【摘要】：番茄(Solanum lycopersicum)作為我國乃至世界上重要的蔬菜作物,其在農(nóng)業(yè)產(chǎn)業(yè)結(jié)構(gòu)以及人們的日常生活中扮演著重要的角色。然而在番茄保護(hù)地栽培日常生產(chǎn)過程中易受到多種病原物的侵?jǐn)_,尤其在我國中部番茄主產(chǎn)區(qū)夏季高溫高濕的栽培環(huán)境下極易引起番茄葉霉病的大面積發(fā)生,造成果實(shí)產(chǎn)量、品質(zhì)的嚴(yán)重下降,威脅當(dāng)?shù)胤训陌踩a(chǎn)。葉霉病菌(Cladosporium fulvum[syn.Passalora fulva])與番茄植株發(fā)生的親和性互作是番茄葉霉病發(fā)生的根源,因此以葉霉病抗性基因Cf為主導(dǎo)的抗葉霉病育種工作便成為工作的重點(diǎn)。目前,已鑒定出Cf-1、Cf-2、Cf-4、Cf-4E、Cf-5、Cf-9、Cf-11、Cf-9DC以及多個Cf-ECPs抗性基因,并成功的將Cf-4、Cf-5、Cf-9應(yīng)用于生產(chǎn)。含Cf基因的番茄能夠抵抗C.fulvum的關(guān)鍵在于,Cf基因編碼的類受體蛋白能夠特異性識別C.fulvum所分泌的效應(yīng)因子,從而介導(dǎo)植株產(chǎn)生HR(Hypersensitive response)。但目前存在的新問題是,隨著具不同Cf基因的商業(yè)番茄品種的長期、大面積推廣栽培,C.fulvum生理小種也發(fā)生了選擇性的激烈分化,其中新近發(fā)現(xiàn)的C.fulvum小種2.4克服了Cf-2、Cf-4基因的抗性,C.fulvum小種2.5和2.5.9克服了Cf-5、Cf-9基因的抗性,這些具有廣譜或者復(fù)合毒性的C.fulvum生理小種使其對應(yīng)的番茄抗病品種失去抗病性,同時(shí)也使番茄主產(chǎn)區(qū)C.fulvum小種的流行趨勢變的更加多態(tài)化。為此,急需挖掘新的、抗性更廣更強(qiáng)的Cf基因應(yīng)用于生產(chǎn)。已經(jīng)發(fā)現(xiàn)的Cf基因都來源于野生番茄,因此挖掘新的Cf抗性基因,并通過轉(zhuǎn)基因或者遠(yuǎn)緣雜交等手段,將Cf基因?qū)敕殉Ｒ?guī)栽培種,繼而培育出具葉霉病抗性的植株是番茄抗葉霉病育種的關(guān)鍵。通過多年的、多地的抗病性鑒定,CGN7495材料具有優(yōu)良的C.fulvum抗性,由Kanwar的鑒定結(jié)果得知該材料含有Cf-12基因,來源于醋栗番茄(L.pimpinellifolium),該基因目前并沒有研究基礎(chǔ)。因此,本試驗(yàn)主要以番茄葉霉病新抗性候選基因Cf-12的挖掘和篩選,以及Cf-12對C.fulvum的抗性應(yīng)答機(jī)制為主要研究重點(diǎn),同時(shí)也對Cf-12與C.fulvum非親和互作的組織學(xué)特征和抗性遺傳規(guī)律進(jìn)行了分析。主要研究結(jié)果如下:(1)對Cf-12抗性番茄材料與C.fulvum的非親和互作過程的詳細(xì)的顯微觀察,明確了Cf-12抵御C.fulvum侵染的組織學(xué)特征,如C.fulvum孢子在抗性和感病材料上的萌發(fā)以及從葉背氣孔侵入的過程并沒有表現(xiàn)出差異性,但是當(dāng)C.fulvum菌絲侵入Cf-5、Cf-12等的抗性材料氣孔2d后便停止生長,且菌絲腫脹、彎曲,與寄主葉肉細(xì)胞接觸的菌絲和葉肉細(xì)胞彼此都相繼崩潰,從而將C.fulvum侵入的菌絲限制在一個很小的有限空間內(nèi),使其無法再侵染,且葉片上壞死斑點(diǎn)的產(chǎn)生逐漸增多和擴(kuò)大。將Cf-12與Cf-5等基因?qū).fulvum的抗性特征進(jìn)行了比較分析后,發(fā)現(xiàn)不同的Cf基因?qū).fulvum的抗性組織學(xué)特征差異表現(xiàn)在過敏性壞死斑點(diǎn)的發(fā)生數(shù)量和面積等方面。(2)Cf基因?qū).fulvum的抗性范圍調(diào)查結(jié)果表明,不同的Cf基因能夠抵抗的C.fulvum生理小種的種類也具有較大差異,含Cf-12基因的番茄材料能夠抵御1.2、1.2.4、1.5、1.2.4.5、1.2.5、1.2.4.9等多種C.fulvum生理小種的侵染,但并不抗生理小種1.2.3.4,抗性范圍的明確對含Cf-12基因番茄有針對性的在不同C.fulvum生理小種流行區(qū)合理種植提供參考。(3)依據(jù)構(gòu)建的S.pennellii LA0716與CGN7495的6世代群體,對群體中Cf-12基因的抗性遺傳規(guī)律的分析結(jié)果表明,F2群體中Cf-12基因的分離比符合3:1的分離定律,BC1P1群體中Cf-12基因的實(shí)際分離比符合1:1的孟德爾分離定律,由此可知Cf-12基因?qū).fulvum的抗性符合單基因顯性遺傳規(guī)律,這與已鑒定的其它Cf基因?yàn)閱位蝻@性遺傳的規(guī)律相一致。(4)在明確Cf-12基因?yàn)閱位蝻@性遺傳的基礎(chǔ)上,通過采用父母本重測序,F2代抗、感群體SLAF簡化測序技術(shù)進(jìn)行基于ED(Euclidean Distance)和SNP-index的聯(lián)合分析,對番茄葉霉病抗性基因Cf-12進(jìn)行篩選,并成功將其關(guān)聯(lián)到番茄第6號染色體,對目標(biāo)關(guān)聯(lián)區(qū)域所包含基因進(jìn)行NR、NT、trEMBL、Swiss-prot、GO、KEGG和COG等多數(shù)據(jù)庫的功能注釋和LRR-TM-sCT結(jié)構(gòu)預(yù)測確定了3個候選基因,在進(jìn)一步通過Proparam、pfam、SMART、SignalP、ProtComp、SOPMA、Swiss-Model等程序?qū)蜻x基因編碼蛋白的結(jié)構(gòu)域、理化性質(zhì)、亞細(xì)胞定位、二級和三級結(jié)構(gòu)、PPI網(wǎng)絡(luò)進(jìn)行了詳細(xì)分析,以及基于qRT-PCR的表達(dá)模式分析和共表達(dá)分析的基礎(chǔ)上,確定位于CGN7495抗性材料上Solyc06g008270.2基因位點(diǎn)的72 bp堿基片段插入后的基因,即Ccg2基因?yàn)樗Y選的Cf-12基因。此外,通過對番茄全基因組進(jìn)行GO分類中定義為結(jié)合活性功能(GO:0005515)且具有eLRR-TM結(jié)構(gòu)的基因進(jìn)行番茄全基因組搜索,結(jié)果發(fā)現(xiàn)已報(bào)道的所有Hcr2s、Hcr9s及Cf-Ecp基因均被覆蓋到,這一思路可為研究其它未知的Cf基因的定位工作提供新的途徑。