants

2019 ◽  
Author(s):  
Weiwen Wang ◽  
Robert Lanfear
Keyword(s):  
Chloroplast Genome ◽  
Genome Structure ◽  
Single Copy ◽  
Land Plant ◽  
Inverted Repeats ◽  
Individual Plant ◽  
Single Individual ◽  
Equal Frequency ◽  
Flip Flop ◽  
Chloroplast Genomes

Abstract The chloroplast genome usually has a quadripartite structure consisting of a large single copy region and a small single copy region separated by two long inverted repeats. It has been known for some time that a single cell may contain at least two structural haplotypes of this structure, which differ in the relative orientation of the single copy regions. However, the methods required to detect and measure the abundance of the structural haplotypes are labour-intensive, and this phenomenon remains understudied. Here we develop a new method, Cp-hap, to detect all possible structural haplotypes of chloroplast genomes of quadripartite structure using long-read sequencing data. We use this method to conduct a systematic analysis and quantification of chloroplast structural haplotypes in 61 land plant species across 19 orders of Angiosperms, Gymnosperms and Pteridophytes. Our results show that there are two chloroplast structural haplotypes which occur with equal frequency in most land plant individuals. Nevertheless, species whose chloroplast genomes lack inverted repeats or have short inverted repeats have just a single structural haplotype. We also show that the relative abundance of the two structural haplotypes remains constant across multiple samples from a single individual plant, suggesting that the process which maintains equal frequency of the two haplotypes operates rapidly, consistent with the hypothesis that flip-flop recombination mediates chloroplast structural heteroplasmy. Our results suggest that previous claims of differences in chloroplast genome structure between species may need to be revisited.

scholarly journals The complete structure of the cucumber (Cucumis sativus L.) chloroplast genome: Its composition and comparative analysis

2007 ◽  
Vol 12 (4) ◽  
Author(s):  
Wojciech Pląder ◽  
Yasushi Yukawa ◽  
Masahiro Sugiura ◽  
Stefan Malepszy
Keyword(s):  
Chloroplast Genome ◽  
Single Copy ◽  
Trna Genes ◽  
Inverted Repeat Region ◽  
Cucumis Sativus L ◽  
Stem Loop ◽  
Protein Coding ◽  
Chloroplast Genomes ◽  
Trna Species ◽  
Rrna Species

AbstractThe complete nucleotide sequence of the cucumber (C. sativus L. var. Borszczagowski) chloroplast genome has been determined. The genome is composed of 155,293 bp containing a pair of inverted repeats of 25,191 bp, which are separated by two single-copy regions, a small 18,222-bp one and a large 86,688-bp one. The chloroplast genome of cucumber contains 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes (4 rRNA species), and 37 tRNA genes (30 tRNA species), with 18 of them located in the inverted repeat region. Of these genes, 16 contain one intron, and two genes and one ycf contain 2 introns. Twenty-one small inversions that form stem-loop structures, ranging from 18 to 49 bp, have been identified. Eight of them show similarity to those of other species, while eight seem to be cucumber specific. Detailed comparisons of ycf2 and ycf15, and the overall structure to other chloroplast genomes were performed.

scholarly journals The complete chloroplast genome of Diarthron linifolium (Thymelaeaceae), a species found on a limestone outcrop in eastern Asia

2021 ◽  
Vol 51 (4) ◽  
pp. 345-352
Author(s):  
Sang-Tae KIM ◽  
Sang-Hun OH ◽  
Jongsun PARK
Keyword(s):  
Phylogenetic Analysis ◽  
Chloroplast Genome ◽  
Single Copy ◽  
Evolutionary Status ◽  
Complete Chloroplast Genome ◽  
Chloroplast Genomes ◽  
Potential Marker ◽  
Anticancer Treatment ◽  
Annual Herb ◽  
Unique Species

