Isolation and expression analysis of differentially expressed genes in stem tissue of the Greek lemon cv. Adamopoulou

Lemon (Citrus limon (L.) Burm. f.) is susceptible to mal secco, a serious vascular disease caused by the fungus Phoma tracheiphila (Petri) Kant. and Gik., as well as low temperatures. The greek lemon cultivar Adamopoulou, thought to be derived from the Portuguese cultivar Lisbon, exhibits enhanced resistance to mal secco and cold as opposed to cv. Lisbon. Suppression subtractive hybridization (SSH) was employed for the isolation of differentially expressed genes in lemon stem tissue. A subtractive cDNA library was constructed and a total of 296 clones were sequenced. The obtained sequences were edited, resulting in 56 non-redundant ESTs. Sequence analysis revealed homology to previously identified genes involved in defense mechanisms against biotic and abiotic stresses, as well as sequences with no significant similarity in the GenBank. Selected ESTs were analyzed by real-time PCR for confirmation purposes. This analysis revealed significant expression differences between the two cultivars for genes expressing allantoinase, ultraviolet-B-repressible protein, 4-coumarate:CoA ligase and other proteins that are known to be upregulated under biotic and abiotic stress conditions.     

紫外线照射对梁平柚果皮基因表达的影响

 采用SSH技术以紫外照射的梁平柚[Citrus grandis（L.）Osbeck]果皮作为实验方（tester）,以未被照射的正常果皮作为驱动方（driver）,构建了一个梁平柚果皮紫外诱导基因的正向差减文库。经菌液PCR检测后随机挑取200个阳性克隆测序,获得168条表达序列标签（ESTs）。比对这168条ESTs,发现有分属于57个基因的114条ESTs与已知基因高度同源,54条ESTs同源性较低或没有同源性。功能分析发现,这些ESTs主要参与抗逆防御、生长发育、细胞凋亡、转录与翻译、细胞分化、信号传导、能量代谢、糖类及氨基酸代谢以及次生代谢等。     

苯并噻二唑处理提高采后沙糖橘对指状青霉菌的抗性

对沙糖橘(Citrus reticulata Blancdo‘Shiyueju’)果实进行苯并噻二唑(S-methyl benzo thiadiazole-7-carbothioate,BTH)渗透处理后接种指状青霉菌孢子,12h后即可明显诱导果皮中抗病相关蛋白CR-CT1和CR-BG3基因表达并在贮藏期间维持较高水平;不同浓度BTH处理果实的发病率和病斑面积均比对照低,其中以0.025g·L-1BTH处理的效果最好;BTH处理提高了果皮几丁质酶(CT)、β–1,3–葡聚糖酶(GUN)、苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)、过氧化物酶(POD)活性和H2O2含量,表明BTH诱导的沙糖橘果实抗病性与果实CR-CT1和CR-BG3基因的表达增强,CT、GUN、PAL、PPO和POD等相关酶活性和H2O2含量提高密切相关。     

沙田柚幼果期套袋对果实品质的影响

本试验研究了不同类型的商品果袋在沙田柚幼果期套袋后果实品质的变化,结果表明,套袋显著地改善了柚果的外观品质,防病虫效果显著,并使单果重明显增加,虽然套袋使鲜果的可溶性固形物和维生素Ｃ的含量略有下降,但贮藏后的果实风味更佳,其中以Ｂ型果袋对提高沙田柚果实商品价值较突出。     

柑橘果皮果胶及其酶活性对皱皮果形成的影响

 研究‘红127’甜橙和‘红江橙’的果皮果胶质、果胶甲酯酶( PE) 及皱皮果率的差异。结果表明, 抗皱皮的‘红127’甜橙皱皮果发生率为7.06% , 而‘红江橙’皱皮果发生率高达28.03%; 皱皮出现在阴面果皮占86%以上。‘红江橙’PE在果实成熟时(12月15日) 比成熟前( 10月1日) 增加2.6倍, 水溶性果胶(WP) 增加8.27倍, 总果胶( TP) 增加65% , 盐酸可溶性果胶(HP) 减少54%。‘红127’甜橙果皮的PE变化较小。PE与WP极显著正相关, 与HP极显著负相关。‘红江橙’TP、WP及PE明显高于‘红127’甜橙, 而HP以‘红127’甜橙的高。阴面比阳面果皮有较多的TP、WP和PE, HP则以阳面果皮为高。皱皮果的TP、WP、PE高于正常果, 而HP则少于正常果。六偏磷酸盐可溶性果胶( PP) 在两种果实上没有差异。     

