【作者】 刘珂珂；

【导师】 巩振辉；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2009， 博士

【摘要】 辣椒是一种世界性的蔬菜,也是我国重要的经济作物。辣椒疫病在我国许多省市普遍发生,成为我国辣椒生产的严重障碍。我们通过建立辣椒抗病性鉴定技术体系,分析抗疫病的生理生化基础,利用cDNA-AFLP技术研究不同生理小种的疫霉菌侵染辣椒后表达谱的差异,结合RACE技术获得阳性片段的全长,利用生物信息学分析这些基因的序列信息及它们所推测蛋白的二级结构、跨膜结构、信号肽和系统进化信息,利用半定量RT-PCR和实时定量技术研究这些基因的表达和调控,以期揭示辣椒抗疫病的抗性机理,为寻找培育广谱、高效、稳定的抗病品种奠定基础。本论文取得的主要研究成果如下:1.建立了辣椒抗疫病离体侧枝接种鉴定技术体系。以高抗品种CM334,中抗品种N3和感病品种EC为材料,研究了辣椒抗疫病的离体侧枝接种鉴定技术。以辣椒第二次分枝后,由第一次分支处剪下的侧枝作为接种对象,在疫霉菌游动孢子悬浮液浓度为1×10~4个/mL,温度为28℃,4000lx,12h/d光照条件下接种,抗、感品种差异最为明显。用该方法对34份材料进行鉴定,并与已报导的离体叶片接种法,切茎接种法和灌根接种法进行了比较,统计分析表明,该方法与其他3种方法均呈显著正相关,相关系数分别为:r=0.9150** r=0.8730**和r=0.8384**,说明该方法能真实反映辣椒对疫病的抗性。2.分析了保护酶活性与辣椒抗疫病的关系。对不同抗性品系A5、A3、EC受到疫霉菌ph3侵染后和专化抗性品系A3受到不同生理小种ph1和ph3侵染后,植株过氧化物酶、苯丙氨酸解氨酶、多酚氧化酶、β-1,3-葡聚糖酶和几丁质酶活性进行测定和比较。经SAS分析表明,在供试的品种中,不同抗性品系的辣椒接种疫霉菌后,抗病与感病类型的保护酶具有明显差异,且这种差异具有明显的规律性。辣椒抗疫病的性状与植株体内的过氧化物酶、多酚氧化酶、苯丙氨酸解氨酶、β-1,3-葡聚糖酶和几丁质酶活性呈正相关,这几种酶可以作为抗病性鉴定的间接指标。3.利用cDNA-APLP技术研究专化抗性品系A3分别接种不同生理小种的辣椒疫霉菌ph1和ph3后的表达差异,获得了差异表达片段80个。登录了其中22个,登录号为:GO496263,GO496264,GO496265,GO496266,GO496267,GO496268,GO496269,GO496270,GO496271,GO496272,GO496273,GO496274,GO496275,GO496276,GO496277,GO496278,GO496279,GO496280,GO496281,GO496282,GO496283,GO496284。4.获得六个抗疫病相关的新基因。利用RT-PCR选择阳性片段结合BLAST的信息选择感兴趣的片段,通过RACE技术获得六个新基因的全长,它们分别是CanTF基因(登录号FJ617518),CanPOD基因(登录号FJ596178),CanBPM4基因(登录号FJ617520),CanNADPH基因(登录号FJ617519 ),CanZf基因(登录号FJ596179),CanOBP基因(登录号FJ617521)。生物信息学分析表明,这六个基因都具有完整的开放阅读框架。其中CanPOD蛋白同时具有信号肽和跨膜结构,CanOBP蛋白仅具有跨膜结构而没有信号肽;其他几个基因所推测的蛋白不含信号肽,也不是跨膜蛋白。进化分析表明,CanNADPH与大戟科蓖麻属蓖麻NADPH:quinone oxidoreductase(EEF49550.1)的亲缘关系最近,辣椒CanZf与禾本科植物水稻锌指蛋白(Os03g0788800)亲缘关系最近。CanOBP被单独聚为一类,其次与低等的真核生物OBP蛋白合并,推测CanOBP可能是编码与辣椒疫霉菌互做相关蛋白的基因。5.利用半定量RT-PCR技术对CanPOD基因的表达进行了研究。不同抗性品系A5、A3、EC受到疫霉菌ph3侵染后,在高抗品种A5中表达最早,反应最为迅速,2h即达到顶峰;而在感病品种中,24h以内表达量变化不大,但表达的持续时间较长。专化抗性品系A3接种不同生理小种的疫霉菌ph3和ph1后,非亲和组合中,CanPOD基因表达迅速;而亲和组合中直至24h才达到略低于非亲和组合水平。研究结果表明,CanPOD基因与辣椒对疫病的抗性密切相关。6.利用实时定量技术对CanZf基因和CanOBP基因的表达进行了研究。荧光定量PCR分析表明在同一疫霉菌生理小种ph3侵染A3后,CanOBP基因和CanZf基因的表达量在根部和叶部有所区别,在根部表达高峰较早,为8h;在叶部稍微推迟,在12h达到高峰。CanOBP基因在叶部的表达量为对照的2万多倍,而CanZf基因在叶部的表达量为对照的28倍,说明这两个基因主要在叶部表达。分别在A3根部接种不同的生理小种ph3和ph1后,CanZf基因在非亲和组合中24h内表达量变化不大,在36h出现表达高峰;在亲和组合中,8h即达到最高峰值,且在24h内高于非亲和组合。非亲和组合中,CanOBP基因的表达量分别在2h和36h出现两个峰;而在亲和组合中,仅在8h出现一个峰值。这两个基因受不同辣椒疫霉菌生理小种的诱导后表达模式不同,说明其与辣椒疫病专化型抗性有关。7.利用实时定量技术对CanZf基因和CanOBP基因的表达调控进行了研究。分别用400mmol/L的甘露醇,400mmol/L的NaCl,5 mmol/L的水杨酸(SA),50 mmol/L的甲基茉莉酮酸(MeJA)和10 mmol/L H2O2和4℃低温处理辣椒材料A3。荧光定量PCR分析表明,CanOBP基因的表达受到SA和MeJA的强烈抑制,早期(8h前)受到H2O2抑制,但是该基因受到低温、干旱和高盐的诱导,提示它可能介导了辣椒对这些胁迫的反应。CanZf基因的表达受到低温、干旱胁迫的诱导和H2O2的抑制。SA和MeJA处理后,CanZf基因的表达也有差异,在2h受SA抑制,8h上升;而在12h内受到MeJA抑制。说明该基因可能参与了多种植物抗逆性途径,也可能该基因位于这些途径的交叉点。8.建立了辣椒遗传转化单倍体受体系统。在22个不同基因型材料,共有13个材料被诱导出胚状体,诱导成功率为59.09%。其中B19出胚率最高,达到22.86%。以P51和P53为实验材料,研究不同培养基和预处理温度对出胚率的影响。不同基因型对培养基的要求有差异。1mg/L 6-BA最适合P51,而4mg/LNAA+1mg/L6-BA更适合P53。变温处理(4℃,3d;35℃,4d)可以明显提高这两个基因型材料的诱导率。创制了一种辣椒小孢子诱导获得胚状体的培养方法,并已获得国家发明专利(专利号ZL200610042616.0)。更多还原

