【作者】 庄云龙；

【导师】 张举仁；

【作者基本信息】 山东大学 ， 细胞生物学， 2007， 博士

【摘要】 玉米不仅是重要的粮食和饲料作物,也是重要的食品工业原料和能源植物。玉米是我国第二大粮食作物,玉米生产在我国国民经济中占有十分重要的地位。我国有60%的玉米种植面积受到干旱胁迫,每年因旱灾而减产20%～30%,直接影响国民经济发展及灾区人民生活。在玉米的生育期中,从孢子母细胞到受精和种子形成的早期阶段对包括干旱胁迫在内的各种胁迫最为敏感,其中有两个敏感高峰期:第一个是从减数分裂到四分体时期,这期间的胁迫可引起配子的不正常发育;第二个高峰期是从开花到籽粒发育的起始,这期间的胁迫可影响授粉或受精,诱导了花的脱落或新形成籽粒的败育。因此搞清楚干旱对开花期玉米的伤害及玉米在此期对干旱的适应和调节机制,对于培育抗旱玉米品种具有重要意义,也是国内外学者们关注的重要课题之一。要探索某一植物对某种胁迫所产生的应答,需要对胁迫应答的基因表达变化有一个整体的认识。由于基因芯片具有一次可以同时分析成千上万基因表达的强大优势,近年来己被用来分析多种植物在多种胁迫或刺激下的基因表达谱。对拟南芥在干旱、冷、盐、强光、损伤、氧化、紫外线等胁迫下的表达谱分析已有较多报道。利用芯片技术研究水稻和其它作物在胁迫条件下基因表达变化的工作也报道较多。玉米在逆境下的基因表达谱研究已有较多的报道。在干旱胁迫下,玉米幼苗叶片和根有着不同的基因表达模式,一个乙烯信号途径可能参与了玉米对干旱的反应。在盐胁迫处理3h时玉米根的转录组变化最为剧烈,这些表达变化的基因中最为显著的是参与运输和信号转导途径的基因。对抽丝前和抽丝后雌穗在遮阴胁迫下的基因表达研究发现,参与ABA信号途径的基因表达上调;参与淀粉合成的基因表达下调,而参与淀粉降解的基因表达没有变化。对抽丝后未受粉的雌穗和授粉后8天的籽粒在缺水胁迫下的基因表达谱研究发现,不同的组织对胁迫的敏感程度不同,共有17个基因在不同的组织中都呈现差异表达,这些基因参与广泛的生物学反应。对授粉后15天的玉米籽粒在干旱和盐胁迫下的基因表达谱分析发现,在这两种胁迫下都上调表达的基因主要是参与非生物胁迫、损伤和病菌侵染的应答基因,而共同表达下调的主要是参与能量代谢的基因。从以上结果来看,芯片表达谱的研究主要集中在玉米苗期和在生殖期中对胁迫敏感的第二高峰期,而在玉米生殖期对干旱胁迫敏感的第一个高峰期的基因组表达谱研究未见报道。本实验以处于对干旱敏感的第一高峰期的雄穗和处于小花分化期的雌穗为材料,利用玉米基因组芯片(microarray)检测了雌雄穗在玉米植株受到缺水胁迫1天和7天时的基因表达谱。该芯片包含了57452个转录本,代表了3万多个基因。我们以处理和对照样品的信号值的比值不低于3倍的转录本为有意义的差异表达转录本。经过1天的缺水胁迫,雌穗中表达上调的转录本有70个,表达下调的有89个(占差异表达转录本的56%);在雄穗中表达上调的转录本有191个,表达下调的有681个(占差异表达转录本的78%)。在7天的缺水胁迫下雌穗中表达上调的转录本有169个,表达下调的有33个,表达上调的转录本占差异表达转录本的84%;在雄穗中表达上调的转录本有939个,表达下调的有574个,表达上调的转录本占差异表达转录本的62%。在雌雄穗中差异表达的转录本共有2552个。雌雄穗的表达谱差异较大。在雌雄穗中都表达上调的转录本有73个,都表达下调的有17个。通过对这些差异表达的转录本的生物信息学分析,发现参与代谢的转录本的比例最大,其中比较突出的是参与蔗糖、棉子糖和海藻糖代谢途径的转录本。另外,在雄穗中参与信号转导和细胞壁代谢的转录本也占据了相当大的比例。这些差异表达的基因可能是对缺水胁迫的应答基因。利用来自叶片差减文库的514个单一(unique)ESTs制作了尼龙膜cDNA宏阵列(macroarray),研究了在缺水胁迫下这些ESTs在雌雄穗中的表达变化。以处理和对照样品的信号值的比值不低于3倍的ESTs为有意义的差异表达ESTs。在1天和7天缺水胁迫下,雌穗中表达上调的ESTs分别有58和106个:雄穗中表达上调的ESTs分别有79和151个。在雌雄穗中表达上调的ESTs共有222个。对这222个ESTs进行功能分类分析,发现参与代谢和信号转导的ESTs占据了主要份额,这与芯片的实验结果相一致。我们把这222个ESTs与玉米芯片上oligo所代表的序列进行比对,序列相匹配的有184个,约占82%,其中有125个在芯片和宏阵列实验中都表现上调,约占68%。表明这两个检测试验的结果一致性较好。在用玉米基因组芯片和宏阵列进行的检测试验中都出现一个受干旱胁迫诱导表达的候选谷氨酸脱羧酶(GAD)基因,我们克隆了该基因的cDNA全长,并在大肠杆菌中实现表达。通过对融合蛋白酶活性分析,证明该基因的确为谷氨酸脱羧酶基因,命名为ZmGAD1。序列分析表明,ZmGAD1编码蛋白具有谷氨酸脱羧酶保守的两个结构域,即序列中部的磷酸吡哆醛(PLP)结合位点和C端的钙调素(CaM)结合位点。Southern blotting分析表明,该基因在基因组中存在一个拷贝。利用相似性搜索获得了ZmGAD1的基因组基因序列,并发现ZmGAD1基因定位在1号染色体上,由7个外显子和6个内含子组成。利用电子克隆,获得了玉米中谷氨酸脱羧酶基因的另一个成员全序列,命名为ZmGAD2。序列分析发现ZmGAD2的C端没有结合CaM的结构特点。分析ZmGAD2的基因组基因序列,发现ZmGAD2基因定位在2号染色体上,由4个外显子和3个内含子组成。ZmGAD1和ZmGAD2分别与水稻OsGAD1和OsGAD2的进化关系较近,而且它们的基因组基因的结构特点相似,推测它们可能是不同物种中的直向同源基因(orthologs)。表达谱分析表明,ZmGAD1在根、叶片、茎、雌穗和雄穗中都有表达,在雌穗中表达强度最高,在叶片中表达强度最低。在PEG、盐、冷、ABA和MeJA处理下,玉米苗期的叶片中ZmGAD1呈现上调表达;在根中,ZmGAD1的表达变化与其在叶片中有差异。分析克隆的ZmGAD1编码区的上游序列,发现含有较多的胁迫应答元件,如ABRE等,表明ZmGAD1基因的表达受转录水平的调节。测定胁迫下玉米叶片和根GAD酶活性,发现随着处理时间的延长,GAD的酶活性持续升高,与该基因在转录水平的变化不完全一致,推测ZmGAD1的酶活性也受翻译后调节。此外,本工作构建了含有ZmGAD1的植物表达载体,为进一步研究ZmGAD1在植物处于逆境中所起的作用打下了基础。谷氨酸脱羧酶(GAD,EC 4.1.1.15)是GABA合成的关键酶,GABA在植物对逆境的反应中可能有重要的生理作用,克隆的ZmGAD1基因可能有较好的应用前景。更多还原

【Abstract】 Maize(Zea mays L.)is not only an important economic crop in the world,but also a vital resource for forage and food industry.In our country about 60%of maize area is under water-deficit stress,which causes 20%-30%of reduction every year and affects the development of national economy and the normal life of people in the arid field.The extent to which crop productivity is affected depends largely on the development stage at which the plants encounter stresses.Among these stages,reproductive development from meiosis in the spore mother cells to fertilization and early seed establishment