黄龙病菌侵染晚锦橙叶脉和根早期病原蛋白组学的比较分析

[目的]黄龙病(Huanglongbing,HLB)是世界柑橘生产上最具毁灭性的病害,由于黄龙病病原菌无法离体培养,其关键的致病机制仍不清楚.为此,通过蛋白组学分析探究黄龙病菌侵染晚锦橙早期时病原蛋白的表达情况.[方法]利用叶圆片嫁接技术对晚锦橙(Citrus sinensis Osbeck)进行嫁接传毒,在传毒2个月...     

柑橘响应黄龙病侵染的韧皮部蛋白2基因CsPP2B15的克隆与表达分析

黄龙病病原菌诱导柑橘筛孔胼胝体过度积累是其致病的一个重要因素。柑橘韧皮部蛋白（Phloem Protein,PP）在筛孔胼胝体积累中起着重要作用。为研究柑橘韧皮部蛋白响应黄龙病亚洲种（Candidatus Liberibacter asiaticus,CLas）侵染的功能,克隆获得了一个响应黄龙病侵染差异表达的PP基因CsPP2B15的编码区和长1.5 kb的启动子序列,并以高感品种锦橙和耐病品种酸柚老叶和嫩叶为材料,进行基因表达分析。结果发现,CsPP2B15在高感品种锦橙嫩叶中受CLas诱导显著上调表达,而在耐病品种酸柚嫩叶中受CLas诱导显著下调表达,且在叶肉中的表达量显著高于叶脉。嫩叶和嫩梢被认为是柑橘响应黄龙病侵染的早期原初侵染部位。因此,CsPP2B15可能在CLas侵染柑橘早期中起重要作用。CsPP2B15启动子的顺式作用元件预测分析发现,CsPP2B15启动子含有许多与逆境和激素有关的顺式作用元件。进一步构建了CsPP2B15启动子控制GUS报告基因的植物表达载体,转化枳[Poncirus trifoliata（L.）Raf.]上胚轴。GUS组织化学染色和GUS酶活性测定均表明,CsPP2B15启动子具有较强的韧皮部组织特异性。     

温州蜜柑线粒体基因的克隆与表达分析

 根据其它植物atp6、cob和coxⅡ基因保守区设计特异引物, 利用RT - PCR技术从‘国庆1号’温州蜜柑cDNA中分别扩增出特异性片段, 将其克隆至pBluescrip t ( sk + ) 载体上进行测序。同源性分析表明, 所克隆的atp6、cob和coxⅡ与其他植物的对应氨基酸区域同源性分别达到85%、98%和96%以上。RT - PCR分析表明它们在叶片、花瓣以及不同时期的花蕾中都有表达, 在幼果中没有表达。Southern分析表明它们在温州蜜柑基因组DNA中为多拷贝。     

脐橙果实发育过程中有机酸合成代谢酶活性的变化

 以红桔砧10 年生罗伯逊脐橙为试材, 研究了果实发育过程中有机酸代谢相关酶活性的变化及其与有机酸积累的关系。结果表明: 在整个果实发育过程中, 柠檬酸合成酶(CS) 和磷酸烯醇式丙酮酸羧化酶(PEPC) 活性与有机酸含量呈极显著正相关。这不仅证明脐橙果实中存在通过CO₂ 固定合成有机酸的途径, 还说明CS、PEPC 是有机酸合成的关键酶, 抑制CS 的活性能降低果实中有机酸的生成量。     

