【作者】 程晓霞；

【导师】 韩烈保；

【作者基本信息】 北京林业大学 ， 草业科学， 2010， 博士

【摘要】 日本结缕草(Zoysia japonica Steud.)是一种具有极大应用潜力的草坪草,特别在我国,日本结缕草资源位居世界首位,它具有适合地域广、抗逆性强,低维护及发达的地下根系等特征,在现代城市绿化和运动场建设及水土保持中,都占有重要地位。但日本结缕草也有苗期生长缓慢、绿色期很短等缺点,尤其绿期短已经成为限制其大量应用的瓶颈。因此,本文对延长结缕草的绿期进行研究和探索,发现GA。(赤霉素)在缓解结缕草叶片衰老中的作用,并分离GA3调控叶片衰老相关基因,对于揭示赤霉素抗衰老机理,改良草坪草特性,缓解草坪草叶片衰老都具有重要的理论意义和实践价值。首先,选用从同一克隆分化而来的结缕草,进行黑暗诱导前的GA。喷施处理。结果发现黑暗可以有效诱导结缕草的叶片衰老,具体表现在叶绿素含量降低、可溶性糖含量升高、叶片细胞死亡的加剧,同时出现MDA的增加和H2O2的累积。然而,对于GA。喷施后的结缕草叶片,以上指标都出现了显著的改善,表现出缓解黑暗诱导的叶片衰老,并且由黑暗诱导引起的氧化伤害也明显的减少。这个结果说明,尽管GA3缓解结缕草叶片衰老的原因尚不明确,但是确定了GA。在结缕草叶片衰老中的负调控作用。为了深入研究GA3缓解结缕草叶片衰老的分子事件,寻找参与过程的功能基因,利用抑制性差减杂交(SSH)技术,以GA3喷施后黑暗处理的结缕草叶片的RNA为"tester",未经处理的RNA为"driver",构建参与GA3缓解黑暗诱导叶片衰老相关基因的差减cDNA文库,共筛选到22个与GA3缓解黑暗诱导衰老有关的基因。根据blast分析结果进行基因功能预测,可将这22个基因分为5类,包括：(1)基本代谢相关基因；(2)参与信号转导过程基因；(3)细胞骨架相关基因；(4)无相似性EST；(5)未知功能基因。根据候选基因的功能推测和前人有关叶片衰老的研究结果,选取了5个可能与GA。缓解黑暗诱导衰老有关的基因(AN—氨连接合成酶基因、V-ATPase-液泡膜上H-ATPas e基因、SAMs-S-腺苷甲硫氨酸合成酶基因、CRY2-隐花色素2基因,OMT—-0-转甲基酶基因),采用RT-PCR技术分析了它们的时空表达模式(衰老的内部诱因)和外界胁迫(衰老的外部诱因)下的表达模式。从实验中初步发现,CRY2基因在幼嫩的叶片中上调表达,同时在外界胁迫的后期出现上调表达,并且在GA3和黑暗处理下出现了单因素上调表达,双因素下调表达的有趣调控模式。因此,认为CRY2基因是一个与叶片衰老最为相关的基因,并挑选其作为感兴趣的基因进行深入的功能分析。通过RACE-PCR技术扩增到CRY2基因的cDNA全长,对其编码的蛋白质氨基酸序列进行生物信息学分析,认为该基因属于隐花色素基因家族,它含有三个保守结构域：DNA光裂解酶,DNA光裂解酶FAD结合区和隐花色素C端,蓝光紫外光感受区域。同时,发现该基因与水稻的CRY2相似性最高；从进化关系上看,与玉米的CRY2的亲缘关系最近。因此,认为克隆到的基因为日本结缕草的ZjCRY2基因,通过对其进行生物信息学分析,发现其无信号肽及跨膜结构域,推测它是一个广谱表达而非膜定位的蛋白。由于前人没有报道CRY2基因与叶片衰老有关联,故利用拟南芥cry2突变体,从发育过程和黑暗诱导的叶片衰老这两个方面进行研究,证实CRY2基因参与调控自然发育过程中的叶片衰老进程。最后,为了对ZjCRY2基因的功能进行深入研究,并获得具有生产实践意义的抗衰老转基因结缕草,分别构建了ZjCRY2基因的过表达载体和RNAi敲除载体,对结缕草的胚性愈伤进行基因枪轰击转化。通过4个月的筛选继代,已经获得抗性愈伤,现在正在进行再生筛选,预计2个月之后获得转基因结缕草植株。综上所述,本文通过对GA。缓解黑暗诱导的结缕草叶片衰老现象进行深入的功能基因组学研究,发现了5个参与衰老调控的基因,并分析他们的表达模式,最后确定了其中CRY2基因参与GA3和黑暗作用下的衰老调控过程。利用RACE技术成功获得结缕草CRY2的全长cDNA序列,并且通过生物信息学的分析证明其是结缕草的ZjCRY2基因,并且利用拟南芥突变体cry2进行了功能预测。最后,构建了ZjCRY2基因的过表达和RNAi载体,并进行了转化,并即将获得转基因结缕草植株。因此,本研究的发现,将对未来结缕草抗衰老和园林植物叶片持绿的理论研究和生产应用奠定基础。更多还原

