Molecular cloning of Brassica napus TRANSPARENT TESTA 2 gene family encoding potential MYB regulatory proteins of proanthocyanidin biosynthesis
Molecular cloning of Brassica napus TRANSPARENT TESTA 2 gene family encoding potential MYB regulatory proteins of proanthocyanidin biosynthesis
复制标题
DOI：
10.1007/s11033-006-9024-8
复制
发表时间：
2007-04
影响因子：
2.8
通讯作者：
Y. Wei;Jia-Na Li;Junxing Lu;Zhanglin Tang;Dong-Chun Pu;Y. Chai
中科院分区：
文献类型：
--
作者：
Y. Wei;Jia-Na Li;Junxing Lu;Zhanglin Tang;Dong-Chun Pu;Y. Chai
分离出甘蓝型油菜透明测试 2 (BnTT2) 基因家族的三个成员，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，有两个内含子，并且具有 938 bp 全长 cDNA，具有 260 个氨基酸的开放阅读框。它们彼此具有 98.2–99.3% 的核苷酸和 96.5–98.5% 的氨基酸同一性，并且与拟南芥 TT2 (AtTT2) 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 并编码早熟蛋白。它们都有一个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 相比，它们的 5' 非翻译区 (UTR) 非常保守，并且在 3' UTR 中也发现了两个保守区域。寡核苷酸缺失导致它们的双起始密码子。与 AtTT2 类似，BnTT2 蛋白是位于核的 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B中可能存在三个BnTT2成员。 napus，低于基于三倍的预测。半定量逆转录-聚合酶链反应(RT-PCR)显示BnTT2-2的表达大多与AtTT2相似，在幼苗中表达强烈，但在根部也有表达，AtTT2无表达。BnTT2-1表现出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，近等基因系L1和L2种子颜色具有相当的BnTT2基因，L2种子颜色发黄可能是由BnTT2以外的位点/位点引起的。我们的研究结果为进一步研究BnTT2基因在类黄酮途径中的调控机制以及新型黄籽B的转基因创造奠定了基础。油菜花。
Three members ofBrassica napus TRANSPARENT TESTA 2(BnTT2) gene family encoding potential R2R3-MYB regulatory proteins of proanthocyanidin biosynthesis were isolated.BnTT2-1,BnTT2-2, andBnTT2-3are 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 toArabidopsis thaliana TT2(AtTT2) with 74.1–74.8% nucleotide and 71.1–71.8% amino acid identities. An mRNA type ofBnTT2-2was found to contain unspliced intron 2 and encode a premature protein. They all have an alternative polyadenylation site.BnTT2-1andBnTT2-3also have an alternative transcription initiation site. Aligned withAtTT2, 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 threeBnTT2members inB. napus, lower than triplication-based prediction. Semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) revealed that the expression ofBnTT2-2is mostly likeAtTT2with intensive expression in young seeds, but it is also expressed in root in whichAtTT2has no expression.BnTT2-1shows lower tissue specificity and transcription levels, whereasBnTT2-3is the lowest. Comparative cloning and RT–PCR indicated that seed color near-isogenic lines L1 and L2 have equivalentBnTT2genes, and the yellow seed color in L2 might be caused by locus/loci other thanBnTT2. Our results lay the basis for further investigating the regulatory mechanism ofBnTT2genes in flavonoid pathway and for transgenic creation of novel yellow-seededB. napusstocks.