琯溪蜜柚高分量核DNA提取方法的建立

以琯溪蜜柚[Citrus grandis(L.)Osbeck'Guanximiyou']叶片为材料,比较了两种提取方法对高分子量核DNA(HMW-DNA)制备效果的影响。结果表明,改良Zhang法与Peterson法相比,前者更适合于琯溪蜜柚HMW-DNA的提取。改良Zhang法用温和的物理方法破碎细胞壁,在一定的渗透压下保证细胞核的完整性,运用miracloth和低速离心去除大部分的多糖和细胞壁碎片,经显微观察,所提取的细胞核完整、纯净、质量高;将这些细胞核用低熔点琼脂糖包埋,以琼脂糖小块的方式保存细胞核,以保持核的稳定;琼脂糖小块的最适消化时间为48h,脉冲场凝胶电泳分析结果表明所获得的HMW-DNA大于1Mb,这些核DNA能够被限制性内切酶消化,适合于琯溪蜜柚BAC基因组文库的构建。     

根癌农杆菌介导绿色荧光蛋白基因转化印度酸桔的研究

 通过根癌农杆菌介导将绿色荧光蛋白基因转入印度酸桔的胚性愈伤组织中, 经潮霉素筛选,获得抗性愈伤组织, 并再生植株。对这些植株进行GUS 染色、PCR 分析、绿色荧光检测和Sourthern 杂交验证, 结果表明绿色荧光蛋白已经在转基因植株中表达。     

Production and evaluation of transgenic sweet orange (Citrus sinensis Osbeck) containing bivalent antibacterial peptide genes (Shiva A and Cecropin B) via a novel Agrobacterium-mediated transformation of mature axillary buds

Transgenic plants of Jincheng orange (Citrus sinensis Osbeck) and Newhall navel orange (C. sinensis Osbeck) containing antibacterial peptide genes Shiva A and Cecropin B were successfully obtained by a novel Agrobacterium tumefaciens-mediated transformation of the mature axillary buds. PCR and Southern blot analysis of the transgenic plants verified that the Shiva A and Cecropin B genes were integrated into the citrus genome. The transgenic plants began to blossom and bear fruit in the 2nd year after grafting on trifoliate orange (Poncirus trifoliata Raf) rootstock in greenhouse. Water-soluble extracts from transgenic citrus leaves exhibit in vitro suppressive effects on Xanthomonas axonopodis pv. citri, suggesting that the expressed products of Shiva A and Cecropin B in citrus retain their native antibacterial activities. Artificial inoculation in greenhouse and open field further indicates significantly increased resistance of transgenic plants to X. axonopodis pv. citri when compared with non-transgenic lines. No significant difference was found in the content of total soluble solids, total acidity, reduced sugar content and other fruit characteristics between transgenic and non-transgenic plants. In this present study, 11 transgenic lines were obtained from 40 transgenic lines, showing enhanced resistance to citrus canker disease.     

根癌农杆菌介导的CG1-400-RNase基因转化创制锦橙转基因再生植株

【目的】为了快速有效地利用基因工程的方法获得柑橘无核新种质,【方法】以我国特色多核柑橘优良品种锦橙实生苗上胚轴切段为外植体,采用根癌农杆菌介导法进行能导致种子败育基因CG1-400-RNase的转化;为快速有效地筛选出转化子,在实生苗上胚轴切段转化再生过程中,根据不同发育阶段的组织或器官对抗生素的敏感程度不同采用不同的选择压。【结果】结果表明,在抗性芽再生过程中卡那霉素质量浓度设定为50 mg.L-1,获得的362个抗性芽转入卡那霉素质量浓度为100 mg.L-1的伸长培养基中进行伸长培养后,进行早期PCR检测,获得28个阳性芽;经过不定芽诱导生根或试管嫁接,获得22株完整植株。【结论】再生植株经PCR和Southern杂交检测,获得2株目的基因以单拷贝的形式插入锦橙基因组的转基因植株,为最终获得具有无核性状且可稳定遗传的柑橘新种质奠定了基础。     

