【作者】 龚小庆；

【导师】 刘继红；

【作者基本信息】 华中农业大学 ， 园艺学， 2014， 博士

【摘要】 干旱、盐、低温、病虫害等逆境严重影响着柑橘的生长发育,继而影响产业发展。传统的育种方法需要投入大量的时间和精力以获得抗性较好的品种,依托基因工程对柑橘进行遗传改良获得抗性品种相对而言非常高效。金柑在柑橘类果树中虽不如枳耐低温,但是其综合抗性比较优良,本研究在实验室已有的研究基础上,从金柑中筛选得到3个逆境响应的基因,分析它们各自的功能及潜在的作用机制,得到的结果如下：1.FcWRKY70全长1196bp,编码328个氨基酸,蛋白的相对分子量及等电点分别是36.78kD和5.89. FcWRKY70包含一个保守的WRKY结构域,定位于细胞核。除干旱、盐以外,FcWRKY70还可以响应生物逆境(柑橘溃疡病菌接种)及不同的激素处理(水杨酸SA及脱落酸ABA)。FcWRKY70超表达可明显增强转基因烟草的抗旱性。同时发现,转基因烟草中多胺含量高于野生型,特别是腐胺(Put),多胺合成相关的基因表达也明显比较活跃。Put合成抑制剂D-Arg处理抑制了转基因烟草的气孔关闭,降低了其干旱抗性。在柠檬中超表达FcWRKY70,与野生型相比转基因柠檬抗脱水能力增强,腐胺含量及介导腐胺合成的ADC表达水平均高于野生型。同时在金柑FcWRKY70干涉系中腐胺含量及ADC的表达被抑制,干涉系相对失水增多。分离金柑ADC启动子,分析表明其上分布数个W-box元件或其核心元件,利用酵母单杂及双荧光素酶检测的方法证明FcWRKY70可与ADC启动子结合。2. FcWRKY40属于IIa类WRKY蛋白,编码257个氨基酸,所组成的蛋白相对分子量及等电点分别为28.7kD和8.83。FcWRKY40包含一个卷曲螺旋结构域和一个保守的WRKY结构域,定位于细胞核。该基因可被低温、盐、ABA和SA诱导表达,但被干旱抑制。超表达FcWRKY40可明显示增强转基因烟草对氧化胁迫的抗性。转基因烟草与野生型相比,H202积累较少,02-积累没有差异性；在转基因烟草中与H202清除相关的过氧化氢酶CAT和过氧化物酶POD的表达上调非常显著。CAT和POD启动子上均存在W-box元件,酵母单杂及双荧光素酶检测分析表明FcWRK40可分别与CAT及POD启动子结合,调控它们的表达。3. FcSISP (Fortunella crassifolia stress-inducible small protein)编码47个氨基酸,其蛋白相对分子量和等电点分别是4.94kD和3.76。该基因定位于细胞核,不具备转录激活活性。FcSISP可被干旱、低温、盐、外源激素(ABA和SA)以及柑橘溃疡病菌诱导表达,其中盐对其诱导效果非常显著。FcSISP在烟草中超表达,提高了转基因植株的耐盐性。转基因烟草中脯氨酸含量、脯氨酸合成相关P5CS和P5CR的表达均高于野生型。盐处理后,转基因烟草根和叶中的Na+量均低于野生型,叶中差异尤其明显。根中K+量较高,由此导致较低的Na/K比值。此外,发现转基因烟草中与Na+外排及区域化隔离相关的SOS基因和NHX基因表达水平均比野生型高。更多还原