(5)對Cf-12對C.fulvum的抗性應(yīng)答機(jī)制研究,采用基于高通量測序的RNA-seq技術(shù),對多時(shí)間點(diǎn)的非親和互作樣本進(jìn)行轉(zhuǎn)錄組學(xué)分析,通過對測序樣本間差異表達(dá)基因(DEGs)的k-mean聚類分析,總共發(fā)現(xiàn)了3類表達(dá)模式,其中最值得注意的是subcluster_4中持續(xù)高表達(dá)的34個DEGs,以及subcluster_3中先低表達(dá)后又劇烈高表達(dá)的41個DEGs,GO分析顯示它們富集于氧化還原、氮代謝、磷代謝、結(jié)合活性、轉(zhuǎn)移酶活性、激素應(yīng)答、離子轉(zhuǎn)運(yùn)以及防御反應(yīng)相關(guān)等類別,這對于后續(xù)試驗(yàn)中篩選特異性表達(dá)的基因起到重要的輔助作用;可變剪接分析(AS)顯示,非親和互作過程主要發(fā)生SE和MXE事件,隨著時(shí)間的延長SE和MXE兩AS事件的總數(shù)量呈現(xiàn)增加的趨勢,說明這些顯著差異的可變剪接在Cf-12基因調(diào)控對C.fulvum抗性的轉(zhuǎn)錄水平的基因表達(dá)的復(fù)雜程度以及蛋白質(zhì)功能的多樣性正在發(fā)生巨大的變化;GO和Pathway分析表明苯丙氨酸代謝途徑、植物—病原菌互作、苯丙烷生物合成、谷胱甘肽代謝,次生代謝產(chǎn)物的生物合成等生物過程中的基因被上調(diào)顯著富集,對植物激素信號轉(zhuǎn)導(dǎo)途徑分析表明SA和ET對Cf-12的抗病防衛(wèi)反應(yīng)起重要的調(diào)控作用,而JA可能在Cf-12對C.fulvum的抗病防衛(wèi)反應(yīng)信號轉(zhuǎn)導(dǎo)途徑中起到交叉、輔助的作用。此外,還富集到大量的氧化物酶體,谷胱甘肽過氧化物酶基因,植保素合成基因,與苯丙素和黃酮類代謝途徑相關(guān)的苯丙氨酸解氨酶基因、4CL、TC4M、F5H,有關(guān)黃酮類合成的CHSs、CHI、DFR、FLS,與細(xì)胞壁合成相關(guān)的纖維素合成酶基因和果膠酶活性的果膠(甲)酯酶基因等也均被富集到呈現(xiàn)上調(diào)表達(dá)的趨勢,與蛋白泛素化相關(guān)的E1、E2、E3等多數(shù)基因呈上調(diào)表達(dá)趨勢,說明蛋白的泛素化被積極響應(yīng),總的來看與SA和ET、PR蛋白、植保素、防衛(wèi)素、硫堇、木質(zhì)素、滲調(diào)蛋白、過氧化物酶以及黃酮類等的大量合成以及細(xì)胞壁的加強(qiáng)都使得Cf-12番茄對C.fulvum產(chǎn)生較好的抗性;在轉(zhuǎn)錄因子(TFs)的分析中,首次發(fā)現(xiàn)多個新的ARF、MADS、G2-like、G3H、C2H2、b HLH等TFs家族參與到Cf-12對C.fulvum的抗性防衛(wèi)反應(yīng)中,且檢測到的這60個TFs家族呈現(xiàn)兩種表達(dá)模式,如bZIP、C2H2、HSF、MADS、NAC等在早期階段先上調(diào)表達(dá),然后在后期下調(diào)表達(dá),而bHLH、GRAS、HB、SBP、MYB、WRKY等在抗性反應(yīng)的后期呈高表達(dá)的趨勢;通過全面的Cf-12對C.fulvum非親和互作的轉(zhuǎn)錄組學(xué)分析,對Cf-12與C.fulvum的抗性應(yīng)答機(jī)制也有了一定的了解。(6)對Cf-12與C.fulvum抗性應(yīng)答反應(yīng)中的部分基因的VIGS的初步功能驗(yàn)證,Ccg2候選基因以及NPR1(Solyc07g044980.2)、CDPK(Solyc03g113390.2)基因沉默后的部分植株出現(xiàn)了感病癥狀,病情指數(shù)統(tǒng)計(jì)發(fā)現(xiàn)沉默NPR1和CDPK的植株仍表現(xiàn)為中抗,而沉默Ccg2的植株表現(xiàn)為中感,說明Ccg2、NPR1(Solyc07g044980.2)、CDPK(Solyc03g113390.2)基因與Cf-12番茄對C.fulvum的抗性應(yīng)答反應(yīng)過程相關(guān)。
[Abstract]:Solanum lycopersicum, as an important vegetable crop in our country and in the world, plays an important role in the structure of agricultural industry and people's daily life. However, it is easily disturbed by various pathogens during the daily production of tomato cultivated land, especially in the main tomato producing area in the middle of our country. The large area of tomato leaf mould was easily caused by the cultivation environment, resulting in the fruit yield, the serious decline in quality and the safety of the local tomato. The affinity interaction between Cladosporium fulvum[syn.Passalora fulva] and tomato plants was the root of tomato leaf mold disease. Therefore, the leaf mould resistance gene Cf was used as the root cause. At present, Cf-1, Cf-2, Cf-4, Cf-4E, Cf-5, Cf-9, Cf-11, Cf-9DC and multiple Cf-ECPs resistance genes have been identified, and Cf-4, Cf-5, and Cf-9 should be used in production. The key to the resistance of tomato containing genes is that the gene encoded receptor protein can be specific. Sex recognizes the effect factors secreted by C.fulvum, which mediates the production of HR (Hypersensitive response) in the plant, but the new problem is that, with the long term of the commercial