被子植物基部类群细辛AP3亚族基因表达、功能与进化的研究

项目名称： 被子植物基部类群细辛AP3亚族基因表达、功能与进化的研究

项目编号： No.30860130

项目类型： 地区科学基金项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 赵银河

作者单位： 云南农业大学

项目金额： 25万元

中文摘要： 通过用RACE方法获得在细辛花器官中表达的AP3亚族cDNA全长，对蛋白质C-末端结构域进行比较发现AcAP3-like不保守，有碱基的插入与缺失而导致提前终止。比较基因组序列，存在可变剪接造成移码突变而导致提前终止。对AP3亚族MADS-box基因用原杂交、RT-PCR和Real-Time PCR对花器官进行表达分析，发现这些基因在花器官中的表达不一致，并且在心皮中有异常表达的基因；用系统树对AcAP3-1，AcAP3-A，AcAP3-B，AcAP3-C，AcAP3-D以及AcAP3-E MADS-box基因进行进化分析，它们是属于B-class的AP3-like基因。通过转基因方法在拟南芥中进行功能验证，与拟南芥中对应的同源基因相比较，获得在心皮中有异常表型效应的基因。本研究结果发现从被子植物基部类群细辛中分离出来的B-class基因比真双子叶植物中分离出来的基因具有更广泛的表达，从而ABC模型在被子植物基部类群取代的是以更加简单的方式发生，在更高等或者高等真真双子叶植物中逐渐进化到典型的ABC模型, 这对正确理解被子植物基部类群花器官发育在进化过程中的遗传变化具有重要的意义。

中文关键词： 尾花细辛；AP3–ike MADS-box基因；基因表达；转化；系统分析

英文摘要： We used RACE to obtain the full-length coding sequences of AP3 subfamily MADS-box transcription factors from Asarum caudigerum (Aristolochiaceae). Alignment with the putative B-class homologs and putative Bsister (Bs) homologs showed that the putative B-class AP3-like gene isolated from A. caudigerum shared the PI and paleoAP3 motifs of other AP3 B-class genes in the C-terminal sequence besides AcAP3-B transcription factor.Compared with other AP3 subfamily MADS-box transcription factors, there were bases inserted in the K1 and K2 dormain of AcAP3-C and resulted premature translation termination in the K1 dormain. To furthern obtain genomic sequences and found that AcAP3-D there is premature translation termination in the C-terminus with a 4-nucleotide deletion, which located to the position of the deletion matches the spliced sites 5′end of the seventh exon comparing with genomic sequence. To obtain information on the expression pattern of the putative AcAP3-like isolated from A. caudigerum, RNA in situ hybridization, RT-PCR and qRT-PCR were performed and indicated that AcAP3-1 and AcAP3-D was upregulated in carpels, AcAP3-A expression was mainly detected in stamens, AcAP3-B, which signal was detected in perianth and carpels，whereas AcAP3-C and AcAP3-E were expressed in whorls 1, 2 and 3 (Perianth, stamens and carpels respectively). Phylogenetic analysis was performed on putative A. caudigerum AP3-like genes to infer evolutionary relationships within the B-class MADS-box gene family formed a clade with AP3-1 of A. europaeum and AeAP3-like A. splendens with a high bootstrap support. However, it did not fall anywhere near AeAP3-2 and the other Bsister genes. To further identify the function of putative the AP3 subfamily of B-class genes, the overexpression of those genes in Arabidopsis will be performed and obtained 2 genes different phenotypes by ectopically expression of those genes in the wild-type Arabidopsis background. Compared with orthology of Arabidopsis, we obtain genes with different phenotypes isolated from A. caudigerum. In the typical ABC model based on A. thaliana, the B-class genes, APETALA3 (AP3) and PISTILLATA (PI) control the specification of petals in conjunction with A-class genes and stamens in conjunction with C-class genes in the second and third whorls, respectively. Based on our study, the expression domain of B-class AP3-like genes in A. caudigerum was found to be broader than for their counterparts in eudicots. The broader gene expression of B-class genes among the flowers of the basal angiosperm lineages suggests that the antagonistic gene interactions observed in Arabidopsis thaliana, Antirrhinum majus, and other higher eudicots may not occur in the basal angiosperms. Therefore, we support the contention that the ABC model was not established at the outset of angiosperm evolution, but instead occurred in a simpler form in the basal angiosperms, and gradually evolved into the canonical ABC model in more the derived higher eudicot lineages, It is very important to understand that the mechanism of the floral organs initiation in the basal angiosperms, and genetic variations of the floral development in the process of evolution of angiosperm flowers.

英文关键词： Asarum caudigerum; AP3-like MADS-box genes; Genes expression; Transgene; Phylogenetic analysis