【Abstract】 The abiotic and biotic stresses, including drought, salt, extreme temperature and pathogens, severely affect the normal growth and development of citrus, restricting the development of citrus industry world wild. It is time-consuming to obtain desirable cultivars with enhanced stress tolerance via traditional breeding methods, while genetic improvement mediated by modern biotechnology has been shown to be crucial in coping with the adverse environmental cues. Meiwa kumquat (Fortunella crassifolia) is not tolerant to low temperature as well as trifoliate orange (Poncirus trifoliata (L.)), but it displayed moderate resistance to multiple stress. In this study, we isolated three stress responsive genes from meiwa kumquat based on the previous study. For the sake of revealing the function of the three genes under various stress and their potential mechanisms, all of them were over expressed or suppressed in tobacco and citrus, respectively. The main results are as follows:1. The full length cDNA of FcWRKY70was1196bp, encoding a putative protein of328amino acids, with calculated molecular mass of36.78kD and theoretical isoelectric point of5.89. FcWRKY70contained a conserved WRKY domain, localized in the nucleus. Besides drought and salt treatment, the expression level of FcWRKY70can be induced by hormones, salicylic acid (SA) and abscisic acid (ABA), and citrus canker pathogen. Overexpression of FcWRKY70in tobacco (Nicotiana nudicaulis) led to enhanced tolerance to dehydration and drought stress. Meanwhile, transgenic tobacco accumulated much more polyamines (PAs) in comparison with wild type (WT), especially putrescine (Put), coupled with activated expression of genes involved in PAs synthesis. When treated with D-Arg, an inhibitor of Put synthesis, the stomatas of transgenic tobacco were prevented from closure, and transgenic plants showed to be a little more sensitive to dehydration treatment compared with WT. Overexpression of Fc WRKY70in lemon (Citrus limon Burm.f.) led to enhanced tolerant to dehydration treatment in comparison with WT. The accumulation of Put in transgenic plants was much higher than in WT, consisting with increased expression level of ADC which involved in the synthesis of Put. Knock-down of FcWRKY70in meiwa kumquat repressed the synthesis of Put, and the transgenic kumquat displayed higher relative water loss under dehydration. Isolation of ADC promoter from kumquat revealed that several W-box and the core elements distributed on it. The yeast one-hybrid assay and transient dual luciferase assay demonstrated that FcWRKY70interacted with the promoter of ADC.2. FcWRKY40was classified into Group Ⅱa WRKY gene in meiwa kumquat, encoding a putative protein of257amino acids with calculated molecular mass of28.7kD and theoretical isoelectric point of8.83. FcWRKY40consisted of a coiled-coil domain and a WRKY domain, localized in the nuclei. Transcript levels of FcWRKY40were upregulated by cold, salt, ABA and SA, but repressed by dehydration. Transgenic tobacco plants overexpressing FcWRKY40were shown to be more tolerant to the oxidative stresses than the WT. The accumulation of H2O2in transgenic tobacco were much lower than in WT, while the O2levels were almost the same between them. In addition, mRNA abundances of two genes related to H2O2scavenging, POD and CAT, were higher in the transgenic plants than in WT. The promoters of POD and CAT contained W-box elements. Both yeast one-hybrid and transient expression assay demonstrated that FcWRKY40could interact with the two promoters, regulating the expression of POD and CAT.3. FcSISP encoded a putative protein of47amino acids, with calculated molecular mass of4.94kD and theoretical isoelectric point of3.76, and was localized in the nucleus without trans-activation activity. Transcript levels of FcSISP were induced by various treatments, such as dehydration, cold and salt, hormones (SA and ABA), and citrus canker pathogen, with the greatest induction being observed under salt treatment. Transgenic tobacco plants overexpressed FcSISP displayed ehanced tolerance to salt stress compared with WT. The accumulation of proline was much more in transgenic lines than in WT, coupled with higher expression levels of P5CS and P5CR. After salt treatment, the Na+levels were significantly lower in the transgenic lines relative to the WT, the dramatic difference being particularly observed in the leaves. The difference in K+contents can be only observed in roots between transgenic lines and WT. As a result, the Na+/K+ratios in the leaf and root of transgenic lines were significantly lower than those of WT. QRT-PCR analyses demonstrated that the expression levels of the examined genes, SOS1-3and NHX1-4, related to efflux or compartmentation of Na+, were almost higher in the two transgenic lines in comparison with WT, excepted for NHX4.更多还原