【作者】 李世国；

【导师】 侯和胜；

【作者基本信息】 辽宁师范大学 ， 细胞生物学， 2011， 博士

【摘要】 本研究在实验室内进行裙带菜配子体的发育诱导,筛选得到了配子体短时间内由营养生长向生殖生长阶段转换的适宜条件,结果显示在光强60～70μmol/m2·s,光周期14：10h(L：D),温度18±1℃培养条件下的配子体,发育诱导10d左右时间精子和卵即可大量释放。DAPI染色法是裙带菜配子体细胞核染色的一种有效方法,染色后细胞核清晰,背景干扰小,是配子体细胞分裂过程研究的有效技术。采用抑制消减杂交(SSH)技术构建了裙带菜配子体发育诱导后上调表达基因的消减cDNA文库,并对文库中的相关基因信息进行初步分析。测序结果共得到可利用的EST片段110条,占总测序样品的91.67%。去除冗余部分后得到共106条非冗余序列(NRS),非冗余序列中全部为独立EST片段(Singleton)。结合软件分析与比对结果可知,片段长度集中在100-2000bp之间,平均长度612bp。与其他物种相关的已知功能基因的氨基酸序列相似度均大于20%,主要集中在20-60%之间,部分序列无对比结果。参与比对的EST片段按其功能注释可分为以下几大类：细胞生长与发育相关18条,占16.70%；代谢相关11条,占10.00%；光合作用与光诱导相关4条,占3.60%；信号转导相关5条,占4.50%；其他功能2条,占1.80%；未知功能70条,占63.40%。密码子偏好性计算结果显示裙带菜密码子第三位碱基G和C的使用偏好无显著差别,GC使用总频率为53.05%。所分析的序列中裙带菜终止密码子的使用偏好TGA。对比显示,红藻第三位碱基对GC的使用频率较大,而褐藻和绿藻较低。通过对文库中相关EST片段的信息分析,利用基因工程手段,分离得到与减数分裂相关的裙带菜UpMND1基因。该基因全长868bp,其中编码区开放阅读框(ORF)序列长度609bp,编码202个氨基酸,序列分析表明该基因具有Mnd1蛋白的保守结构域。UpMND1基因在编码区具有4个内含子,将其分离成5个外显子部分。对该基因编码的氨基酸序列分子量、理论等电点、信号肽序列、跨膜结构、磷酸化位点、激酶磷酸化位点、亚细胞定位和二级结构的预测,以及系统进化分析的结果都进一步证实所获得的序列为裙带菜UpMND1基因。该基因编码的氨基酸序列与褐藻同源基因氨基酸序列的相似度较高,特别是与水云的序列一致性最高达到72%,进化关系最近。该序列提交到Genebank后获得登录号JF357961。利用实时荧光定量PCR技术分析显示,UpMND1基因在配子体发育诱导的不同时期以及组织表达特异性上存在差异。在裙带菜假根、茎、叶片、孢子叶等各组织器官中表达较为广泛,但在生殖器官孢子叶中相对表达量最高。配子体发育诱导初期(发育诱导2d)和精子、卵形成时期(发育诱导8d)相对表达量也会显著提高。运用农杆菌介导的基因转化法和PEG介导法分别获得了整合UpMND1基因的野生型拟南芥过表达植株、突变体拟南芥互补表达植株和酵母突变体互补表达菌株,并对拟南芥花粉母细胞减数分裂过程中染色体进行了DAPI染色。结果显示,在拟南芥植株体内过表达UpMND1基因后,染色体联会和重组过程不能够顺利完成,染色体行为紊乱,形成的小孢子中染色体分配失去应有的规律,最终导致花粉育性下降,转化植株的表型与Atmnd1突变体植株表型相似。在Atmnd1突变体植株体内表达UpMND1基因后,染色体联会和重组过程能够顺利完成,染色体行为恢复到正常减数分裂模式,形成的小孢子中染色体分配均匀,最终形成的花粉可育,植株的表型与野生型拟南芥植株相似。证实该基因功能与减数分裂过程中染色体联会和重组相关。转UpMND1基因酵母的生长曲线与Scmnd1突变体和转化空质粒的酵母生长曲线没有显著的区别,没有影响酵母有丝分裂过程。UpMND1基因并不参与到普通营养生长的配子体细胞有丝分裂调控,但在裙带菜配子体生殖细胞(精子和卵)形成过程和减数分裂过程中会产生影响。MND1基因的功能具有进化的保守性,在高等植物拟南芥(显花植物)和在低等的海藻裙带菜(隐花植物)中功能都是相似的。利用高效热不对称交错PCR(hiTAIL-PCR)方法分离得到了UpMND1基因上游638bp启动子序列。利用在线分析程序预测,该启动子区域包含CAAT-Box和TATA-Box等普通真核生物启动子共有的核心元件,并具有3个光敏感型顺式作用元件GATA-motif、GA-motif和G-Box,2个激素敏感型顺式作用元件TGA-element和ABRE,1个分生组织特异性激活元件CCGTCC-Box。用5’缺失后的UpMND1启动子序列调控报告基因在拟南芥原生质体细胞中瞬时表达,根据检测的报告基因表达活性,检验不同的启动子顺式作用元件对下游基因表达的影响。结果表明,启动子的-296--166bp区域顺式作用元件对下游基因的表达有促进作用,而-621～-296bp区域启动子序列顺式作用元件对下游基因的表达起到抑制作用。更多还原

