【作者】 郑尚永；

【导师】 裴炎；

【作者基本信息】 西南农业大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 植物花器官发育研究是植物发育分子生物学的重要内容。自Coen等(1991)首次提出花器官发育的“ABC”模型以来，花器官发育的研究取得了突破性进展。以拟南芥、金鱼草等模式植物为代表的与花器官发育相关的基因相继被克隆。研究表明，控制花器官发育的绝大部分基因属于MADS盒基因家族成员。该家族成员是一种重要的转录因子，都含有一个保守性很强的MADS盒，最早在酵母(MCM1)、拟南芥(AGAMOUS)、金鱼草(DIFICIENS)和血清学应答因子(SRF)中发现，是广泛存在于动植物中的调节基因，在花器官发育以及同源异型转换中起着非常重要的作用。分离植物花器官发育相关基因，将有助于探明植物生殖发育的分子模式，并为观赏植物和花卉品种的基因工程改良奠定坚实的基础。 棉花是一种重要的经济作物，其花器官发育也很有特点：营养器官与生殖器官同时形成，每一朵花均为顶花，由果枝顶端分生组织形成一片真叶和一片先出叶后形成。因此，研究棉花花器官的发育具有一定的理论和实践意义，但目前还未见棉花花器官发育研究的相关报道。突变体是发育生物学研究的重要材科，我们在棉花组织培养再生植株中发现了一批花器官突变体，为研究棉花花器官的发育提供了良好的科研素材。我们对其中一个花器官均突变为苞叶状叶性器官的突变体进行了分析。根据其表型，初步判断可能是其A、B、C三组功能基因均未发挥作用所致。为弄清涉及这些突变体的基因以及相关基因在棉花花器官突变体中的作用，我们利用棉花EST数据库资料，通过EST序列整合，从陆地棉徐州142花蕾中克隆出一个含MADS结构域的蛋白编码区段。该片段与葡萄、烟草、矮牵牛、拟南芥和金鱼草等属AGL2类的MADS结构域蛋白亚家族有较高的同源性。经对该基因的初步分析，得到如下主要实验结果： 1．棉花MADS盒基因的克隆 通过对同源EST序列的整合和延伸，得到一条含MADS盒的整合序列。根据该序列设计引物， 内而衣业人学二皿三而厂卜学位论义经扩增、十四和测序，得剑一个 MADS盒基因的完整的编码区段，其核昔酸序列与整合的序列相比基本一致，将其命名为 GNI（Gmp hbi’uln’ L）。该基因在 GenBank的登录号为 AF38965。2．棉花GW－1基因的序列与同源性分析 核百酸序列分析表明，GN－IK 715hp，包含一个 7llbp的开放阅读框，编码 236个氨基酸。经在NCBI中进行蛋白质相似性分析，结果表明，GhMADS－且与AGLZ类MADS盒亚家族的同源性较高，与葡萄的VVMADS4的同源性高达82％；与金鱼草的DEFH3X）的同源性为76％。而与其它特征性ipS盒基冈的同源性则仅在30＄左右。3．GhMADS－l的系统进化与结构分祈 系统进化聚类分析结果表明，该基因属于植物MIKC型MADS盒的AGLZ类亚家族成员，其功能与花器官的发育相关，主要调控与花瓣、雄蕊和心皮发育相关基因的转录和表达。该基因属植物m正C型MAD s盒结构蛋白，第1至第62个氨基酸为MAD s盒结构，第奶至第160个氨基酸为二级纬构较为保守的K盒，最后是不保守的C端，在WoS盒和K盒之间约30个氨基酸左右为不保守的！区（intervening region）。4．RTPCR表达分析 RT．PCR分析结果表明，该基因（GNS－1）在陆地棉的花瓣、雄蕊、胚珠和纤维中表达，特别是在花瓣中表达量最高，而在根、茎、叶等营养器官不表达。说明GW．1在棉花的生殖生长器官中特异表达，可能参与棉花花器宫发育的控制c5．GhMADS－l与CHVI的关系的初步分析 突变体CHVI的遗传学特性比较稳定。通过对其进行表型和扫描电镜分析，CHVI花蕾中所有花器宫均变成苞叶，与拟南芥花器官变成薯片相类似（拟南芥花器宫无苞叶结构）。GhMADS－l属于AGLZ类ioS盒亚家族成员，其功能是调控花器宫内三轮的发育。RT－PCR分析显示该基因在突变体CHVI的变异花蕾中不表达。说明GhMADLI基因在突变体中的表达可能受到抑制或该基因本身发生了夹变，从而可能与茫变体CHVI的形成有一定的联系。更多还原

【Abstract】 As an important transcription factor, MADS-box protein plays an important role in various cellular processes, especially in regulation of floral organ development. The genes related to this identity are conserved MADS-box genes whose name initially came from the first letter of MCMl(from yeast), AGAMOUS (from Arabidopsis), DEFICIENS (bom Antirrhinum) and S/ZF(from human). The MADS region linked specifically to the coalescent site of target gene and formed a homogenous or heterogenous dimer to regulate it, which can make different kinds of-floral organs for many flowering plants. So they are important factors to control the development of floral organs and to make homeotic transformations. It is very useful to clone and analyze the floral organ identity genes in order to understand the molecular model of plant floral organ development and to improve the flower varieties using gene engineering.Cotton is an important economic plant because of its fiber and its floral organ is also very specific. The floral organ mutant (CHV1) found by our lab is a precious material for us to understand the development of floral organs of cotton. Using the land cotton xuzhou 142 and the mutant CHV1 as the materials, the coding region of a cotton MADS-box protein was obtained by RT-PCR from buds of cotton based on the contig analysis and the cotton ovule & fiber EST database. Sequence comparison and functional analysis have been made and the results are as follows: 1. Cloning of GhMADS-l:The cloned fragment of 715 bp contains an open reading frame of 711 bp, coding a polypeptide of 236 amino acids designated as GhMADS-l. It has been submitted to the GenBank database and the accession number is AF538965.2. Homologous analysis of GhMADS-1:Sequence analysis shows that the deduced GhMADS-1 protein was highly homologous to the AGL2-typed-MADS proteins of Vitis vinifera, Nicotiana sylvestris, Petunia hybrida, Arabidopsis thaliana and Antirrhinum majus. Phylogenetic analysis also indicates that GhMADS-1 belongs to the novel MADS box protein type_AGL2.3. Structure analysis of GhMADS-1:The GhMADS-1 is a classical MADS-box gene of plant MIKC-type. The MADS box (l-62aa) is in the N-end and the K box (90-160aa) in the middle. The I region (intervening region) is between the MADS box and the K box which encodes a keratin-like protein structure and it is very important to make heterogenous dimer of two MADS box genes. The C-end is a non-conserved region and its function is not very clear.4. RT-PCR expression analysis of GhMADS-1:RT-PCR expression analysis shows that GhMADS-1 gene expressed in petal, anther, ovule and fiber, but not in root, stem, leaf, bract and sepal. The strongest expression of GhMADS-1 gene was detected in petals, but not in the floral buds of a somatic mutant(CHVl). It was proposed that GhMADS-1 gene would be crucial for the development of cotton floral organs and the reproductive organ development.5. The relationship between GhMADS-1 and CHV1:According to the function of AGL2 subfamily and the RT-PCR analysis of GhMADS-1 in CHV1, GhMADS-1 gene is not expressed in CHV1 shows that the gene is probably related to the construction of CHV1. To make clear the molecular mechanism of floral organ development of cotton, the continous research is ongoing.更多还原