Efficiency of ligation-mediated PCR and TAIL-PCR methods for isolation of RbcS promoter sequences from green microalga Ankistrodesmus convolutus

Thanh, Tran; Quynh, Vu Thi; Abdullah, Md. Pauzi; Omar, Hishamuddin; Napis, Suhaimi

doi:10.1134/s0026893312010220

Cited by 15 publications

(11 citation statements)

References 18 publications

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“…Two gene specific primers i.e. GSP1 and GSP2 (Sukganah et al 2013) were used as reverse primers with an arbitrary degenerate primer AD4 (Thanh et al 2012) as the forward primer (Table 1). TAIL-PCR was adapted from Liu and Whittier (1995) and briefly described below.…”

Section: Methodsmentioning

confidence: 99%

A pseudogene of caffeic acid-o-methyltransferase (COMT) in Acacia mangium: Comparative analysis with other COMT plant promoters

Abdul¹,

Tam

Ratnam³

2018

Preprint

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Acacia mangium is a prominent tree species in the forest plantation industry of Southeast Asia, grown mainly to produce pulp and paper, and to a lesser extent wood chips and solid wood products. Lignin, a natural complex polymer used by plants for structural support and defence, has to be chemically removed during the production of quality paper. Delignification is very expensive and moreover, is an environmental pollutant. Understanding the complex mechanisms that underlie the regulation of lignin biosynthetic genes requires in-depth knowledge of not only the genes involved but also their regulatory elements. Using Thermal Asymmetric Interlaced PCR, a 770 bp promoter sequence with 93% identity with COMT1 gene from Acacia auriculiformis × A. mangium hybrid was isolated from A. mangium. Bioinformatics analysis revealed the presence of cis acting elements commonly found in other lignin biosynthesis genes such as TATA box, CAAT box, W box, AC-I and AC-11 elements. However, a nonsense mutation that created a premature stop codon was found on the first exon. Modelling of MYB transcription factor binding site on this newly isolated pseudogene shows it has binding sites for important transcription factors involved in lignin biosynthesis both in Arabidopsis thaliana and Eucalyptus grandis. Given the remarkable structures of its regulatory region, the possible structure of its transcript was detected using Mfold. Results show the transcript are capable of forming stem loop structures, a characteristic commonly attributed to presence of miRNA. Possible functions of pseudoAmCOMT1 were discussed.

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Section: Methodsmentioning

confidence: 99%

A pseudogene of caffeic acid-o-methyltransferase (COMT) in Acacia mangium: Comparative analysis with other COMT plant promoters

Abdul¹,

Tam

Ratnam³

2018

Preprint

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“…The promoter sequences of HtMYB2 were isolated from 'QY1' and 'QY3', based on thermal asymmetric interlaced (TAIL)-PCR [35]. According to the nucleotide sequence differences between the promoters of HtMYB2 of 'QY1' and 'QY3', a polymorphic PCR marker HtproS was designed to distinguish between 'QY1' and 'QY3' (Table S5).…”

Section: Genotyping Of a Natural Population Of Helianthus Tuberosusmentioning

confidence: 99%

Functional MYB transcription factor gene HtMYB2 is associated with anthocyanin biosynthesis in Helianthus tuberosus L

et al. 2020

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Background: Tuber color is an important trait for Helianthus tuberosus L. (Jerusalem artichoke). Usually, purple tubers with high anthocyanin content are more nutritious than white tuber. But, the molecular mechanism underlying it is unknown. Results: In the current study, high-throughput RNA-sequencing was used to compare the transcriptomes between plants with tubers with red or white epidermis. Compared with the white-skinned tubers of cultivar QY3, anthocyanin biosynthesis structural genes had greater expression in the red-skinned tubers of cultivar QY1, indicating that the anthocyanin biosynthesis pathway was activated in 'QY1'; quantitative PCR confirmed this difference in expression. HtMYB2 (Unigene44371_All) was the only MYB transcription factor, homologous to the MYB transcription factor regulating anthocyanin biosynthesis, expressed in the red tuber epidermis of 'QY1'. The anthocyanin concentration in the root, stem, leaf, flower, and tuber epidermis of 'QY1' was higher than in 'QY3', especially tuber epidermis. Correspondingly, HtMYB2 had greater expression in these tissues of 'QY1' than in 'QY3'. The expression of HtMYB2 was associated with anthocyanin accumulation in the different tissues. Overexpression of HtMYB2 activated the anthocyanin biosynthesis pathway, accumulating the pigment in leaves of transgenic tobacco, supporting the model that HtMYB2 regulated anthocyanin biosynthesis. Further experiments found that HtMYB2 had the same coding sequence and genomic sequence in 'QY1' and 'QY3', but that there were several single nucleotide polymorphisms and one insertiondeletion (indel) mutation of 21 nucleotides in the promoter region between the two alleles. The deletion of three nucleotides "AAA" made the promoter of 'QY1' predicted to contain one more possible promoter region. A specific primer, based on the indel, could differentiate between cultivars with red or white tuber epidermis. The genetic variation in HtMYB2 was associated with the tuber skin color in a natural population. Conclusions: RNA-seq can successfully isolate the candidate gene (HTMYB2) controlling anthocyanin biosynthesis in purple epidermis of Jerusalem artichoke tuber. HTMYB2 can regulate anthocyanin biosynthesis in plants and is closely related to the formation of purple phenotype in tubers. This study should be useful in understanding the genetic mechanism underlying different tuber skin colors and in breeding new H. tuberosus cultivars with different tuber skin colors.