tomato varieties with different Cf genes, large area cultivation and the selective intense differentiation of the C.fulvum physiological races, of which the newly discovered 2.4 grams of C.fulvum species are newly discovered. The resistance of Cf-2, Cf-4 gene, C.fulvum small species 2.5 and 2.5.9 overcame the resistance of Cf-5 and Cf-9 genes. These broad-spectrum or compound toxic C.fulvum species make their corresponding tomato resistant varieties lose their resistance to disease, and also make the flow trend of C.fulvum small species in tomato main producing area become more polymorphic. The Cf gene, which has a more extensive and stronger resistance, is used in production. The Cf gene has been found from the wild tomato. Therefore, it is the key to develop a new Cf resistance gene and transfer the Cf gene into the conventional cultivation of Tomato by transgene or distant hybridization. After many years of identification of resistance to disease, CGN7495 material has excellent C.fulvum resistance. It is found that the material contains Cf-12 gene from the results of Kanwar identification, which is derived from gooseberry tomato (L.pimpinellifolium). This gene has no research basis at present. Therefore, this experiment is mainly based on the mining and screening of the candidate gene of tomato leaf mould resistance candidate gene Cf-12. Selection, and the main research focus on the resistance response mechanism of Cf-12 to C.fulvum, and the analysis of the histologic characteristics and resistance genetic rules of Cf-12 and C.fulvum incompatibility interaction are also analyzed. The main results are as follows: (1) the detailed microscopic observation of the non affinity interaction process of Cf-12 resistant tomato materials and C.fulvum is made clear that Cf-12 is offset. The histological features of the C.fulvum infection, such as the germination of the C.fulvum spores on the resistant and susceptible materials and the invasion of the stomata from the back of the leaf, were not different, but when the C.fulvum mycelium invaded the Cf-5, Cf-12 and other resistant materials, the stomata stopped growing, and the mycelium swelled and curved, and the mycelium contacted with the host mesophyll cells. The mesophyll cells collapse one after another, thus limiting the C.fulvum intruded mycelium to a small limited space, which can not be reinfected, and the production of necrotic spots on the leaves gradually increases and expands. The resistance characteristics of the Cf-12 and Cf-5 genes to the resistance of C.fulvum have been compared, and the resistance of the different Cf genes to the C.fulvum is found. The differences in the characteristics of sexual histology showed in the number and area of the occurrence of anaphylactic necrosis spots. (2) the investigation of the resistance range of the Cf gene to C.fulvum showed that the species of C.fulvum physiological races which were resistant to the Cf genes were also different, and the Cf containing the gene of the gene could resist the 