【作者】 陈明辉；

【导师】 李杨瑞；

【作者基本信息】 广西大学 ， 作物栽培学与耕作学， 2013， 博士

【摘要】 甘蔗是中国乃至世界第一大糖料作物,起源于热带及亚热带地区,属喜温作物。甘蔗宿根矮化病(Ratoon stunting disease, RSD)是由Leifsonia xyli subsp.xyli(Lxx)引起的,是目前世界所有植蔗地区危害性极大的病害之一。RSD已经成为甘蔗产量的主要限制因素。现在对RSD的研究主要集中在检测方法和病原菌的研究,而对RSD病原菌和甘蔗的互作研究较少。本研究从接菌诱导入手,观察和测定了RSD侵染甘蔗引起的甘蔗产量性状、蔗糖分、内源激素含量以及茎、叶超微结构的变化；构建了RSD病原菌诱导的甘蔗茎抑制消减杂交(SSH)文库；利用qRT-PCR技术对部分应答RSD病原菌诱导的基因差异表达进行验证；选取5个重要的应答RSD病原菌诱导的基因进行克隆和表达分析。主要研究结果如下：1.以新台糖22号(ROC22)和果蔗(Badila)健康种茎为实验材料,通过接菌研究发现,感染RSD种茎的出苗率比对照减少了2.94和8.50个百分点；株高比对照下降16.7和19.4个百分点；茎径比对照小0.28cm和0.60cm；节间长度比对照减短3.50cm和3.00cm；单茎重比对照低0.36kg和1.10kg；感染RSD植株的蔗糖分低于对照0.9和1.1个百分点(绝对值)；感染RSD处理植株的GA3、IAA含量明显低于对照和温汤脱菌处理的,而ABA含量则相反,感染RSD处理的ABA含量明显高于对照和温汤脱菌处理的：对照和温汤脱菌处理激素含量差异不显著,温汤脱菌处理可有效地防治甘蔗宿根矮化病：利用透射电镜技术对感染RSD植株茎、叶细胞超微结构进行观察表明,叶片叶肉细胞、维管束鞘细胞及茎细胞内的细胞器及细胞核都发生了明显的病理变化。与健康叶片相比,叶绿体变形,叶绿体基质片层大部分消解,基粒结构消失,叶绿体外膜和内膜剥离。线粒体形态异常,有的肿大、内嵴模糊,严重者内嵴消失并空泡化,仅剩未被消解的残骸；细胞核形态变为不规则,核膜破裂,染色质分布不均匀,呈降解状态。在感染RSD甘蔗茎维管束导管细胞内积累有大量的电子致密物质,细胞壁有不同程度的溶解和断裂,这可能和RSD病原细菌侵染有关。以上结果表明,RSD侵染甘蔗后,可能导致光合效率下降,对水分和营养物质的运输能力降低,从而导致甘蔗品质和产量的降低。2.以ROC22健康种茎为实验材料,构建了RSD病原菌诱导的甘蔗茎正向SSH文库,富集筛选到应答RSD相关差异表达基因195个。通过BLASTN功能比对发现,195个差异表达基因中,41个属于抗病与防卫反应基因,数量最多,占全部的21%；另外,蛋白质合成、降解14个,占7%；新陈代谢17个,占9%；核糖体生物合成12个,占6%;细胞结构10个,占5%;翻译相关9个,占5%；转录调控18个,占9%；信号传导12个,占6%；膜转运蛋白8个,占4%；54个为有待验证的未知功能假定蛋白基因和未分类基因,占全部的28%。基于GO分类标准对所得序列进行分类,结果表明：参与生物学过程最多的依次为胁迫应答、代谢过程和转录与翻译,分别占基因总数的25.1%、17.7%和16.6%。最少的是甲基化作用和糖酵解,均只有0.06%；从分子功能看,最多的是蛋白质结合和核糖体结构组分,分别占15%、11%,最少的是泛素蛋白连接酶活性和钙离子绑定,只占0.6%；从细胞组分看,组成细胞质最多,占36%,其次是细胞核和线粒体结构,分别占15%和13%。最少的是内质网膜和转录阻遏因子,占0.6%。3、应用qRT-PCR技术对其中14个应答RSD侵染的差异基因进行了表达分析,在RSD诱导下都呈上调表达。4、克隆获得了甘蔗Remori膜蛋白基因,cDNA全长为778bp；糖原合成激酶3基因,cDNA全长为936bp；甘蔗Ras-related蛋白基因,cDNA全长为935bp；甘蔗新生肽复合物基因,cDNA全长为901bp；甘蔗剪接调节蛋白基因,cDNA全长为1119bp。这5个基因在RSD病原菌诱导下都呈上调表达。在PEG、NaCl、H2O2和4℃低温胁迫下的表达量不尽相同。更多还原

