【作者】 袁广胜；

【导师】 潘光堂；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 玉米是国民经济重要的粮食和经济作物,各种病害的发生限制了玉米产量,造成大量减产。玉米穗粒腐病(Maize Ear Rot)就是其中的一种,该真菌病害由串珠镰刀菌(Fusarium moniliforme)引起,特别是在我国高温高湿的西南地区该病害流行甚广,危害玉米的生产。因此,积极寻找抗玉米穗粒腐病的相关基因并探知其功能,不仅对培育广谱、高效、稳定、持久抗病性品种,而且对玉米穗粒腐病的抗病机理研究的深入,均具有重要的理论和实践意义。本研究以抗玉米穗粒腐病的玉米自交系Bt-1和感病自交系掖478为材料,玉米乳熟初期采用人工接高致病性融合菌Fusarium moniliforme于果穗中上部的籽粒与苞叶之间,套袋、保湿,分别取抗、感材料的内层苞叶为实验材料。对接菌后两个自交系材料的不同时间段的部分防御酶活性进行分析,扫描电镜观察侵染病菌的组织病理学变化,提取苞叶的不同时间段总RNA,利用抑制性消减杂交技术(SSH)构建差减文库,结合cDNA芯片技术进行抗病基因筛选。获得的主要结果如下：1.对接种鉴定的抗病自交系Bt-1和感病自交系掖478各单穗的发病情况进行统计分析。结果显示：按抗感程度的划分标准,Bt-1病穗率为1.5%,属于高抗玉米穗粒腐病自交系；掖478病穗率为59.4%,属于高感玉米穗粒腐病自交系。2.取抗(Bt-1)、感(478)材料间隔24小时的6个时间段接菌部位苞叶组织进行扫描电镜观察(设处理0 h为对照),结果表明：在抗、感材料中,接种后2-3天,菌丝在苞叶细胞表面延伸增长并生成分支,接着菌丝大量增长,形成更加明显的网状结构,已有少量菌丝形成侵染；接种96 h后可见已经完全形成侵染,菌丝开始进入气孔；接种后120 h,侵染继续增大,菌丝穿过气孔,并向前继续侵入；接种后144 h,苞叶组织的气孔侵入的大量菌丝。另外,研究发现病菌能通过气孔直接进入苞叶组织内,在接菌后的不同时间段里,随着时间推移,苞叶表面组织的菌丝体量逐渐增多,而且进入感病材料要稍稍早于抗病材料,说明致病菌丝直接通过气孔进行侵染而导致玉米穗粒腐穗粒腐病发生的重要途径。3.对接菌后抗(Bt-1)、感(478)材料6个时间段的苯丙氨酸解氨酶(PAL)过氧化物酶(POD)、超氧化物歧化酶(SOD)、丙二醛(MDA)的活性进行测定和POD同工酶酶谱分析,在感病材料中PAL的活性要比抗病材料中增加的更快、更高；同样对于POD来说,在感病材料中的活性要比抗病材料中要高,但变化趋势在两个材料中相似；而MDA的含量则相反,在感病材料中的活性要比抗病材料中要低；SOD在两个材料中差异不明显,无明显规律可循；对于POD同工酶酶谱分析看,在两个材料中都增加了3～4个条带,这说明玉米感病后会通过增加POD的活性和条带来抵御外源病菌的侵入。总体而言PAL和POD水平与材料抗性呈负相关；MDA与材料抗性呈正相关关系。4.利用抑制性消减杂交(SSH)分别构建了抗(Bt-1)和感(478)两个自交系受病菌诱导后的正向和反向共4个差减文库,有效地富集6,560多个单克隆,其中3,560克隆来自于抗病材料,3,000克隆来自于感病材料,此举为进一步分离克隆抗玉米穗粒腐病特异表达基因的研究搭建一个材料平台。利用PCR检测文库的克隆片段长度,发现克隆片段的平均长度在400 bp,大都介于200～1000 bp之间。将所有的克隆以反向Northern杂交筛选,共获得杂交信号差异显著的阳性克隆145个测序后,经生物软件同源性比对,共获得93条唯一的EST序列,其中来源于抗病材料(Bt-1)的正向文库的EST序列28条,来源于反向文库的EST序列12条；而来源于感病材料(478)的正向文库的EST序列26条,在反向文库中有EST序列27条。5.将上述93条EST序列在公共数据库Genbank中进行生物信息学分析,在抗、感材料中共获得68条已知基因功能的EST序列,24条未知功能EST序列和1条为新发现EST序列。在抗病材料(Bt-1)中,对已知基因功能进行分类后发现：基因参与抗病或防御途径(20.0%)、基因转录调控(17.5%)、信号传导(12.5%)、蛋白质修饰加工(7.5%)、代谢功能(5.0%)、能量调控(2.5%)和未知功能(35.0%)在感病材料(478)中,对已知基因功能进行分类后发现：基因参与抗病或防御途径(24.5%)、基因转录调控(16.9%)、信号传导(9.4%)、蛋白质修饰加工(13.2%)、代谢功能(5.7%)、能量调控(5.7%)、蛋白质转运(3.8%)和未知功能(22.6%)6.将抗(Bt-1)、感(478)材料接种后96 h的玉米苞叶样品用于Affymetrix公司玉米全基因组生物芯片的大规模筛选,在抗病材料中共获得482条显著上调差异表达基因序列,其中372条可推知起功能,110条未知功能,可能为芯片筛选出来新基因；而在感病材料中只获得7个上调差异表达序列,其推测的功能都包含于抗病材料中的基因。对这些大量的特异表达基因序列初步推测功能分析表明,主要涉及抗病相关蛋白(15%)、初级和次级代谢功能和能量代谢(14%)、信号转导(12%)、转录调控(11%)、活性氧化物(11%)、蛋白质修饰加工(7%)、蛋白质转运(5%)、胁迫蛋白(3%)和未知功能(22%)等多个生理生化途径中的相关基因。这说明玉米抗穗粒腐病的抗病机制是一个复杂的关系网,需要大量的基因参与其中来协同作用。7对从SSH中获得的序列和从基因芯片中获得的差异代表序列序列进行GO注释结果表明,对应的基因注释包括分子功能、生物过程和细胞组分3个层次。另外,将从SSH筛选出的93条EST和芯片中得到的482条差异表达序列行进一步生物信息学分析,共获得340条单一拷贝序列,将单一拷贝序列与他人研究的玉米穗粒腐相关抗性QTL位点共定位在玉米染色体图谱上,共有36条单拷贝序列在一致性QTL区间内,大多数序列在染色体上的位点聚集在一致性QTL位点附近,说明通过SSH和生物芯片技术筛选出来的抗玉米穗粒腐病差异表达基因与前人QTL研究结果具有诸多的一致性。8.重点筛选其中几个重要相关功能的基因进行时空表达验证,以RT-PCR对SSH和生物芯片筛选得到的抗性相关EST的进行半定量分析,从分析结果看,在病原菌胁迫下这几类功能的EST在各个时间段的表达量均有变化,总体而言,相对于对照来说诱导后的抗性EST都有上调表达趋势。9.综合分析本研究的结果,对玉米感染穗粒腐病后的组织病理学电镜观察,探讨了穗粒腐病菌丝侵染的途径,通过生理生化指标的测定,了解到其生理功能变化,同时通过SSH技术及结合生物芯片大规模筛选,获得了大量与玉米穗粒腐病抗性相关的基因,并对穗粒腐病与这些抗病相关基因的关系进行初步探讨,此举也为分离克隆玉米穗粒腐病抗性基因创造了条件,最后为并进一步深入阐述玉米对穗粒腐的抗性机制和培育抗玉米穗粒腐新品种奠定基础。更多还原

【Abstract】 Ear rot caused by Fusarium moniliforme (FM) is a destructive disease for its decrease of grain yield in maize, one of the important crops for food in Asia. Especially, a high incidence of ear rot occurs in the moist and warm regions of Southwest China, as well as other regions with similar longitude in other countries. To this end, it is great important to isolate resistant genes with potential function in maize, and to use these genes to breed efficient, broad-spectrum and stable-resistant cultivars to