is extremely sensitive to various stresses, such as drought.Two peaks of sensitivity are encountered within this period.The first is centered on the period from meiosis to tetrad break-up in anthers.The second peak occurs during anthesis and initial stages of grain development.Stresses in this period cause a variety of abnormalities in floral organs which interfere with pollination or fertilization,and induce abscission of flowers or abortion of newly formed grains.Understanding the mechanism of plant tolerance to environmental stresses and the molecular basis of plant responses to water stress,especially at extremely sensitive stage might provide new strategies to improve the stress tolerance of agriculturally important plants.Understanding maize responses to water stress requires a comprehensive evaluation of stress-induced changes in gene expression and is expected to advance our insight into crop improvement.Microarray provides an analytical tool by which thousands of genes can be studied at one time.cDNA or Oligo microarray has recently been used to monitor global gene expression in response to several abiotic stresses in higher plants especially in Arabidopsis and rice.Recently,this technology has been applied to analyze maize gene expression profiles under various stress conditions.The leaves and roots of maize seedlings had different expression profiles under drought treatment,and an ethylene signaling pathway might be involved in the maize response to this stress.The response of the transcriptome of maize roots to salt stress was rapid and transient,leading to a burst changes after three-hour salt treatment.Of the salt induced ESTs,genes involved in the transport and signal transduction pathways were prominent.The results of gene expression profiles in ears before and after silking under shade stress showed that genes concerned in the ABA signal pathway were up-regulated,and genes involved in starch synthesis were down-regulated while genes of starch degradation didn’t have significant changes.Oligonucleotide microarrays were used to examine genes expression at 4 days after silking and 8 days after pollination in maize ear and kernel in response to water-deficit stress.The result showed that the sensitivity to the extent of stress were different in different tissues.Only 17 genes,involved in many responses,expressed differentially in all the tissues.During water- and salt-stress treatments of developing kernels at 15 days after pollination,the genes involved in various stress responses(abiotic,wounding and pathogen attack)were up-regulated and the gene predominantly involved in energy generation were down-regulated in both stresses.Collectively,those researches were focused on the maize seedling and reproductive stage at the second peak of sensitivity to abiotic stress.However,the mechanistic bases of cellular response at the first sensitive peak to water deficit are still not been reported previously.To advance our understanding of the response to water deficit stress in the maize reproductive organs at the first peak sensitive to water deficit,we monitored gene expression in the developing immature tassels and ears under water deficit stress using oligo microarray slides containing 57,452 transcripts.After 1 day and 7 days of stress,immature tassels and ears differed considerably in their transcriptional responses,and the majority of changes were