【Abstract】 Pepper (Capsicum annuum L) which was widely planted around the world is an important economic vegetable crop in our country, while the pepper blight caused by Phytophthora capsici has been epidemic in many provinces and areas and has become the major limiting factor of pepper production. In this research, the evaluation technique of pepper resistance to Phytophthora capsici was development, the resistant mechanisms in physiology and biochemistry was investigated, and the difference in gene expression profile of Capsicum annuum infected by different physiological races of Phytophthora capsici was analyzed by cDNA-AFLP. After its full length was cloned via RACE technology, the sequence and structure characters of positive cDNA and corresponding protein were analyzed using bioinformatics, including the secondary structure , transmembrane zones, signal peptide and evolution pattern. The expression and regulation of these were analyzed by semi-quantitative RT-PCR and real-time quantitative techniques. The aim of this study is to reveal the resistant mechanism of pepper to P. capsici and establish basis for breeding of new pepper cultivar with resistance. The main results of our research are showed as follows:1. First, in vitro evaluation technique with lateral shoots of pepper resistance to Phytophthora capsici using CM334 with high resistance, N3 with moderate resistance and EC with sensitiveness. When the lateral shoots from the first branch after the second branch shot were inoculated with P. capsici, the difference of disease symptom between resistant and sensitive cultivars showed mostly significant under zoospore concentration with 1×104mL-1, temperature with 28℃, light intensity with 4000lx and photoperiod with 12h/d. Compared to in vitro leaf-inoculation, stem-inoculation and root-irrigating method, the evaluation results of 34 pepper materials using this technique showed marked positive correlation with these three common methods, and the correlation coefficient were0.9150** , 0.8730** and 0.8384**. These results suggested that the new technique could effectively evaluate the disease resistance of pepper to P. capsici. 2. The changes of protective enzymes’activities were studied. The results showed that the activities of peroxidase (POD) were significantly higher in resistant cultivars than those in susceptible ones, though activities increased both in resistant and susceptible cultivars after inoculation. Results analysis showed that there was a positive correlation between the activities of phenylalanine ammonia lyase (PAL),polyphenol oxidase (PPO) ,β-1,3-Glucanase and chitinase and the pepper resistance to Phytophthora capsici.3. 80 ESTs of different expression gene were got from pepper tread with Phytophthora capsici. 22 ESTs were submitted to GenBank, whose accessed number are as follows: GO496263,GO496264,GO496265,GO496266,GO496267,GO496268, GO496269,GO496270,GO496271,GO496272,GO496273,GO496274,GO496275,GO496276,GO496277,GO496278,GO496279,GO496280,GO496281,GO496282,GO496283,GO496284.4. Six TDFs were selected and got full-length cDNA, they are CanPOD(FJ5961 78), CanZf(FJ596179), CanTF(FJ617518), CanNADPH(FJ617519),CanBPM4(FJ617 520), CanOBP(FJ617521). Bioinformatics analysis showed that these six genes all have a complete ORF.The deduced amino acid sequence of CanPOD and CanOBP