the maize ear rot or understand the defense mechanisms to combat invasion in maize ear rot. In our study, we used two maize cultivars, Bt-1 and Ye478 as completely resistant and highly susceptible to FM due to many years evaluation of field in southwest of China to characterize the specific host response to FM infection. Milky stage maize plants were inoculated with high pathogenic fungus anastomosis groups Fusarium moniliforme on each bract by injection. The inoculated plants were grown under controlled conditions at Maize Research Institute of Sichuan Agricultural University, and bract tissues were collected six times with a 24 h interval after inoculation. To better know the processes involved of defense in maize ear rot upon the FM infection, the time-course infected bracts were observed through Scanning Electron Microscope (SEM) to demonstrate pathogen progression in maize bracts. Then, the biochemical and physiological enzymes activities were analyzed of the both cultivars. In the follow, we identified the differentially expressed genes responding to FM-infection in maize by suppression subtractive hybridization (SSH) and Affymetrix GeneChip Maize Genome Array. The main results are as follows:1. The disease incidence of the both cultivars after FM-infection was investigated in the field. The results showed that the disease incidence was accounted for 1.5%in Bt-1 and mean that this inbred line is belonged to high resistance to FM according to resistant standard. The Ye478 is belonged to high highly susceptible to FM for its 59.4%disease incidence.2. The invasion procedure of fungus on bracts was investigated through SEM. Inoculated and mock-inoculated bract tissues were picked randomly by collecting at 24 h intervals for three independent biological replicates, each consisting of the independent maize bract. FM invasion on the maize bracts of resistant Bt-1 cultivar and the susceptible Ye478 cultivar were observed through SEM at 24 h intervals after inoculation for six stages. During the thin and weak early stage (around 2-3 d), the hyphae of FM developed and expanded on the bracts surfaces of both cultivars. After becoming significantly strong and fine cylindrical, the hyphae gradually moved and infected the maize bract through the stoma at approximately 72 hpi. With the increasing invasion of FM, more and more hyphae assembled as mass into stoma by 120 hpi in both cultivars. However, the invasion of hyphae into stoma was delayed in resistant cultivar Bt-1. This phenomenon reflects more possible defense mechanisms in cultivar Bt-1 were activated than that in Ye478.3. To study the production of enzymes involved in the defense of FM infection, the phenylalanine ammonia-lyases (PAL), peroxidase (POD) and malondialdehyde (MDA) activities were detected during a 6-d time period. In addition, patterns of POD isozymes were also detected to demonstrate POD isozymes for specific different two maize cultivars. PAL and POD were increased promptly higher and quicker in Ye478, comparing to that in resistant cultivar Bt-1. The content of MDA was higher in Bt-1 than Ye478. Finally, the patterns of POD isozyme changed dramatically and increased three or four belts in both cultivars after infection. The result demonstrated that the host might be increase POD activity and isozyme bands to resistant the exogenous pathogen attack for protecting host organism. In a word, our results implies that the relationship