【作者】 于彦丽；

【导师】 郑成超； 张凌云；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 在显花植物授粉受精过程中,具顶端极性生长特性的花粉管是雄性生殖单位的载体,也是研究细胞生长分子调控机理的理想体系。与被子植物相比,裸子植物具有生长周期长,花粉管生长缓慢、易分叉等特点,具有不同于被子植物花粉发育的独特发育模式。对于裸子植物花粉萌发和花粉管生长的调控机理,目前尚不十分清楚。本文以松类植物中比较有代表性的裸子植物青杄(Picea willsonii)花粉为试材,构建青杄花粉和花粉管的cDNA文库,并通过RT-PCR方法克隆到微管蛋白基因TUA1,从Ca2+诱导青杄花粉cDNA文库中分离得到CCAAT框结合蛋白类型的转录因子基因HAP5。对这两个基因进行表达分析和功能鉴定,结果如下:1.利用Clontech SmartTM cDNA Library Construction Kit构建青杄花粉及花粉管的cDNA文库,根据同源序列设计兼并引物,通过RT-PCR的方法获得微管蛋白基因TUA1的中间片段,经5’-和3’-RACE PCR,得到其全长cDNA,为1721bp,编码区全长为1353bp,编码含451个氨基酸的多肽,分子量为49.58kDa。在Ca2+诱导的青杄花粉cDNA文库中筛选得到基因PwHAP5,该基因编码一个CCAAT框结合蛋白类型的转录因子,cDNA全长为985bp,编码区全长为603bp,编码含201个氨基酸的多肽,分子量为22.44kDa。2.同源序列比较发现,青杄的微管蛋白基因TUA1与拟南芥、水稻、棉花和花旗松的微管蛋白有高度的序列同源性,分别为88.47%、96.9%、97.78%和99.78%。聚类分析表明PwTUA1属于I类α-微管蛋白。青杄的转录因子PwHAP5与拟南芥、水稻、酵母和人类HAP5的关键结构域都比较保守,与拟南芥的AtNF-YC9(AtHAP5C)和水稻的OsHAP5A/B亲缘关系较近。3.半定量RT-PCR分析表明PwTUA1在青杄花粉中特异表达,且表达水平随着花粉萌发过程逐渐升高,12小时达最高值,随后逐渐下降,并且在花粉萌发的各阶段均受到Ca2+和硼的诱导。PwHAP5在青杄花粉、针叶、茎和根中均有表达,随着花粉萌发时间延长,表达量逐渐升高,18小时达峰值后一直保持不变,在不同萌发时段PwHAP5表达均受Ca2+的诱导。4.在青杄花粉中超表达PwTUA1可以提高花粉的萌发率和花粉管的生长速度,而超表达PwHAP5可以改变花粉管生长方向。5.在拟南芥中组成型超表达PwTUA1使得转基因植株生长迟缓,出现螺旋生长,甚至由于不能够完成形态建成而死亡。在拟南芥花粉中专一表达PwTUA1可以促进花粉的萌发和花粉管生长,即使是在Ca2+和硼浓度非最适培养基中同样可以起到促进作用,并且PwTUA1在拟南芥花粉中的表达改变了TUA蛋白(微管蛋白α亚基)在花粉管中的分布模式以及花粉管的超微结构。野生型拟南芥中TUA蛋白主要定位于除花粉管尖端区域以外的整个花粉管中,而在转基因拟南芥花粉管的顶端区域有大量的TUA蛋白存在。野生型拟南芥花粉管壁远远厚于转基因拟南芥,在转基因拟南芥花粉管顶端存在许多短的游离的胞质微管,并且富集小泡,而在野生型拟南芥花粉管的相应位置中没有发现这些结构。6.在拟南芥中超表达PwHAP5后,会影响果荚的排列,但不影响花粉管的生长方向。用青杄转录因子PwHAP5的N末端77个氨基酸、C末端130个氨基酸及全长分别做诱饵筛选青杄花粉和花粉管cDNA文库,共得到约80个克隆,经重新验证活性后,选择结合活性较强的克隆测序。在测序结果中筛选到7个与PwHAP5全长、N端和C端均相互作用的克隆。GenBanK中Blast结果发现这些克隆与拟南芥基因组中的同类基因有较高同源性。选择其中的PwFKBP12蛋白与PwHAP5通过双分子荧光互补实验进行体内互作的验证。更多还原

【Abstract】 Pollen tube, a carrier of sperm nuclei during the process of sexual plant reproduction in flowering plants, is a highly polarized, rapidly tip-growing plant cell, and thus is an ideal system for studying the molecular mechanism involved in the regulation of cell growth. As compared with angiosperm, pollen tubes of coniferous species grow slowly, tend to ramify, and lack a tip-to-base zonation of organelles, implying that tubes of the two types of plant differ in developmental mechanism and wall construction. However, the mechnisms of pollen germination and tube growth in coniferous species remain enigmatic. Thus, in this paper, we constructed pollen and pollen tubes cDNA library and cloned a cDNA fragment ofα-tubulin gene (TUA1) in coniferous species (Picea wilsonii). In addition, a Ca2+-induced cDNA library of Picea wilsonii pollen was constructed and a CCAAT-box binding factor gene (HAP5) was obtained by differential hybridization. A series of studies have been conduced on the sequence and expression analysis and functional identification of PwTUA1 and PwHAP5.The obtained results are as follows:1. A cDNA library of Picea wilsonii pollen and pollen tubes was constructed with Clontech SmartTM cDNA Library Construction Kit. Two mixed primers were designed to amplify specific DNA fragment using cDNA library on the homologous sequences from other plants. The middle fragment of interested cDNA was obtained by RT-PCR. The full length cDNA was further isolated by 5’-RACE and 3’-RACE. The isolated cDNA is 1721 bp in length and harbors an opening reading frame of 1353 bp encoding a 49.58 kDa protein of 451 amino acids. A Ca2+-induced cDNA library of Picea wilsonii pollen was constructed and a CCAAT-box binding factor gene (HAP5) was obtained by differential hybridization. The isolated