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题名: 竹类植物开花时间相关基因的克隆与功能分析
作者: 田波
学位类别: 博士
答辩日期: 2005
授予单位: 中国科学院昆明植物研究所
授予地点: 中国科学院昆明植物研究所
导师: 李德铢
关键词: 竹类植物 ; MADS-box基因 ; 原位杂交 ; 转基因植物 ; CO-hke基因
学位专业: 植物学
中文摘要: 花发育是有花植物生活史中一个极其重要而复杂的事件,它既涉及到细胞的分裂与分化,又涉及到组织和器官的形成,它既受环境因子的影响,又受自身生理信号的调控,它是一系列基因表达综合作用的结果。本论文在本实验室已克隆到一些麻竹MADS-box基因的基础上,对其中两个基因进行了功能分析;同时,通过TD-PcR方法从草丝竹的总DNA中克隆了其CO-like基因的全长,并利用GENSCAN在线预测了其结构。主要研究结果如下:IDIMADS的表达模式以引种栽培的麻竹的花为材料,进行RT-PCR和RNA原位杂交实验,结果表明,DIMADSS在麻竹成熟花的雄蕊表达强烈,主要集中在花粉囊和花粉粒,在雌蕊和内释有微弱的表达,而在外释和颖片中不表达。该表达模式与水稻的OSMADS5的表达模式极为相似。DIMADS8和OSMADS5均为sEP-like基因,DIMADS8可能只是麻竹中多个SEP-like基因中的一个。ZDIMADS8和DIMADS8基因的异位表达利用双元载体pCambia2301和pWM101构建了一个含GuS报告基因和NPT 11基因的中间表达载体pCambia23ol-101。将DIMADS8和DIMADS18分别置于CoMV355启动子下转化拟南芥355D劫了注DSS转基因的拟南芥植株叶卷曲,开花时间提前,抽苔后表现出三种不同的表型。表型I植株不产生二级花序,Northern杂交只检测到很弱的信号,DI彻DSS可能与拟南芥的内源基因发生了共抑制;表型n植株茎生叶严重卷曲,花瓣和花曹发育迟缓,不能完全覆盖雌雄蕊;表型111植株抽苔后产生顶花,顶端优势明显减弱,二级花序发达。表型n、m植株均检测到很强的D从似DSS表达信号。35斗D从“Ds18转基因高表达植株开花时间明显提前、叶卷曲、植株矮小、产生顶花。总之,D动翻DSS和力从侧DS18可能参与调控麻竹的开花时间,其表达可能受BA的诱导。3竹类植物CO-like基因的克隆以灌木状竹类植物草丝竹(Yushaniaandropogonoides)的总DNA为模板,通过TD-PCR和常规PCR方法克隆到一个CO-like基因YaCO了。GENSCAN在线预测结果表明,该基因有一个长达567bp的内含子,开放读码框长IO32bp,推导编码344个氨基酸,与小麦的TaHd的氨基酸相同率最高(64%)。在氨基酸序列的氨基端,有一个类似锌指蛋白的B一box结构域(cxZcxscx7CxZCx4Hx8H),梭基端有一个由43个氨基酸组成的CCT结构域。
英文摘要: Floral development is a very important and complicated event in the life cycle of flowering plants. It involves the cell division and differentiation, as well as tissue and organ formations and is controlled by both environmental and endogenous physiological inputs. All are regulated by an intricate gene network.. Our laboratory had cloned some MADS-box genes from the sweet bamboo, Dendrocalamus latiflorus. In this paper, we analysed the function of two bamboo MADS-box genes, DIMADS8 and D1MADS18. In addition, we cloned a CO-Iike gene from the genomic DNA of another bamboo species, Yushania andropogonoides by TD-PCR and predicted its structure by using the online with the GENSCAN program. The major results are as follows. 1 The expression pattern of DIMADS8 The flower materials were collected from cultivated plants. The results of RT-PCR and in situ hybridization indicated that D1MADS8 was strongly expressed in the stamens of D. latiflorus mature flowers, mainly in anthers and pollens, with very low expressions in pistils and paleas, but without expressions in lemmas and glumes. This patterns was very similar to that of OsMADSS isolated from rice, Oryza sativa. DIMADS8 and 0sMADS5 belonged to SEP-like genes. Maybe there were many SEP-like genes in D. latiflorus as in Oryza sativa, 2 Ectopic expression oiDlMADS8 and D1MADS18 Using binary vectors pCambia2301and pWMlOl, we constructed a middle expression vector pCambia2301-101 which contained GUS report gene and NPT II gene. cDNAs of DIMADS8 or D1MADS18 driven by the CaMV 35S promoter were transformed into Arabidopsis plants for functional analyses, respectively. 35S: DIMADS8 transgenic Arabidopsis plants showed the phenotypes of early flowering and little curled leaves. After flowering, they were separated into three different phenotypic transgenic groups. First, phenotype I plants without producing secondary inflorescences. Only very weak D1MADS8 northern hybridization signals were detected in this group. It was possible that there were co-suppressions between DIMADS8 and other endogenous genes. Second, phenotype II plants producing severe curled cauline leaves. Their petals and sepals were shorter, and did not covere the pistil completely. Third, phenotype III plants producing terminal flowers at the end of the bolting. More secondary inflorescences were induced because of reduced apical dominance, Strong D1MADS8 northern hybridization signals were detected in phenotypell and phenotypelll plants. Transgenicc plants of DIMADS18 exhibited the phenotypes of curled leaves, dwarfism, and early flowering with clustered terminal flowers. These results indicated that DIMADS8 and D1MAJDS18 probably be involved in controlling the floweing time of D. latiflorus. Because the two genes were cloned from the young spikelets from in vitro flowering cultures of D. latiflorus, it was very likely that they could be induced by a cytokine, BA. 3 Isolation of a bamboo CO-like gene Template genomic DNA was extracted from a shrubby bamboo Yushania andropogonoides. A CO-like gene named as YaCOl was isolated by TD-PCR and normal PCR. We predicted its structure using online GENSCAN program. The results indicated that the gene have an intron of 567bp long, and the ORF was 1032bp long encoding a putative 344-amino acid protein which showed 64% identity to TaHdl of wheat. There was a zinc-finger B-box (CX2CX8CX7CX2CX4HX8H) near the amino terminus and a region of 43 amino acids near the carboxy terminus termed the CCT(CO, CO-like, TOCl) domain.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/788
Appears in Collections:昆明植物所硕博研究生毕业学位论文_学位论文

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竹类植物开花时间相关基因的克隆与功能分析.田波[d].中国科学院昆明植物研究所,2005.20-25
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