1.2,1.2.4,1.5,1.2.4.5,1.2.5,1.2. .4.9 and other C.fulvum physiological races were infected, but they were not resistant to the physiological race 1.2.3.4, and the range of resistance was clear for the rational planting of the Cf-12 gene tomato in different C.fulvum physiological races. (3) the resistance inheritance of the Cf-12 gene in the population based on the constructed S.pennellii LA0716 and the 6 generation of CGN7495. The results of regular analysis show that the separation ratio of Cf-12 gene in F2 population is consistent with the law of separation of 3:1. The actual separation ratio of Cf-12 gene in BC1P1 population conforms to the Mendel law of 1:1, thus the resistance of the Cf-12 gene to C.fulvum is consistent with the dominant inheritance law of the single gene, and the other Cf genes identified are the dominant inheritance of the single gene. (4) (4) on the basis of the explicit genetic dominance of the single gene, the genetic analysis of ED (Euclidean Distance) and SNP-index was carried out by the combined analysis of ED (Euclidean Distance) and SNP-index by the parental re sequencing, the SLAF sequencing technology of the susceptible population and the susceptible population, and was screened for tomato leaf mould resistance based on Cf-12, and was successfully associated with tomato No. sixth. Chromosomes, the functional annotations of NR, NT, trEMBL, Swiss-prot, GO, KEGG, and COG, including the functional annotations and the structural prediction of LRR-TM-sCT, determine the 3 candidate genes. The properties, subcellular localization, two and three level structures, PPI networks were analyzed in detail, and based on qRT-PCR expression pattern analysis and co expression analysis, the base of the 72 BP base fragment inserted at the Solyc06g008270.2 locus on the CGN7495 resistant material was determined, that is, the Ccg2 gene was selected as the Cf-12 gene. The whole genome of tomato was classified by GO classification, which was defined as a gene that combines active function (GO:0005515) with eLRR-TM structure to search the whole genome of tomato. The results showed that all of the reported Hcr2s, Hcr9s and Cf-Ecp genes were covered. This idea can provide a new way for the study of other unknown Cf genes. (5) In the study of the resistance response mechanism of Cf-12 to C.fulvum, the RNA-seq technology based on high throughput sequencing was used to make a transcriptional analysis of the non compatible interaction samples of multiple time points. Through the K-mean clustering analysis of the differentially expressed genes (DEGs) among the sequencing samples, 3 types of expression patterns were found, among which the most noteworthy was in subcluster_4. Continuous high expression of 34 DEGs, and 41 DEGs with low expression and intense high expression in subcluster_3, GO analysis shows that they are enriched in redox, nitrogen metabolism, phosphorus metabolism, binding activity, transferase activity, hormone response, ion transport and defense response related categories, which can be used to