【Abstract】 The suitable culture conditions for the conversion from vegetative growth to reproductive growth of the gametophytes in short time were screened in this investigation. The results showed that a large number of sperms and eggs can be released from the gametophytes about 10 days under the conditions that the light intensity was 60～70μmol/m2·s, the photoperiod was 14:10 h (L:D) and temperature was 18±1℃. Furthermore, DAPI staining was employed for gametophytes nuclei observation in the experiment and the clear chromosomes and weak background could be obtained under microscope using this method.The suppression subtractive hybridization cDNA library of up-regulated genes from U.pinnatifida gametophytes under the development induction was constructed.110 ESTs were obtained after sequencing the positive clones, and accounted for 91.67% of the total sequencing samples. Among these ESTs,106 fragments were non-redundant sequences. PCR analysis indicated that the length of the ESTs were ranged from 100 to 2000bp and the average length was 612bp. Based on the functional similarity with amino acids coded by homologous genes in algae and higher plants, the ESTs could be divided into several functional groups:cell growth and development (accounting for 16.70%), metabolism (accounting for 10.00%), photosynthesis and photo-induction (accounting for 3.60%), signal transduction (accounting for 4.50%) and other functions (accounting for 1.80%).70 sequences with unknown function or no comparative result accounted for 63.40% of the total ESTs. The similarity values distributed between 20% to 60% and the deduced amino acid sequences of the ESTs have higher similarity with the sequences of algae and higher plants. It was shown that the G and C usage in the third position of the codons have no significant difference in U. pinnatifida and the GC frequency of codon usage was 53.05%. The stop codon usage preferred to TGA for U. pinnatifida. The GC usage frequency in the third codon position of red algae was higher than that in brown and green algae.A homologous sequence of meiosis-related gene in U. pinnatifida was cloned with the full length cDNA of 868bp from the library by using of RACE method. The length of open reading frame is 609bp, encoding 202 amino acids that with conserved Mndl protein domain. 4 introns and 5 exons were found in the coding region of this gene. The results of the prediction for molecular weight, theoretical isoelectric point, signal peptide, transmembrane structure, phosphorylation sites, kinase phosphorylation sites, subcellular localization and secondary structure, as well as phylogenetic analysis indicated that the isolated sequence was MND1 gene of U. pinnati/ida (UpMND1). The amino acid sequence of UpMND1 gene has a high similarity to the genes of other brown algae, especially to Ectocarpus siliculosus with the similarity up to 72%. The UpMND1 gene sequence has been submitted to the Genebank with the accession number JF357961.The real-time quantitative PCR results demonstrated that the relative expression abundance of UpMNDl gene were different in all developmental stages and different tissues. It was expressed ubiquitously in many organs, such as rhizoid, stem, leave and sporophyll. However, the highest expression was detected in the sporophyll, which was a reproductive organ. The relative expression level increased significantly during the early period of gametophyte development and also in the period of sperms and eggs formation.In order to investigate biological functions of UpMND1 gene, the expression vectors of this gene were constructed and transformed into wild and mnd1 mutant plants of A. thaliana and mndl mutant of Saccharomyces cerevisiae by the method of Agrobacterium-mediated and PEG-mediated transformation, respectively. Here the phenotypes and chromosomes stained by DAPI during meiosis of wild type, overexpression type, complement expression type and mutant type of A. thaliana were observed to analyze the role of the UpMNDl gene. The growth curve of transgenic yeast was plotted and compared with the mutant. The data revealed that the overexpression of UpMNDl gene in A. thaliana in vivo resulted in the synapsis and recombination of chromosomes cannot be successfully completed and the chromosome behavior disordered in the process. As well as the pollen fertility decreased in this state. The chromosome behavior in male gametes of A. thaliana that the UpMND1 gene overexpressed was similar to the mutant plant. Similar to the wild plants, the synapsis and recombination of the chromosomes in Atmndl mutants that the UpMNDl gene complementary expressed could be successfully completed and the chromosome behavior returned to normal level. The pollen fertility also returned to normal level in these plants. It was indicated that the fuction of the UpMND1 gene has correlation with synapsis and recombination in the meiosis of U. pinnatifida. The growth curve of transgenic yeast mutant was not significantly different from the controls, showing that the UpMNDl gene was not concerned with the regulation of normal mitosis. However, this gene played regulatory function during meiosis and the formation of reproductive cells of U. pinnatifida. The evolution of MND1 gene function was relatively conservative both in the higher plants A.thaliana (flowering plants) and the algae U. pinnatifida (cryptogamic plants).A 638bp promoter sequence on upstream of UpMNDl gene was cloned by high thermal asymmetric interlaced PCR (hiTAIL-PCR) method. The results that analyzed using online programs showed that the promoter contained CAAT-Box, TATA-Box and other cis-acting elements, for example, three light-sensitive cis-acting elements GATA-motif, GA-motif and the G-Box, two auxin-sensitive cis-acting elements TGA-element and ABRE and a meristem specific activation component CCGTCC-Box. The expression levels of reporter gene in transient expression system of A.thaliana protoplast cells controlled by the promoter with different length of 5 ’deletion sequences were different. The results suggested that the distinguishing cis-acting elements could enhance or suppress gene expression in the regulation. The cis-acting elements in -296～-166bp region of the promoter showed a promoting effect on the downstream gene expression, and the cis-acting elements in -621～-296bp region could inhibit the expression of downstream gene.更多还原