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以结球甘蓝(Brassica oleracea L.)‘519’品系为试材,通过结球期茎尖和叶片以及莲座期茎尖和叶片的转录组对比分析,筛选出4个显著差异表达HB类基因,分别为BoHAT2、BoHB12、BoHB7和BoHB27。进一步对上述4个基因以及调控拟南芥(Arabidopsis thaliana)叶片卷曲但在结球甘蓝转录组中未检测到表达差异的基因BoPHB、BoPHV和BoREV进行了同源克隆。序列分析表明上述7个基因都含有同源异型域(homeodomain,HD),具有同源异型盒(homeobox,HB)蛋白家族的典型结构特征。BlastP(蛋白序列与蛋白库做比对)表明它们与拟南芥的同源蛋白相似性高,来自结球甘蓝的BoHAT2、BoHB12、BoHB7、BoHB27、BoPHB、BoPHV、BoREV与拟南芥中的HAT2、ATHB12、ATHB 7、ATHB27、PHB、PHV、REV蛋白同源,同源性分别为86%、80%、79%、60%、94%、95%和96%。BlastP比对和进化树分析结果表明,BoHAT2属于HD-ZipⅡ类,BoHB12和BoHB7属于HD-ZipⅠ类,BoHB27属于ZF-HD,BoPHB、BoPHV和BoREV属于HD-ZipⅢ类。BoHAT2、BoHB12、BoHB7、BoHB27、BoPHB、BoPHV、BoREV在结球甘蓝莲座期到结球期的茎尖和叶片中均有表达,BoHB12和BoHB7在结球期叶片中表达量显著增高,分别是莲座期叶片的38.1倍和6.2倍,其余基因表达量差异不明显,表明BoHB12和BoHB7可能是结球甘蓝球叶自然卷曲过程中的主效调控基因。
Brassica oleracea L. 519 strain was used as materials to screen four differentially expressed HB genes by comparing the transcriptome of shoot tips and leaves at the heading stage and the shoot tips and leaves at the rosette stage. Respectively BoHAT2, BoHB12, BoHB7 and BoHB27. Further, homologous cloning was carried out on the above four genes and BoPHB, BoPHV and BoREV, genes that curl Arabidopsis thaliana leaves but no difference in expression in the cabbage transcriptome. Sequence analysis showed that the above seven genes all contained the homeodomain (HD), which had the typical structural characteristics of the homeobox (HB) protein family. BlastP (protein sequence alignment with protein library) showed that they are highly homologous to Arabidopsis thaliana homologues and that BoHAT2, BoHB12, BoHB7, BoHB27, BoPHB, BoPHV, BoREV from Cabbage and HAT2 from Arabidopsis , ATHB12, ATHB7, ATHB27, PHB, PHV and REV, respectively. The homology was 86%, 80%, 79%, 60%, 94%, 95% and 96% respectively. BlastP alignment and phylogenetic tree analysis showed that BoHAT2 belonged to HD-Zip class II, BoHB12 and BoHB7 belonged to HD-Zip class I, BoHB27 belonged to ZF-HD, and BoPHB, BoPHV and BoREV belonged to HD-Zip class III. BoHB2, BoHB12, BoHB7, BoHB27, BoPHB, BoPHV and BoREV were all expressed in the shoot tips and leaves of the cabbage at rosette to anthesis stage. The expression levels of BoHB12 and BoHB7 in the leaves at the stage of heading stage were significantly increased, 38.1 folds and 6.2 folds of the leaves, the rest of the gene expression was not significantly different, indicating that BoHB12 and BoHB7 may be the main effect of the natural curl of cabbage leaf curl regulatory genes.