screen genes for specific expression in follow-up tests. The variable splicing analysis (AS) showed that the SE and MXE events occurred mainly in the incompatible interaction process, and the total number of SE and MXE two AS events increased with time, indicating that the variable splicing of these significant differences in the Cf-12 gene regulates the complex process of gene expression at the transcriptional level of C.fulvum resistance. The diversity of degree and protein function is undergoing great changes; GO and Pathway analysis show that phenylalanine metabolic pathway, plant pathogen interaction, phenylpropane biosynthesis, glutathione metabolism, and biosynthesis of secondary metabolites are significantly enriched, and the plant hormone signal transduction pathway is divided. The analysis shows that SA and ET play an important role in regulating the resistance and defense response of Cf-12, and JA may play a cross and auxiliary role in Cf-12 on the signal transduction pathway of the resistance defense response of C.fulvum. In addition, a large number of oxide enzymes, glutathione peroxidase (GSH PX), pod synthesis gene, and phenylpropanol and flavonoid generation are also enriched. The phenylalanine ammonia lyase gene, 4CL, TC4M, F5H, related to the flavonoid synthesis of CHSs, CHI, DFR, FLS, and the cellulose synthetase gene related to cell wall synthesis and pectinase activity of pectin (a) esterase gene are also enriched to present the trend of up-regulated expression, and most of the genes related to protein ubiquitination, E1, E2, E3, etc. The up-regulated trend indicates that the ubiquitination of protein is actively responsive. In general, a large number of synthesis of SA and ET, PR protein, flocking, defensin, thio, lignin, peroxidase, and flavonoids, and the enhancement of cell wall, make Cf-12 tomatoes have a better resistance to C.fulvum; in the analysis of the transcription factor (TFs), It is the first time that a number of new ARF, MADS, G2-like, G3H, C2H2, B HLH and other TFs families are involved in the resistance defense response of Cf-12 to C.fulvum, and the detected 60 TFs families present two kinds of expression patterns. A high expression trend in the later stage of sexual response; the transcriptional analysis of C.fulvum incompatibility interaction by a comprehensive Cf-12 also has a certain understanding of the resistance response mechanism of Cf-12 and C.fulvum. (6) preliminary functional verification of the VIGS of some genes in the response to Cf-12 and C.fulvum responses, the Ccg2 candidate genes and NPR1 (Solyc07g0449). 80.2) a part of the plant with CDPK (Solyc03g113390.2) gene was silent, and the disease index showed that the plants with silent NPR1 and CDPK still showed moderate resistance, while the silent Ccg2 plants showed medium sense, indicating that Ccg2, NPR1 (Solyc07g044980.2), CDPK (Solyc03g113390.2) gene and Cf-12 tomato responded to the resistance response to C.fulvum. Course related.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S436.412