between protective enzymes activity and resistant cultivars are negative correlated, while a positive correlation between the content of MDA and resistance of the cultivars.4. To study the genes expressed differentially in resistance of FM in maize, we constructed four cDNA libraries by suppression subtractive hybridization (SSH) method using the RNAs from the bracts of resistant maize cultivar Bt-1 as well as susceptible maize cultivar Ye478. The obtained cDNA fragments were cloned into pMD 18-T easy vector and checked the recombination efficiency of 4 SSH libraries by PCR. The lengths of PCR products were 0.2-1.0 kb and clones containing no or more than one insert were removed from further investigation. A total of 6,560 (efficiency rate 93%) clones with 3,560 clones from the resistant two libraries in Bt-1 cultivar and 3,000 clones from the susceptible two libraries in Ye478 cultivar were screened and re-examined by PCR. The re-amplified PCR products were dot-blotted on membrane and hybridized with four kinds of DIG-labeled probes. As a result,145 positive clones were obtained and sequenced from all 6560 clones through reverse dot-blot hybridization screening. The DNA sequences of the 145 positive clones were analyzed against GeneBank database, resulting in identification of 93 unique genes.28 cDNA clones were obtained from forward libraries and 12 were obtained from reverse libraries in the resistant cultivar Bt-1.27 subtracted genes were obtained from forward libraries and 26 were obtained from reverse libraries in the susceptible cultivar Ye478.5. Base on the 93 unique genes, the amino acid sequences of 68 genes were identified to exhibit high homology to the genes with function known, while 24 were genes with function unknown. Moreover, one gene did not match any known sequences. These 68 genes were categorized according to the functions of the proteins. The cellular functions of these identified in Bt-1 can be classified into disease defense (8 distinct proteins encoded by 8 ESTs:20.0%), gene destination and transcription (6 distinct proteins encoded by 7 ESTs:17.5%), signal transduction (5 distinct proteins encoded by 5 ESTs:12.5.0%), protein destination and storage (3 distinct proteins encoded by 3 ESTs:7.5%), metabolism (2 distinct proteins encoded by 2 ESTs:5.0%), energy (1 distinct proteins encoded by 1 EST:2.5%), unknown (35.0%including 14 ESTs). The cellular functions of these genes identified in Ye478 can be classified into disease and defense (10 distinct proteins encoded by 13 ESTs:24.5%), gene destination and transcription (8 distinct proteins encoded by 9 ESTs:16.9%), signal transduction (4 distinct proteins encoded by 5 ESTs:9.4%), protein destination and storage (6 distinct proteins encoded by 7 ESTs:13.2%), metabolism (3 distinct proteins encoded by 3 ESTs:5.7%), energy (3 distinct proteins encoded by 3 ESTs: 5.7%), intracellular trafficking (1 distinct proteins encoded by 2 ESTs:3.8%), unknown (22.6%including 12 ESTs).6. Using the GeneChip Maize Genome Array platform, we investigated gene expression changes in the maize bract between resistant inbred Bt-1 and susceptible inbred Ye478 at 96 h post-inoculation with FM. In total, there were 482 unique genes up-regulated more than 1.5 fold in resistant inbred Bt-1 (ANOVA, p<0.05) when compared to mock-inoculated bract tissues. However, only seven of these genes were up-regulated in susceptible inbred Ye478, indicating that the gene