黑芥和埃塞俄比亚芥PAP2基因克隆及B基因组PAP2功能验证

Cloning of PAP2 genes in Brassica nigra and Brassica carinata and functional validation of PAP2 in the B genome

PAP2(Production of anthocyanin pigment 2)编码MYB转录因子,与bHLH、WD40形成MYB-bHLH-WD40复合体来调控花青素合成。本研究从埃塞俄比亚芥和黑芥中分别克隆出2个和1个PAP2基因拷贝,分别命名为BcaB.PAP2、BcaC.PAP2、BniB.PAP2,这三个基因均由3个外显子和2个内含子组成,均编码247个氨基酸。基于本研究克隆的PAP2基因序列和已报道的其他芸薹属PAP2基因序列,设计了3对能分别检测A、B、C基因组PAP2基因的引物,通过等位特异PCR可以区分芸薹属禹氏三角中的6个物种PAP2的基因组来源。选取芥菜型油菜B基因组的BjuB.PAP2基因构建过表达载体,转化拟南芥,其叶片变紫,表明PAP2基因正调控花青素合成。遮光处理10天后紫叶埃塞俄比亚芥的叶片紫色变淡,花青素含量下降了41.22%。荧光定量PCR和转录组研究表明,遮光处理植株叶片中BcaB.PAP2和BcaC.PAP2表达量均下降,花青素合成的结构基因查尔酮合成酶基因(CHS)、查尔酮异构酶基因(CHI)、黄烷酮3-羟化酶基因(F3H)、二氢黄酮醇还原酶基因(DFR)、花青素合成酶基因(ANS)、类黄酮3-葡糖基转移酶基因(UFGT)等基因的表达量也下降。本研究完成了对芸薹属植物中埃塞俄比亚芥和黑芥PAP2基因的克隆,并对其进行进化分析。根据本实验中克隆及其他已报道的PAP2基因序列设计等位基因特异性PCR引物,为芸薹属种间杂交后代PAP2基因的基因组传递鉴定提供了分子标记。通过BjuB.PAP2基因转拟南芥植株结果表明PAP2基因参与花青素的积累。紫叶埃塞俄比亚芥经遮光处理后,发现BcaPAP2基因及花青素合成结构基因的表达受光照的诱导。本研究在芸薹属中克隆PAP2基因,并初步验证PAP2的功能,为进一步解析芸薹属植物花青素合成的调控机制提供参考。

PAP2(Production of anthocyanin segment 2)encodes MYB transcription factor,and regulates anthocyanin synthesis by forming MYB-bHLH-WD40 complex with bHLH and WD40.In this study we cloned PAP2 gene in Brassica,and preliminarily verified the function of PAP2,providing a reference for further understanding the regulatory mechanism of anthocyanin synthesis in Brassica.Two and one copies of PAP2 gene were cloned from Brassica carinata and Brassica nigra,respectively,and named as BcaB.PAP2,BcaC.PAP2,BniB.PAP2.These three genes are composed of 3 exons and 2 introns,encoding 247 amino acids.Based on the PAP2 gene sequences cloned in this study and other reported PAP2 gene sequences of Brassica,three pairs of primers were designed to detect the PAP2 gene of A,B and C genomes respectively,and the genomic origin of PAP2 of six species in the U’s Triangle of Brassica could be distinguished by allele specific PCR.The BjuB.PAP2 gene from the B genome of Brassica jun-cea was selected to construct an overexpression vector and transformed into Arabidopsis thaliana.The transgenic Arabidopsis leaves turned purple,indicating that PAP2 gene is regulating anthocyanin synthesis.After shading treatment for 10 days,the purple color of Brassica carinata lightened,and the anthocyanin content decreased by 41.22%.Quantitative PCR and transcriptomic studies showed that the expression of BcaB.PAP2 and BcaC.PAP2 in the leaves of shading plants decreased.The expression of structural genes for anthocyanin synthesis,such as chal-cone synthase gene(CHS),chalcone isomerase gene(CHI),flavanone 3-hydroxylase gene(F3H),dihydroflavonol reductase gene(DFR),anthocyanin synthase gene(ANS),flavonoid 3-glucosyltransferase gene(UFGT)and other genes were also decreased.This study completed the cloning of PAP2 gene of Brassica carinata and Brassica nigra,and carried out their evolutionary analysis.Allele specific PCR primers were designed according to the sequences of PAP2 gene cloned in this experiment and other reported genes to provide molecular markers for the genomic transmission identification of PAP2 gene in Brassica interspecific hybrids.Through the transformation of BjuB.PAP2 gene into Arabidopsis plants,it was found that PAP2 gene was involved in the accumulation of anthocyanins.It was found that the expression of BcaPAP2 gene and anthocyanin synthesis structure gene were induced by light after shading.This study cloned the PAP2 gene in Brassica and preliminarily verified its function,providing a reference for further understanding the regulatory mechanism of anthocyanin synthesis in Brassica plants.