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 史艷梅;魏攀;陳媛媛;楊冉;金立鋒;劉萍萍;李鋒;屈凌波;林福呈;王燃;;煙草ζ-胡蘿卜素脫氫酶基因的克隆和功能分析[J];煙草科技;2015年09期

2 劉慧芹;劉慧平;韓巨才;;葉霉菌粗毒素對番茄幼苗防御酶及活性氧的誘導(dǎo)[J];華北農(nóng)學(xué)報(bào);2009年06期

3 龍書生;曹遠(yuǎn)林;李亞玲;康振生;;小麥抗條銹病過敏性壞死反應(yīng)中的活性氧代謝[J];西北農(nóng)林科技大學(xué)學(xué)報(bào)(自然科學(xué)版);2009年11期

4 杜紅旗;李晨陽;田璐;;一種改進(jìn)大腸桿菌DH5α轉(zhuǎn)化效率的方法[J];安徽農(nóng)業(yè)科學(xué);2009年22期

5 許先國;吳俊杰;洪小珍;朱發(fā)明;嚴(yán)力行;;鑒定9個新的RHD基因mRNA可變剪接體[J];遺傳;2006年10期

6 孟凡娟;許向陽;李景富;洪瑞;;東北三省新的番茄葉霉病菌生理小種分化初報(bào)[J];中國蔬菜;2006年01期

7 程紅焱;宋松泉;;植物一氧化氮生物學(xué)的研究進(jìn)展[J];植物學(xué)通報(bào);2005年06期

8 柴敏,于拴倉,丁云花,姜立綱;北京地區(qū)番茄葉霉病菌致病性分化新動態(tài)[J];華北農(nóng)學(xué)報(bào);2005年02期

9 蔡新忠,徐幼平;水楊酸和乙烯對依賴于Cf基因的過敏壞死的調(diào)控作用[J];植物生理與分子生物學(xué)學(xué)報(bào);2003年01期

10 劉勝毅,許澤永,何禮遠(yuǎn);植物與病原菌互作和抗病性的分子機(jī)制[J];中國農(nóng)業(yè)科學(xué);1999年S1期

相關(guān)博士學(xué)位論文 前6條

1 李欣龍;山葡萄‘雙紅’與葡萄霜霉菌互作的分子機(jī)制研究[D];中國農(nóng)業(yè)大學(xué);2016年

2 趙婷婷;番茄抗葉霉病基因Cf-19的精細(xì)定位及抗病應(yīng)答基因篩選[D];東北農(nóng)業(yè)大學(xué);2016年

3 吳曉軍;玉米抗細(xì)菌性褐斑病候選基因的克隆與鑒定及NIL的轉(zhuǎn)錄組分析[D];中國農(nóng)業(yè)大學(xué);2014年

4 薛飛;小麥抗白粉病基因的分子作圖及其與白粉菌互作的表達(dá)譜分析[D];西北農(nóng)林科技大學(xué);2012年

5 李曉玉;玉米全基因組NBS類抗病基因的進(jìn)化及病原菌誘導(dǎo)表達(dá)模式分析[D];安徽農(nóng)業(yè)大學(xué);2011年

6 王長春;番茄Cf-4和Cf-9基因介導(dǎo)的過敏性反應(yīng)調(diào)控及基因沉默技術(shù)的研究[D];浙江大學(xué);2005年

相關(guān)碩士學(xué)位論文 前2條

1 何艷龍;番茄果實(shí)硬度遺傳規(guī)律的研究及QTL定位[D];東北農(nóng)業(yè)大學(xué);2016年

2 李嬌;玉米苗期干旱脅迫下可變剪接及其調(diào)控因子的研究[D];鄭州大學(xué);2013年

，

本文編號：2162749