expression in the resistant genotype responded strongly to FM. For the genes that were significantly different in both inbreds, we observed that all the seven genes identified in susceptible inbred Ye478 were found in the 482 FM-induced genes of resistant inbred Bt-1. Further analysis of the 482 FM-induced genes indicated that they represented 372 UniGenes and 110 unknown functions based on the bioinformatic analysis. With the information from the genechip, we were able to identify and assign putative function of the FM-induced genes into eight functional categories. The largest category was "defense anti-microbial proteins", including proteins related to elevated disease resistance, which was the most abundant and accounted for 15%of the genes. The second most abundant category was "metabolism and energy" including lipid, carbohydrate and primary metabolism and energy, accounting for 14%of the genes. Other categories included "signal transduction" (12%), "transcriptional regulators" (11%), "reactive oxygen scavengers" (11%), "protein destination" (7%), "transporters (5%), and "stress proteins" (3%). A total of 22%of the genes were "unclassified or with unknown function proteins".7. Molecular annotation system was used to analyze these large number genes differentially expressed from SSH and Genechip FM infection. The results showed that these large reponsive genes were belonged to cellular component, molecular function, biological process with complicated network. In addition,340 single-copy differentially expressed sequences were found from SSH including 93 sequences and Genechip including 482 FM-induced genes after bioinformatics alignment. Then, the the co-localization analysis was performed between the 340 single-copy and ear rot QTL locus which from other researches in the map of maize chromosomes. The result showed that 36 FM-induced genes were located in the Meta-QTLs region and most other genes were closed with the Meta-QTLs. It showed that our results was comformed with others QTLs research conclusion.8. Expression profiles of representative maize ear rot defense-related genes during the time course of infection were indeed confirmed with semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that expression of genes involved in the FM infection were up-regulated in the resistant maize cultivar as well as susceptible maize cultivar.9. In this research, we investigated the processes involved in maize ear rot upon infection by FM, the time-course infection was observed through SEM to demonstrate pathogen progression in maize bract and the biochemical and physiological enzymes activities were analyzed in the two maize cultivars, Bt-1 and Ye478, completely resistant and significantly susceptible to FM respectively. The FM-responsive genes from both cultivars SSH libraries and genechip data presented here are a valuable resource for further functional genomics studies addressing resistant mechanisms in maize ear rot. Further functional analysis of these responsive genes to FM may provide new insights into the molecular mechanisms of the host defense response. In future studies, the involvement of each gene in FM inducement should be investigated and will help us clarify the process of defense mechanisms acquisition combating invasion in maize ear rot. Such information can be used by breeders for selection of candidate genes, and their transfer to agronomically important maize cultivars.更多还原