were have typical trasmembrance protein and CanPOD protein has a typical singal peptide.Phylogenetic analysis showed taht CanNADPH had close relationship with Ricinus communisi NADPH:quinone oxidoreductase(EEF49550.1), CanZf had close relationship with oryza sativa Japonica zinc finger protein(Os03g0788800), the sequence of CanOBP is conservetive, it was conjectured CanOBP gene related to the interaction between pepper and Phytophthora capsici.5. CanPOD mRNA expression were analyzed by semi-quantitative RT-PCR. The results show that intensive expression of CanPOD gene was induced by Phytophthora capsici. Different dynamic changes in the expression of CanPOD gene of incompatible intera -ctions and compatible interactions, which of incompatible interactions the CanPOD was expressed early and quickly ,which of compatible interactions, the up-regulated time of this gene is long, from 8h to72h6. The expression of CanZf and CanOBP were detected by real-time quantitative PCR. After treat with different physiological races of Phytophthora capsici, the CanOBP gene is up-regulated expression in A3’s root of incompatible interactions and compatible interactions. There are two expression peaks of incompatible interactions which were at 2h and 36h; but there is only one expression peak of compatible interactions, which was at 8h. The expression is also different between root and leaf after inoculation with ph3. The expression maximal peak (at 12h) in leaves is later than that in root, but it particularly higher than that in root. Furthermore, low temperature(at4℃), drought(400mmol/Lmannitol), high salt concentration(400mmol/LNaCl) could lead to up-regulated expression of CanOBP, and CanOBP is inhibited by SA(5 mmol/L), MeJA(50mmol/L) and H202(10mmol/L).The expression of CanZf is also up-regulated after treat with different physiological races of Phytophthora capsici. In incompatible interactions, the expression is 63 times at 36h contrast with CK, and in compatible interactions, the expression is 81 times at 8h contrast with CK. In leaves, the expression of CanZf is similar to CanOBP. Low temperature (at 4℃) and drought(400mmol/Lmannitol) could induced to up-regulated expression of CanZf, and H202.(10mmol/L) could suppressed expression of CanZf. After the expression of CanZf inhibition early which treated with SA (5mmol/L), MeJA (50mmol/L) and high salt concentration (400mmol/LNaCl), it is up-regulated expression later.The results indicated that CanOBP and CanZf play important roles in pepper resistant to Phytophthora capsici, and it can responded to many stress.7. Establishment of high efficient pepper haploid cultivation system. Genotype is a limit factor for pepper haploid cultivation. In this article, anther culture of 22 genotypes were tested, the embryoes could induced from 13 genotypes.The best culture medium is 1 mg·L-1 6-BA for P51 and that is 4 mg·L-1 NAA +1 mg·L-1 6-BA for P53. Changing temperature is best for embryos induced, first 4℃,3d,and then 35℃,4d. We created a method of embryoid obtainment from induced microspore of pepper, and have access to national patent.更多还原