【Abstract】 Zoysiagrass (Zoysia japonica Steud.) is a kind of turfgrass with great potential applications. Especially in China, the resource of zoysiagrass lines for the first in the total of the world, and also it is suitable for the regional wide with strong resistance, low cost of maintenance and developed the underground root system. Therefore, it plays important role in the modern construction of urban greening and playgrounds, and even in water and soil conservation. However, the disadvantages of zoysiagrass as short green period has become the bottleneck of its application. In this study, aim for the extension of green stage in zoysiagrass, we found GA3 could alleviate leaf senescence induced by darkness, and separated GA3 regulation related gene, which gives more understanding on antiaging mechanism in GA3 pathway, and also is important theoretical and practical value.Here, we first show that zoysiagrass seedlings treated with GA3 could delay the leaf senescence induced by darkness. The zoysiagrass seedlings subjected to darkness manifested in sharp chlorophyll and protein loss, improved contents of MDA and H2O2, also exhibited higher cell death folds. However, the seedlings treated by GA3 exogenous before darkness stress displayed better phenotype than control. Although mechanism of GA3 on leaf senescence is unknown, it is first time reported that the GA3 plays negative role in the leaf senescence of zoysiagrass.To study expression of genes responsive to staying green in zoysiagrass, suppression subtractive hybridization (SSH) was used to identify differentially expressed genes between non-GA3-treated and GA3-treated seedlings subjected to darkness. A total of 307 ESTs were generated, of which 226 ESTs clustered into 54 contigs and 81 were singlets. Total 22 differentially expressed genes selected by subtractions were classified into five categories according to their putative functions generated by BLAST analysis, they were "General metabolism", "Signal transduction", "Cytoskeleton"; "Sequences without identity" and "Unknown function".Expression of five selected genes, OMT, SAMs, V-ATPase, CRY2 (gene of cryptochrome 2) and An were examined by RT-PCR, when the zoysiagrass were under normal senescence and senescence induced by environment. RT-PCR results demonstrated that the differential expressions of these genes were attributable to delaying senescence. Especially, the CRY2 exhibited higher expression in the young leaves, and up-regulated on the later stage of environment stress, and also display interested expression pattern under GA3 exogenous and darkness stress, so the CRY2 is selected for further research.The full length cDNA of CRY2 was obtained by RACE-PCR, and the protein encoded by CRY2 was analyzed by bioinformatics. The results showed that CRY2 belongs to the family of cryptochrome with three conserved functional domain such as DNA photolyse, FAD-binding superfamily and crypochrome C terminal superfamily. In addition, CRY2 showed high homology with OsCRY2 in rice and the phylogenetic analysis demonstrated similar pattern. Therefore, CRY2 was regarded as the ZjCRY2, a gene of cryptochrome 2 in zoysiagrass. Besides the bioinformatics analysis, the investigation on the senescence of Arabidopsis cry2 mutant was carried out, and the results also proved the CRY2 (AtCRY2) could participate in the regulation of developmental leaf senescence.Further, the over expression and RNAi expression vectors of ZjCRY2 were constructed for transformation. After embryogenic calli were pre-cultured, they were genetic transformated by biolistic bombardment. For about 4 months of subculture, the transgenic calluses with hygromycin resistance were obtained, and the transgenic seedlings would be obtained after another two months.In this study, five genes associated with leaf senescence in zoysiagrass were found through the SSH. The expression patterns of these genes were analyzed, and the CRY2 was selected finally and regarded as the functional gene related to GA3 and darkness crosstalk on the leaf senescence. Also, we obtained the full length cDNA of CRY2, and confirmed the CRY2 is the gene of cryptochrome 2 in zoysiagrass as the ZjCRY2 by bioinformatics analysis. The function of ZjCRY2 associated with leaf senescence was further predicted by analysis of Arabidopsis cry2 mutant. Finally, the vectors of over expression and RNAi expression of ZjCRY2 were constructed, and the transgenic calluses were obtained. All the results in this study, could contribute to the further research on the GA3 signal pathway related to leaf senescence and the application of transgenic turfgrass and ornamental plant with enhanced resistance to leaf senescence.更多还原