organ specific.By 1 day of stress,70 transcripts were up-regulated and 89 transcripts(56%of differential expression transcripts)were down-regulated in the ears;191 transcripts were up-regulated and 681 transcripts(78%of differential expression transcripts)were down-regulated in the tassels.By 7 days of stress,169 transcripts(84%of differential expression transcripts)were up-regulated and 33 transcripts were down-regulated in the ears;939 transcripts(62%of differential expression transcripts)were up-regulated and 574 transcripts were down-regulated in the tassels.There were obvious differences between the expression profiles of the ears and tassels.Only 73 transcripts were up-regulated and 17 were down-regulated in the two organs.Most of these transcripts have not been previously reported to be associated with water-deficit stress and are involved in a broad range of cell metabolisms,predominately in sucrose, trehalose and raffinose metabolism,and in cell wall metabolism and signal transduction in the tassels.These transcripts may be the genes response to water-deficit stress.A cDNA macroarray containing 514 unique ESTs in the subtracted cDNA library from maize leaves under water-deficit stress was constructed and used to analyze their expression profiles in maize immature ears and tassels during water-deficit stress.The results indicated that 58 ESTs by 1 day of stress and 106 ESTs by 7 days of stress were significantly up-regulated in the ears under water stress. Correspondingly,79 and 151 ESTs were significantly up-regulated in the tassels by 1 day and 7 days of stress.All of 222 ESTs,found to be up-regulated for at least one time-course point in either maize ears or tassels,were used for the hierarchical cluster analysis,and the results suggested that most ESTs were involved in cell metabolism, which was a good agreement with the microarray result.When the 222 ESTs were aligned with the sequences represented by 70 mer oligo on microarray using BLASTN program,184 ESTs were shown with good matched sequences.About 68%of these 184 ESTs were co-upregulated in the microarray and macroarray experiments,which showed a good agreement between them.A full-length cDNA,putative glutamate decarboxylase designated ZmGAD1, induced by water stress both in the microarry and macroarry experiments,has been isolated by the combination of bioinformatics and PCR based approaches.The entire cDNA ORF coding for the ZmGAD1 protein was inserted into the pET30a(+) expression vector.The recombinant protein showed the activity of glutamate decarboxylase.Sequence analysis showed that this protein had two conserved domains of glutamate decarboxylase,a pyridoxal-5’-phosphate(PLP)binding domain in the middle region of the peptides and a calmodulin(CaM)-binding domain at the carboxyl terminus.Southern blotting analysis suggested that the ZmGAD1 was a single-copy gene in the maize genome.The genomic sequence of ZmGAD1 was cloned,showed that ZmGAD1 was located on Chromosome 1 and had 7 exons and 6 introns.Based on a short mRNA sequence,we cloned another maize GAD gene in silico,designated ZmGAD2,which also had an entire ORF.Sequence analysis showed that ZmGAD2 didn’t have the CaM site.ZmGAD2 was located on Chromosome 2 and had 4 exons and 3 introns.A homology tree of GADs from various plant species revealed ZmGAD1 had a closer evolutionary relationship with rice OsGAD1,and ZmGAD2 with OsGAD2,更多还原