【Abstract】 Sugarcane is the largest sugar crop, originated in tropical and subtropical regions, and is a thermophilic crop. Ratoon stunting disease (RSD) of sugarcane (Sacchrum spp. Hyb.), caused by bacterium Leifsonia xyli subsp. xyli (Lxx), is one of the most important economically damaging diseases worldwide. RSD has become a major limiting factor for sugarcane production. The current research is mainly focusing on the detection methods and pathogen of RSD, and less on the interaction between the pathogen and host. The present study investigated the effects of RSD on cane yield components, sucrose content, and endogenous hormones content and ultrastructure of stalk and leaf in sugarcane. SSH library was established for Lxx-infected sugarcane stalk, gene differential expressions related to response to Lxx-inoculation were detected by real-time PCR technique, and five important genes responded to RSD pathogen-induction were cloned and their expressions were analyzed. The results showed as follows:1. Two commercial cane varieties (i.e. sugar cane variety of ROC22and chewing cane variety of Badila) were selected as the plant materials. The emergence rate of seedcane, plant height, stalk diameter, internode length, single stalk weight and sucrose content in ROC22and Badila were decreased by2.94%and8.50%,16.7%and19.4%,0.28cm and0.60cm,3.50cm and3.00cm,0.36kg and1.10kg,0.9%and1.1%(absolute value), respectively, in the RSD infection treatment than in the control. The plant height in the RSD infected seedcane treatment was significantly lower than those in the control and thermal treatment; The contents of GA3and IAA were obviously lower in the RSD infected seedcane treatment than those in the control and thermal treatment, in contrast to the content of ABA which was higher in the infected seedcane treatment as compared with the control and thermal treatment. Two cane varieties showed the same pattern and there was no significant difference between the control and thermal treatment in plant height and endogenous hormone levels. The thermal therapy is efficient to control RSD in sugarcane. The ultras tructure of stalks and leaves under transmission electron microscopy showed apparent difference between healthy and infected plants in chloroplast, mitochondria and nucleus of mesophyll cells, bundle sheath cells and stalk cells. The chloroplasts in diseased leaves were deformed, most chloroplast grana were dissolved, and the inner and outer membrane of chloroplast delaminated. The abnormal mitochondria expanded with indistinct cristae. In serious abnormal mitochondria, cristae disappeared and vacuolation of mitochondria with a few remains were found. The abnormal nucleuses were found and envelop of nucleus was split. The chromatin showed asymmetrical and degraded. There was a large amount of electron-dense substances accumulated in the infected stalk cells that might be associated with bacterial infection, and xylem cell walls were degraded and broken in different degrees. The results indicate that sugarcane quality and yield reduced due to RSD infection might associate to the disorder of water and nutrition transportation and photosynthetic efficiency reduce in the plants.2. ROC22was selected as the experimental material for establishing a SSH library for Lxx-infected sugarcane stalks, and195differentially expressed genes responsed to RSD were sceened out. Through the BLASTN functional comparison, it was found that there were41genes having a high degree of similarity with the genes related to disease and defence, accounted for21%of all, and this is the largest number. In addition,14genes were for protein synthesis and degradation, accounting for7%;17for metabolism, accounting for9%;12for ribosome biogenesis, accounting for6%;10for cell structure, accounting for5%;9translation related, accounting for5%;18transcription related, accounting for9%;12for signal transduction, accounting for6%;8for membrane trafficking protein, accounting for4%; and54for hypothetical protein and showed no significant similarity to the found genes, accounted for28%. the gene sequences were acquired and classified based on the GO classification standard. The results indicated that the genes involving in biological processes most were response to stress, metabolic processes, and transcription and translation, which were25.1%,17.7%and16.6%, respectively. The genes for methylation and glycolysis were the least, accounting for only0.06%. For molecular function, the mosts were for protein binding and structural constituent of ribosome, accounting for15%and11%, respectively, the least for ubiquitin-protein ligase activity and calciumion binding, accounting for only0.6%. For cellular components, the mosts were for composing cytoplasm, accounting for 36%; followed by nucleus and mitochondrion,15%and13%. But the least were for endoplasmic reticulum membrane and transcriptional repressor complex, accounting for0.6%.3. The expressions of14differential genes in response to Lxx-induction were analyzed using real-time PCR technology, and found that they showed upregulated expression under Lxx-infection.4. Five genes responded to RSD pathogen induction, remorin membrane protein (REMORIN), glycogen synthase kinase3(GSK3), ras-related protein (RASP), nascent peptide complexes transcription factor3(BTF3), splicing regulatory protein (SRP1) were cloned. Their full-length cDNAs were778bp,936bp,935bp,901bp,1119bp, respectively, which showed upregulated expression under Lxx-infection, and their expression quantity varied under PEG, NaCl, H2O2and4℃stresses.更多还原