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首页> 外文会议>Proceedings of the 12th International Rapeseed Congress: Sustainable Development in Cruciferous Oilseed Crops Production >Molecular cloning of Brassica napus TRANSPARENT TESTA 2 (Bn TT2) gene family encoding potential MYB regulatory proteins of proanthocyanidin biosynthesis
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Molecular cloning of Brassica napus TRANSPARENT TESTA 2 (Bn TT2) gene family encoding potential MYB regulatory proteins of proanthocyanidin biosynthesis

机译：甘蓝型油菜TRANSPARENT TESTA 2（Bn TT2）基因家族的分子克隆，编码原花青素生物合成的潜在MYB调控蛋白

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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 all are 1102 bp with 2 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' UTRs are astonishingly conserved, and 2 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. Semi-quantitative RT-PCR revealed that the expression of BnTT2-2 is most 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, while 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 creating of novel yellow-seeded B. napus stocks.
机译：分离了三个甘蓝型油菜TRANSPARENT TESTA 2（BnTT2）基因家族的成员，该家族编码原花青素生物合成的潜在R2R3-MYB调节蛋白。 BnTT2-1，BnTT2-2和BnTT2-3均为1102 bp，带有2个内含子，并具有938 bp的全长cDNA和260个氨基酸的开放阅读框。它们彼此共有98.2％-99.3％的核苷酸同一性和96.5％-98.5％的氨基酸同一性，并且与拟南芥TT2（AtTT2）同源，具有74.1％-74.8％的核苷酸和71.1％-71.8％的氨基酸同一性。发现BnTT2-2的mRNA类型包含未剪接的内含子2，并编码一种过早的蛋白质。它们都具有替代的聚腺苷酸化位点。 BnTT2-1和BnTT2-3也有一个替代的转录起始位点。与AtTT2对齐后，它们的5'UTR非常惊人地保守，并且在其3'UTR中也发现了2个保守区域。寡核苷酸缺失导致它们的双起始密码子。 BnTT2蛋白类似于AtTT2，是位于核中的R2R3-MYB蛋白，包含预测的DNA结合位点，bHLH相互作用残基和转录激活域。 Southern印迹表明，在油菜中可能存在三个BnTT2成员，低于基于三倍体的预测。半定量RT-PCR显示，BnTT2-2的表达最像AtTT2，在年轻种子中强烈表达，但也表达在不表达AtTT2的根中。 BnTT2-1显示较低的组织特异性和转录水平，而BnTT2-3最低。比较克隆和RT-PCR表明，种子色近等基因系L1和L2具有相同的BnTT2基因，L2中的黄色种子色可能是由BnTT2以外的基因座/基因座引起的。我们的结果为进一步研究类黄酮途径中BnTT2基因的调控机制和转基因创建新的黄色种子油菜油菜奠定了基础。

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