cDNA is 985bp in length with an open reading frame (ORF) of 603bp comprising 201 amino acid residues with the predicted molecular mass of 22.44kDa. 2. Multi-alignment analysis revealed that PwTUA1 was highly related to other plant TUAs, sharing a homology of 88.47% to AtTUA1, 97.78% to GhTUA1, 96.9% toOsTUA1, and 99.78% to PmTUA1. The phylogenetic analysis of various TUAs also indicated that PwTUA1 was attributed to class I . The main functional domains of PwHAP5 were conserved compared with other HAP5s from Arabidopsis, rice, yeast and human. The phylogenetic analysis also indicated that the kinship of PwHAP5 was close to AtNF-YC9(AtHAP5C) and OsHAP5A/B.3. Semi-quantitative RT-PCR was used to examine the expression of PwTUA1 and PwHAP5. The results showed that PwTUA1 was expressed specifically in germinating pollen but was not detected in needles, stems, and roots. PwTUA1 was first detected at 6 h after incubation (just germinated); it was abundant during middle developmental stages (12, 18, and 24 h after incubation) and then continued weakly at later stages (30 and 36 h after incubation). The expression of PwHAP5 was detected in all of the tissues and increased during the pollen germination stages till reached the peak after incubation for 18h and keep to the tubes stop growing.4. Transient expression of PwTUA1 improved pollen germination and pollen tube growth in Picea wilsonii, while transient expression of PwHAP5 changed the orientation of pollen tubes growth.5. Arabidopsis overexpressing PwTUA1 driven by the CaMV 35S promoter showed growth retardation and helical growth to some degree. Some of them were unable to complete morphogenesis, resulting in death. When PwTUA1 was expressed in Arabidopsis pollen exclusively by using the pollen-specific promoter Lat52, both pollen germination and pollen tube growth were enhanced in the transgenic lines when compared to wild-type Arabidopsis, even at the nonoptimal boron or Ca2+ concentrations present in the culture medium. PwTUA1-overexpression changed the subcellular localization of theα-tubulin protein and affected the ultrastructure of pollen tubes in Arabidopsis. Theα-tubulin protein existed throughout the tubes of wild-type Arabidopsis, excluding the tip or apical region. However, highly intense fluorescence was observed in the apical region of transgenic Arabidopsis pollen tubes. The pollen tube wall of transgenic Arabidopsis was only slightly detectable, while the wall of wild-type Arabidopsis was much thicker. Additionally, many short, solitary cytoplasmic MTs were observed in the tip-most portion of the transgenic pollen tubes where the vesicles accumulated, but were rarely observed in the tip region of wild-type pollen tubes.6. Overexpressing PwHAP5 changed the arrangement of transgenic Arabidopsis siliques instead of the orient of pollen tubes growth. Interactive proteins were screened from Picea wilsonii pollen cDNA library with the N, C terminal and full-length of PwHAP5 as bait protein by Yeast Two-Hybrid System. More than 100 positive clones were identified by PCR colony screening and the representative clones were sequenced. Among them 7 clones were selected which interacted with C, N terminal and full-length PwHAP5. By blast in GenBank, the prey cDNA inserts were homologous to mRNA sequences in Arabidopsis. PwFKBP12 protein was selected and we tested its interaction with PwHAP5 in vivo by using bimolecular fluorescenc complementation (BiFC) analysis.更多还原