锦橙叶片镁质量分数与若干光合指标的相关性

选用缺镁程度不同的北碚447锦橙,采用缺镁黄化分级方法,测定不同级别叶片的镁质量分数、叶绿素各成分质量分数和净光合速率,探讨叶片镁质量分数与后2者的相关性。结果表明,不同缺镁级别叶片的镁质量分数存在极显著差异;叶片的叶绿素a(Chla)、叶绿素b(Chlb)、叶绿素(Chl)和类胡萝卜素(Car)与镁质量分数呈显著的线性正相关关系,而与Car/Chl呈显著的指数负相关关系;叶片镁质量分数与净光合速率(Pn)也呈极显著的正相关关系,二项式回归方程为:y=0.0135x2+0.0484x+0.056(n=100,R2=0.9255)。因此,可以采用缺镁黄化分级法对柑橘叶片缺镁程度作定性分析,并通过测定叶片的Chla质量分数、Chlb质量分数、Chl质量分数、Car质量分数、Car/Chl或Pn值,建立与镁质量分数相关的数学模型,定量换算得到叶片的镁质量分数。     

柑桔种质资源对脚腐病的抗性鉴定

采用刻伤接种柑桔脚腐病病原菌(Phytophthora parasitica),观察51个柑桔种质资源对柑桔脚腐病的抗病程度。测定结果表明:枳、枳橙、枳柚、大叶金豆、酸橙等19个种类或品种对脚腐病具有高抗或抗病性;福桔、尤力克柠檬等14种材料为感病或重感病型;其余18个为中抗或中感型。其中,高抗类型大叶金豆(Fortunella hindsii var.chintou)为首次报道。     

滴灌施肥对‘特罗维它’甜橙生长结果的影响

 以卡里佐枳橙砧‘特罗维它’甜橙为材料,以每年撒施10次肥料为对照,研究了年施肥总量一定,不同滴灌施肥频率（每年4次、10次和16次）对树体生长、产量和品质的影响。2002—2007年的结果显示,仅个别年份的处理与对照的树干粗度、树高和冠径存在显著差异以外,到试验结束时,不同滴灌施肥频率对树干粗度、树高和冠径大小无显著影响,滴灌施肥与对照间的树体生长无显著差异。滴灌施肥可显著提高果实产量,处理的累计产量比对照高29.4% ~ 36.5%;增加滴灌施肥频率无显著增产作用;滴灌施肥对果实品质无显著影响。建议碱性紫色土柑橘园滴灌施肥频率为每年4次。     

Identification of reference genes for real-time quantitative polymerase chain reaction based gene expression studies on various Olive (Olea europaea L.) tissues

Reference genes are essential for the normalisation of the expression data of quantitative real-time PCR for the purposes of validation. Although several reference genes have been validated for the olive, comprehensive analyses including an excessive number of candidate reference genes still require study in various olive tissue samples. In this work, a total of 40 candidate reference genes were tested for their stability in 8 different olive tissues (root, apical bud, lateral bud, pedicel, young leaf, mature leaf, fruit mesocarp, and seed) with the utilisation of the most popular software programs including GeNorm, NormFinder, BestKeeper, and ΔC_t. The analyses of expression stability of candidate reference genes using quantitative real-time PCR demonstrated ubiquitin-conjugating enzyme (UBC1) as the most stable reference gene for the studied tissues of the olive. The GeNorm software also calculated the optimum reference gene combinations as two which consist of UBC1 and the Clathrin adaptor complex medium subunit (CLATHRIN) genes. This study provides the most stable reference gene combination for normalisation of target genes for quantitative real-time PCR gene expression studies on the olive.     

红肉脐橙果肉中主要色素的定性及色素含量的变化

 运用薄层层析法及二极管阵列检测器与高效液相色谱联用的方法, 确定红肉脐橙果肉中含有番茄红素和β- 胡萝卜素两种主要色素, 它们在液相色谱图中占总峰面积(计溶剂峰) 的73. 57 %～77. 78 % , 番茄红素占55. 36 %～58. 49 % , β- 胡萝卜素占18. 21 %～19. 93 %。利用反相高效液相色谱仪以及番茄红素和β- 胡萝卜素标样制作标准曲线, 测定果实发育及室温贮存过程中色素的变化动态, 结果表明, 该脐橙果肉中的番茄红素在成熟期含量最高, 达109. 67μg·g^-1DM, β- 胡萝卜素此时也达到最高值(15. 52μg·g^-1DM) ; 番茄红素的含量在采收后基本呈下降趋势(但采后30 d 有显著升高) , 说明果肉中存在类胡萝卜素采后合成的现象; 贮存4 个月后, 番茄红素含量急剧降低至最高时的1/ 10。