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“…The promoter sequences of HtMYB2 were isolated from 'QY1' and 'QY3', based on thermal asymmetric interlaced (TAIL)-PCR [31]. According to the nucleotide sequence differences between the promoters of HtMYB2 of 'QY1' and 'QY3', a polymorphic PCR marker HtproS was designed to…”

Section: Genotyping Of a Natural Population Of Helianthus Tuberosusmentioning

confidence: 99%

Functional MYB transcription factor gene HtMYB2 is associated with anthocyanin biosynthesis in Helianthus tuberosus L.

Gao

Zong²,

Yang

et al. 2020

Preprint

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Background: Tuber color is an important trait for Helianthus tuberosus L. (Jerusalem artichoke), but the molecular mechanism underlying it is unknown. Results: In the current study, high-throughput RNA-sequencing was used to compare the transcriptomes between plants with tubers with red or white epidermis. Compared with the white-skinned tubers of cultivar QY3, anthocyanin biosynthesis structural genes had greater expression in the red-skinned tubers of cultivar QY1, indicating that the anthocyanin biosynthesis pathway was activated in ‘QY1’; quantitative PCR confirmed this difference in expression. HtMYB2 (Unigene44371_All) was the only MYB transcription factor expressed in the red tuber epidermis of ‘QY1’. HtMYB2 resembled an MYB transcription factor, regulating anthocyanin biosynthesis, and possessing an intact SANT/MYB and MYB-like DNA-binding domain. The anthocyanin concentration in the root, stem, leaf, flower, and tuber epidermis of ‘QY1’ was higher than in ‘QY3’, especially tuber epidermis. Correspondingly, HtMYB2 had greater expression in these tissues of ‘QY1’ than in ‘QY3’. The expression of HtMYB2 was associated with anthocyanin accumulation in the different tissues. Overexpression of HtMYB2 activated the anthocyanin biosynthesis pathway, accumulating the pigment in leaves of transgenic tobacco, supporting the model that HtMYB2 regulated anthocyanin biosynthesis. Further experiments found that HtMYB2 had the same coding sequence and genomic sequence in ‘QY1’ and ‘QY3’, but that there were several single nucleotide polymorphisms and one insertion–deletion (indel) mutation of 21 nucleotides in the promoter region between the two alleles. The deletion of three nucleotides “AAA” made the promoter of ‘QY1’ predicted to contain one more possible promoter region. A specific primer, based on the indel, could differentiate between cultivars with red or white tuber epidermis. The genetic variation in HtMYB2 was associated with the tuber skin color in a natural population.Conclusions: RNA-seq can successfully isolate the candidate gene (HTMYB2) controlling anthocyanin biosynthesis in purple epidermis of Jerusalem artichoke tuber. HTMYB2 can regulate anthocyanin biosynthesis in plants and is closely related to the formation of purple phenotype in tubers. This study should be useful in understanding the genetic mechanism underlying different tuber skin colors and in breeding new H. tuberosus cultivars with different tuber skin colors.

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Efficiency of ligation-mediated PCR and TAIL-PCR methods for isolation of RbcS promoter sequences from green microalga Ankistrodesmus convolutus

Cited by 15 publications

References 18 publications

A pseudogene of caffeic acid-o-methyltransferase (COMT) in Acacia mangium: Comparative analysis with other COMT plant promoters

A pseudogene of caffeic acid-o-methyltransferase (COMT) in Acacia mangium: Comparative analysis with other COMT plant promoters

Functional MYB transcription factor gene HtMYB2 is associated with anthocyanin biosynthesis in Helianthus tuberosus L

Functional MYB transcription factor gene HtMYB2 is associated with anthocyanin biosynthesis in Helianthus tuberosus L.

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