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Molecular cloning of Brassica napus TRANSPARENT TESTA 2 gene family encoding potential MYB regulatory proteins of proanthocyanidin biosynthesis

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Abstract

Three members of Brassica napus TRANSPARENT TESTA 2 (BnTT2) gene family encoding potential R2R3-MYB regulatory proteins of proanthocyanidin biosynthesis were isolated. BnTT2-1, BnTT2-2, and BnTT2-3 are 1102 bp with two introns, and have a 938-bp full-length cDNA with a 260 amino acid open reading frame. They share 98.2–99.3% nucleotide and 96.5–98.5% amino acid identities to each other, and are orthologous to Arabidopsis thaliana TT2 (AtTT2) with 74.1–74.8% nucleotide and 71.1–71.8% amino acid identities. An mRNA type of BnTT2-2 was found to contain unspliced intron 2 and encode a premature protein. They all have an alternative polyadenylation site. BnTT2-1 and BnTT2-3 also have an alternative transcription initiation site. Aligned with AtTT2, their 5′ untranslated regions (UTRs) are astonishingly conserved, and two conserved regions were also found in their 3′ UTRs. Oligonucleotide deletion leads to double-start codons of them. Resembling AtTT2, BnTT2 proteins are nuclear-located R2R3-MYB proteins containing predicted DNA-binding sites, bHLH interaction residues, and transcription activation domains. Southern blot indicated that there might be three BnTT2 members in B. napus, lower than triplication-based prediction. Semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) revealed that the expression of BnTT2-2 is mostly like AtTT2 with intensive expression in young seeds, but it is also expressed in root in which AtTT2 has no expression. BnTT2-1 shows lower tissue specificity and transcription levels, whereas BnTT2-3 is the lowest. Comparative cloning and RT–PCR indicated that seed color near-isogenic lines L1 and L2 have equivalent BnTT2 genes, and the yellow seed color in L2 might be caused by locus/loci other than BnTT2. Our results lay the basis for further investigating the regulatory mechanism of BnTT2 genes in flavonoid pathway and for transgenic creation of novel yellow-seeded B. napus stocks.

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Abbreviations

aa:

amino acid

AtTT2/TT2 :

Arabidopsis thaliana TRANSPARENT TESTA 2

BnTT2 :

Brassica napus TRANSPARENT TESTA 2

bp:

base pair

cDNA:

complementary DNA

DAF:

day after flowering

DIG:

digoxygenin

pI:

isoelectric point

Mw:

molecular weight

NCBI:

National Center of Biotechnology Institute

ORF:

open reading frame

PA:

proanthocyanidin

PCR:

polymerase chain reaction

RACE:

rapid amplification of cDNA ends

RT:

reverse transcription

UTR:

untranslated region

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Acknowledgments

This research was supported by the Major Program of Chongqing Municipal Natural Science Foundation (No. 8446) and the Major Program of National Natural Science Foundation (No. 30330400).

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Authors and Affiliations

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

    Yun-Liang Wei, Jia-Na Li, Jun Lu, Zhang-Lin Tang, Dong-Chun Pu & You-Rong Chai

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

    Yun-Liang Wei, Jia-Na Li, Jun Lu, Zhang-Lin Tang, Dong-Chun Pu & You-Rong Chai

  3. Key Lab of Biotechnology and Crop Quality Improvement of Agricultural Ministry of China, Southwest University, 216 Tiansheng Road, Beibei, Chongqing, 400716, People’s Republic of China

    Yun-Liang Wei, Jia-Na Li, Jun Lu, Zhang-Lin Tang, Dong-Chun Pu & You-Rong Chai

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

    Yun-Liang Wei, Jia-Na Li, Jun Lu, Zhang-Lin Tang, Dong-Chun Pu & You-Rong Chai

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  1. Yun-Liang Wei
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  2. Jia-Na Li
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Correspondence to You-Rong Chai.

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Wei, YL., Li, JN., Lu, J. et al. Molecular cloning of Brassica napus TRANSPARENT TESTA 2 gene family encoding potential MYB regulatory proteins of proanthocyanidin biosynthesis. Mol Biol Rep 34, 105–120 (2007). https://doi.org/10.1007/s11033-006-9024-8

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  • DOI: https://doi.org/10.1007/s11033-006-9024-8

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