【作者】 郭林林；

【导师】 李学宝；

【作者基本信息】 华中师范大学 ， 生化与分子生物学， 2005， 硕士

【摘要】 植物肌动蛋白(actin)骨架在高度特化的细胞如花粉管、根毛、纤维等的发育及形态建成中起重要作用。许多研究表明,ADF/cofiun(actin depolymerizing factor/cofilin)对肌动蛋白骨架的组装和功能至关重要,而肌动蛋白骨架对细胞伸长和顶端生长是必需的。本实验室从棉花cDNA文库中筛选出来7个ADF基因,并对该基因进行了分子克隆和表达分析,以此深化对ADF基因结构,基因的组织,保守性及相关功能的认识。 1.序列分析 棉花(Gossypium hirsutum)和其他来源的ADF/cofilin家系12个基因都含有420bp左右的开放阅读框,编码140个氨基酸左右的蛋白质,有着高度的同源性。进化树分析表明棉花(Gossypiumhirsuturn)ADF蛋白质分为四个进化群,GhADF218独立于其他的棉花ADF的进化分支之外,且与目前已经确定的花粉管特异表达的五个ADF基因蛋白序列同源性高,由于蛋白质的基本结构决定蛋白质的功能,因而可以推测GhADF218也许与花粉管的发育有关 2.基因表达分析 提取棉花发育早期各组织的RNA,对各个基因的表达进行Northern杂交分析。其中,GhADF105在胚珠,纤维和下胚轴中表达信号强,而在其他组织中信号弱,表明该基因在棉花各个组织中差异表达。GhADF218在花药中特异表达,表明该基因与花药组织的发育有关。本实验初步表明,ADF在棉花的不同组织中表达量不同,呈组织特异性。 3.载体的构建与转化 利用DNA重组技术,将ADF基因插入到pPZP221和pBI121中,分别构建更多还原

【Abstract】 Actin cytoskeleton is critical for the development and morphogenesis in plant cells such as pollen tubes,root hairs and fiber cells.A lot of studies showed that ADF/cofilin is essential to the function and organization of actin cytoskeleton which is necessary to cell elongation and tip growth. To investigate gene structure, conservation and function in cotton, we isolated seven ADF/cofilin genes which belong to a gene family from cotton cDNA library and do some analysis.1. Sequence analysisAll the 11 ADF/cofilin genes from cotton (Gossypium hirsutum ) and other plants contain about 420 bp open-reading-frame which encode a protein with 140 amino acids or so and share high homology each other. In this study, the 7 cotton ADFs deduced from the isolated GhADF genes have diverged into 4 subclasses, GhADF218 belong to a single clade.There are higher similarity among GhADF218 and those pollen-specific ADF genes from other plants in the DNA and protein sequences, suggesting that those genes may have very close evolutionary relationship,which manifests that GhADF218 probably involves in the pollen tube development.2. RNA gel blot analysisRNA was extracted from young cotton tissues, we analyzed the expression patterns of GhADF218 and GhADF 105 gene by RNA gel blot, using 3’-untranslated cDNA fragments as probes. In these experiments, GhADF218 transcripts were found in anther specificly, while GhADF105 transcripts were detected almost in all organs although their expression levels in ovules, fibers, anthers and hypocotyls were relatively higher.3. DNA cloning and transformationDifferent ADF fragments were cloned into pPZP221 and pBI121 to obtain overexpression vector and supression vector respectively. Cotyledon and hypocotyl explants from Coker 312 were transformed using Agrobacterium-mediated transformation.更多还原