【作者】 倪勇祥；

【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， 2007， 硕士

【摘要】 同源异型域-亮氨酸拉链蛋白(homeodomain-leucine zipper protein，HD-Zip)是高等植物中特有的一类转录因子。研究表明，HD-zip对植物的早期发育起到至关重要的作用，如叶的发育、茎的延长以及在逆境条件下的生长调节等。我们从棉花cDNA文库中筛选出4个编码HD-Zip转录因子的基因，并对该基因进行了分子鉴定和表达分析，获得了如下的研究结果：1．从棉花(Gossypium hirsutum)中分离4个编码HD-Zip转录因子的基因，分别命名为GhHB(Gossypium hirsutum homeobox)1，GhHB2，GhHB3和GhHB4。蛋白质初级结构分析显示4个GhHB蛋白序列中都含有同源异型域(homeodomain，HD)和亮氨酸拉链区域(lecuine zipper region)，且都含有多个蛋白激酶C磷酸化位点和酪蛋白激酶Ⅱ磷酸化位点。2．进化树分析表明4个棉花与来自不同植物的40个HD-Zip蛋白质可分为2个进化大支，GhHB1，GhHB2，GhHB3和GhHB4分别位于这两个进化支上，且它们HD部分同源性比较分析也显示出它们在进化上的保守性。GhHB1，GhHB 2，GhHB 3和GhHB 4分别与OsHox3，HAT4，PpHB3和ANL2有最近的亲缘关系。3．提取棉花不同组织的总RNA，反转录得到cDNA。利用实时荧光定量PCR对4个基因的表达情况进行分析。结果显示，GhHB1，GhHB2，GhHB3初GhHB4都在根和下胚轴有较强的表达，其中GhHB4在叶中也有很大的表达量。这表明这些基因与棉花早期发育有关。同时，GhHB1在根中的表达受到了NaCl，ABA和GA3调控。4．利用DNA重组技术，将GhHB3基因插入到pPZP221和pBI121中，分别构建了过量表达载体(overexpression vector)和RNAi载体(supression vector)。利用农杆菌(Agrobacterium LBA4404)介导的转化方法，成功地将GhHB3转入到棉花中，现已经得到了大量的转化小苗，部分已被鉴定为阳性。更多还原

【Abstract】 Homeodomain leucine zipper (HD-Zip) is one type of transcriptional factor. Its structures consist of a homeodomain and a leucine zipper region. HD-Zip encoding genes are identified only in plant. HD-Zip plays an important role in morphological changes of plant, such as leaf development, stem elongtion, hormone response and so on. In our study, we isolate four HD-Zip encoding genes. Structure, conservation and function four genes are investigated. The results as follows:1. We isolate four HD-Zip protein-encoding genes, named GhHB1 (Gossypium hirsutum homeobox) 1, GhHB2, GhHB3 and GhHB4. The elementary anlysis of four HD-Zip protein sequences from cotton (Gossypium hirsutum) show that all of them have many PKC_PHOSPHO_SITEs and CK2_PHOSPHO_SITEs. The analysis also shows that they possess homedomain and leucine zipper region.2. Phylogenetic analysis indicates that all 44 HD-Zip proteins from different plant are divided into two clades. GhHB1, GhHB2, GhHB3 and GhHB4 are belonged to those two clades, suggesting that those genes may have very close evolutionary relationship. GhHB1, GhHB2, GhHB3 and GhHB4 have the closest relationship with OsHox3, HAT4, PpHB3 and ANL2, respectly.3. RNA was extracted from cotton tissues; we analyzed the expression patterns of GhHB1, GhHB2, GhHB3 and GhHB4 by realtime SYBR-Green fluorescence quantity PCR. The results indicate that GhHB1, GhHB2, GhHB3 and GhHB4 are predominantly expressed in roots and hypocotyls, suggesting that it may function in roots and hypocotyls development. GhHB4 is also expressed in cotyledon. GhHBl expression in roots and hypocotyls is also up-regulated by NaCl, GA3 and ABA, suggestting that the GhHB1 may play an important role in response to salt-stresses and to different phytohormone signalings.4. Different GhHB3 fragment was cloned into pPZP221 and pBI121 to obtain overexpression vector and supression vector respectively. GhHB3 fragment was transformed into cotton by Agrobacterium-mediated transformation method. A great number of transformed seedlings have been obtained. Some seedlings have been indentified.更多还原