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Molecular cloning, characterization and expression of two rapeseed (Brassica napus L.) cDNAs orthologous to Arabidopsis thaliana phenylalanine ammonia-lyase 1

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Abstract

Phenylalanine ammonia-lyase (PAL) plays important roles in determining flavonoid- and lignin-related traits. Two PAL full-length cDNAs, BnPAL1-1 and BnPAL1-2, were isolated from Brassica napus. BnPAL1-1 is 2460 bp encoding a 722-aa protein, while BnPAL1-2 is 2396 bp encoding a 719-aa protein. BnPAL1-1 shares 86.8% nucleotide identities and 94.9%/97.0% identities/positives at protein level to BnPAL1-2. NCBI BLAST, pairwise alignment and preferential amino acid analysis indicated that they are orthologs of Arabidopsis thaliana PAL1 (AtPAL1), suggesting that the divergence of AtPAL1 and AtPAL2 preceded the divergence of genera Arabidopsis and Brassica. Southern hybridization and EST analysis showed that B. napus has 2–6 orthologs of AtPAL1. High ratios of synonymous to non-synonymous substitutions suggest that in Brassicaceae PAL1 orthologs evolve under high pressure of purification selection, while among PAL1 paralogs this pressure is a little lower. Escherichia coli-expressed 6 × His-tagged BnPAL1-1 and BnPAL1-2 were both bioactive, but BnPAL1-2 was much higher in tested catalytic activity. BnPAL1-1 and BnPAL1-2 are transcribed in all the 11 organs tested, but most abundant in flower. BnPAL1-1 shows declined expression in late-stage seed, and BnPAL1-2 shows stronger organ specificity. Transcription of these two genes in middle and late stages of seed development of yellow-seeded line L2 is obviously lower than near-isogenic black-seeded line L1, indicating the correlation of yellow-seed trait to down regulation of PAL transcription, but this is probably caused by suppressed seed-specific upstream regulatory signal or by seed-specific feedback inhibition from downstream pathways.

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Abbreviations

DAF:

Days after flowering

PAL:

Phenylalanine ammonia-lyase

RACE:

Rapid amplification of cDNA ends

RT-PCR:

Reverse-transcriptase-polymerase chain reaction

UTR:

Untranslated region

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Acknowledgements

This research was supported by the National Natural Science Foundation Major Program (30330400), the National “863” Project program (20060110Z1024), and the Chongqing Municipal Natural Science Foundation Major Program (8446) respectively. We thank Dr Rui Wang from Chongqing Rapeseed Technology Research Center for providing the B. napus stock line 5B for this research.

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Authors and Affiliations

  1. College of Agronomy and Biotechnology, Southwest University, 216 Tiansheng Road, Beibei, Chongqing, 400716, People’s Republic of China

    Yu Ni, Hong-Lin Jiang, Bo Lei, Jia-Na Li & You-Rong Chai

  2. Chongqing Rapeseed Engineering Research Center, 216 Tiansheng Road, Beibei, Chongqing, 400716, People’s Republic of China

    Yu Ni, Hong-Lin Jiang, Bo Lei, Jia-Na Li & You-Rong Chai

  3. Chongqing Key Laboratory of Crop Quality Improvement, 216 Tiansheng Road, Beibei, Chongqing, 400716, People’s Republic of China

    Yu Ni, Jia-Na Li & You-Rong Chai

  4. Key Lab of Biotechnology & Crop Quality Improvement of Ministry of Agriculture, 216 Tiansheng Road, Beibei, Chongqing, 400716, People’s Republic of China

    Yu Ni, Jia-Na Li & You-Rong Chai

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  1. Yu Ni
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  2. Hong-Lin Jiang
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Correspondence to Jia-Na Li or You-Rong Chai.

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Ni, Y., Jiang, HL., Lei, B. et al. Molecular cloning, characterization and expression of two rapeseed (Brassica napus L.) cDNAs orthologous to Arabidopsis thaliana phenylalanine ammonia-lyase 1 . Euphytica 159, 1–16 (2008). https://doi.org/10.1007/s10681-007-9448-9

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