Diarthron linifolium Turcz. is an annual herb usually found in sandy soil or limestone areas. Plants in the genus Diarthron are known to have toxic chemicals that may, however, be potentially useful as an anticancer treatment. Diarthron linifolium is a unique species among the species of the genus distributed in Korea. Here, we determine the genetic variation of D. linifolium collected in Korea with a full chloroplast genome and investigate its evolutionary status by means of a phylogenetic analysis. The chloroplast genome of Korean D. linifolium has a total length of 172,644 bp with four subregions; 86,158 bp of large single copy and 2,858 bp of small single copy (SSC) regions are separated by 41,814 bp of inverted repeat (IR) regions. We found that the SSC region of D. linifolium is considerably short but that IRs are relatively long in comparison with other chloroplast genomes. Various simple sequence repeats were identified, and our nucleotide diversity analysis suggested potential marker regions near ndhF. The phylogenetic analysis indicated that D. linifolium from Korea is a sister to the group of Daphne species.

scholarly journals Chloroplast genome analysis and genetic transformation of Parachlorella kessleri-I a potential marine alga for biofuel production

2019 ◽  
Author(s):  
Prachi Nawkarkar ◽  
Sagrika Chugh ◽  
Amita Tanwar ◽  
Surbhi Sharma ◽  
Charli Kaushal ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Chloroplast Genome ◽  
Marine Alga ◽  
Chloroplast Transformation ◽  
Single Copy ◽  
Biofuel Production ◽  
Essential Information ◽  
Complete Chloroplast Genome ◽  
Parachlorella Kessleri ◽  
Species Specific

Abstract Background: Parachlorella kessleri-I produces higher biomass and lipid content suitable for commercial production of biofuels. Sequencing complete chloroplast genome will be instrumental in the constructing species specific chloroplast transformation vectors and generating chloroplast transgenic microalga with the desired traits and greater productivity, essential for commercial sustainability of microalgae based biofuel production. Results: Complete chloroplast genome sequence (cpDNA) of P. kessleri-I was annotated. The 109,642 bp chloroplast genome exhibited a quadripartite structure with two reverse repeat regions (IRA and IRB), a long single copy (LSC) and a small single copy (SSC) region. The genome encodes 117 unique genes, with 70 predicted protein coding genes, 35 tRNAs, 4 rRNAs. The cpDNA provided essential information like codons, UTRs and flank sequences for homologous recombination to make a species specific chloroplast transformation vector that facilitated chloroplast transformation of P. kessleri-I. To optimize chloroplast transformation, two antibiotic resistance makers aminoglycoside adenine transferase (aadA) conferring resistance to spectinomycin and Sh-ble gene from bacteria that conferred resistance to zeocin were tested. Using a aadA gene, transgenic colonies were retrieved on TAP medium containing 400 mg/l spectinomycin. However, no transgenic colonies were recovered in the zeocin supplemented medium. The spectinomycin resistant algal cell lines were analyzed by PCR. Southern blotting confirmed the stable transgenes integration into the chloroplast genome of P. kessleri-I via homologous recombination. Conclusion: The complete chloroplast genome analysis may provide valuable resources for population and evolutionary studies of Parachlorella species and identifying the related species. The chloroplast genome of P. kessleri-I was assembled as a quadripartite structure of 109,642 bp with defined IR regions. Its complete sequencing has provided essential information like codons, UTRs and flanking sequences to generate the species specific chloroplast transformation vector and obtaining the successful site-specific chloroplast transformation in P. kessleri-I via homologous recombination. The optimized chloroplast transformation in marine alga P. kessleri-I should open a new possibilities like controlling the chain lengths of fatty acids for biofuel application, manipulating the RubisCo enzyme for improving photosynthetic process, introducing carbon concentration mechanism (CCM) for fixing higher CO2 that may be instrumental in producing economically viable biofuel molecules.

scholarly journals Complete chloroplast genome sequences of Dioscorea: Characterization, genomic resources, and phylogenetic analyses

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e6032 ◽  
Author(s):  
Zhenyu Zhao ◽  
Xin Wang ◽  
Yi Yu ◽  
Subo Yuan ◽  
Dan Jiang ◽  
...  
Keyword(s):  
Chloroplast Genome ◽  
Phylogenetic Analyses ◽  
Single Copy ◽  
Genome Sequences ◽  
Complete Chloroplast Genome ◽  
Genomic Resources ◽  
Chloroplast Genomes ◽  
The Family ◽  
Small Single Copy

Dioscorea L., the largest genus of the family Dioscoreaceae with over 600 species, is not only an important food but also a medicinal plant. The identification and classification of Dioscorea L. is a rather difficult task. In this study, we sequenced five Dioscorea chloroplast genomes, and analyzed with four other chloroplast genomes of Dioscorea species from GenBank. The Dioscorea chloroplast genomes displayed the typical quadripartite structure of angiosperms, which consisted of a pair of inverted repeats separated by a large single-copy region, and a small single-copy region. The location and distribution of repeat sequences and microsatellites were determined, and the rapidly evolving chloroplast genome regions (trnK-trnQ, trnS-trnG, trnC-petN, trnE-trnT, petG-trnW-trnP, ndhF, trnL-rpl32, and ycf1) were detected. Phylogenetic relationships of Dioscorea inferred from chloroplast genomes obtained high support even in shortest internodes. Thus, chloroplast genome sequences provide potential molecular markers and genomic resources for phylogeny and species identification.

scholarly journals Structural and Comparative Analysis of the Complete Chloroplast Genome of Pyrus hopeiensis—“Wild Plants with a Tiny Population”—and Three Other Pyrus Species

2018 ◽  
Vol 19 (10) ◽  
pp. 3262 ◽  
Author(s):  
Yongtan Li ◽  
Jun Zhang ◽  
Longfei Li ◽  
Lijuan Gao ◽  
Jintao Xu ◽  
...  
Keyword(s):  
Chloroplast Genome ◽  
Population Decline ◽  
Gc Content ◽  
Single Copy ◽  
Wild Plants ◽  
Similar Degree ◽  
Protein Coding ◽  
Chloroplast Genomes ◽  
Close Relationship ◽  
History Of

Pyrus hopeiensis is a valuable wild resource of Pyrus in the Rosaceae. Due to its limited distribution and population decline, it has been listed as one of the “wild plants with a tiny population” in China. To date, few studies have been conducted on P. hopeiensis. This paper offers a systematic review of P. hopeiensis, providing a basis for the conservation and restoration of P. hopeiensis resources. In this study, the chloroplast genomes of two different genotypes of P. hopeiensis, P. ussuriensis Maxin. cv. Jingbaili, P. communis L. cv. Early Red Comice, and P. betulifolia were sequenced, compared and analyzed. The two P. hopeiensis genotypes showed a typical tetrad chloroplast genome, including a pair of inverted repeats encoding the same but opposite direction sequences, a large single copy (LSC) region, and a small single copy (SSC) region. The length of the chloroplast genome of P. hopeiensis HB-1 was 159,935 bp, 46 bp longer than that of the chloroplast genome of P. hopeiensis HB-2. The lengths of the SSC and IR regions of the two Pyrus genotypes were identical, with the only difference present in the LSC region. The GC content was only 0.02% higher in P. hopeiensis HB-1. The structure and size of the chloroplast genome, the gene species, gene number, and GC content of P. hopeiensis were similar to those of the other three Pyrus species. The IR boundary of the two genotypes of P. hopeiensis showed a similar degree of expansion. To determine the evolutionary history of P. hopeiensis within the genus Pyrus and the Rosaceae, 57 common protein-coding genes from 36 Rosaceae species were analyzed. The phylogenetic tree showed a close relationship between the genera Pyrus and Malus, and the relationship between P. hopeiensis HB-1 and P. hopeiensis HB-2 was the closest.

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