【作者】 李大志；

【导师】 邓子牛； 谢丙炎；

【作者基本信息】 湖南农业大学 ， 果树学， 2007， 博士

【摘要】 在农业生产中，植物病害的为害造成了巨大的损失，而生产上采用的高毒、高残留农药对产品和环境均造成严重的污染，已成为制约我国无公害农产品生产可持续发展的突出问题。研究开发可持续、环保型控制技术是解决这一问题的关键。β-氨基丁酸（BABA）是从经暴晒的番茄根系中分离得到的一种次生代谢非蛋白氨基酸，具有高效、广谱的诱抗活性，可诱导多种作物抵抗由卵菌、真菌、细菌、病毒和线虫引起的病害，保护植物免受病害为害。要开发利用BABA诱导植物抗病潜能，创新有害生物综合治理途径与技术，必须进一步认识BABA的诱导抗病机理。从功能基因组学角度分析BABA诱导植物防御反应过程中信号互作功能基因群的表达特征，是揭示寄主—病原物互作的分子机制的新思路。过去，基于技术局限，关于植物诱导抗病的基因转录调控分析大多集中在若干已知PR蛋白和个别SA信号传导路径中的几个基因上。现在，利用功能基因组学的多项技术，可以明确更多应答诱抗时植物体内转录发生改变的基因，从而更准确、全面地认知植物诱导抗病行为机制。本研究以番茄为材料，利用cDNA-AFLP基因转录谱分析技术研究番茄根部组织应答BABA诱导后基因转录谱，取得的结果如下：1．由于植物总RNA提取的质量具有很强的组织特异性，对试验中常用的TRIzol Reagent、RNAplant、TRIpure Reagent等三种RNA提取试剂盒进行了番茄成熟老化组织RNA提取效果的研究，发现RNAplant能有效从多种成熟组织中提取纯度高、完整性好的RNA，符合需要总RNA质量较高的分子试验要求。2．采用25mmol／L BABA溶液处理番茄六叶期幼苗，蒸馏水处理作为对照，运用cDNA-AFLP差别显示技术对基因转录谱进行分析。从显示出的约5000个转录基因片段（Transcript-derived fragments，TDFs）中发现了23个受BABA诱导上调基因片段（BABA Induced Fragment，BIF），6个诱导下调片段。通过克隆测序最终得到12个转录上调基因序列。用生物信息学方法分析这12个cDNA片段的功能，可将其分为5种类型：①防卫反应基因：BIF13，BIF14为病程相关蛋白基因P69家族成员，它们具有蛋白水解酶功能；②转运蛋白基因：BIF2，BIF3、BIF6、BIF17，BIF4，负责细胞内外离子、水和小分子物质的跨膜运输；③信号传导蛋白：BIF5为Ca²⁺绑定蛋白，结合游离Ca²⁺，参与Ca²⁺信号传导；④大分子蛋白相关基因：BIF 1为协助蛋白复性的分子伴侣蛋白编码基因；⑤功能未知基因：BIF10、BIF11、BIF12。3．对上述基因片段进行序列分析，发现BIF3、6、17三个基因片段序列长度一致，同源性高达99％，为编码细胞逆境信号通路中的钙调转运蛋白酶(Calcium transporting-ATPase，Ca²⁺-ATPase)基因片段。利用分子系统发育分析对Ca²⁺-ATPase基因家族进行了细分；在此基础上，依据同源克隆的原理，结合步进PCR和RACE方法获得这个质膜型（Plasma membrane-type，PM-type）Ca²⁺-ATPase全长cDNA序列，基因全长3389bp，开放阅读框为3090bp，编码1029个氨基酸的蛋白质。这是首次在茄科类植物中克隆到PM型Ca²⁺-ATPase。4．利用Northern Blotting分子杂交对靶标Ca²⁺-ATPase基因受BABA诱导的时空规律进行了研究，结果显示，Ca²⁺-ATPase基因在根中表达量最高，叶其次，茎中最弱；基因的转录水平在6—12h达到最高，持续到72h仍有微量表达。5．利用生物信息学手段对靶标基因编码蛋白的理化性质和二、三级结构、代谢途径进行分析，并构建了该蛋白的理论三维模型，发现该基因编码的多肽链有7个基序（motif），4个保守功能结构域（domain），8个跨膜螺旋，都与蛋白的活性有关，该基因参与Ca²⁺信号代谢途径。对靶标基因进行了分子系统发育分析，找到了该基因在生物界进化的地位。6．利用SMART和Primer Extension技术相结合，构建了一个富集BABA诱导抗性相关基因的cDNA全长文库，原始文库的滴度达到为4.4×10⁶，重组率为96％，插入片段大小分布在400—1800bp，平均大小为700—1000bp；为克隆响应BABA诱导的其他重要抗性基因全长cDNA奠定了基础。7．利用了Ca²⁺-ATPase的保守区段设计基因特异引物（Gene Specific Primer，GSP），在两个经受低温锻炼的柑橘品种中进行半定量RT-PCR，试验结果表明此基因广泛存在于植物组织内部，属诱导表达型基因，积极参与了植物逆境信号转导过程。更多还原

【Abstract】 In agriculture, the pathogens cause severe losses in yield throughout the world. And continuousapplications of chemicals lead to heavy contaminations of the products and the environment. Duringthe past 20 years,β-Aminobutyric Acid （BABA） has been proved to be effective to induceresistance against a broad spectrum of pathogens in agricultural crops and vegetables. Moreover,BABA has been verified one of the most effective elicitors to introduce tolerance to abiotic stresses.Although the phenomenon has long been known, the underlying mechanisms are poorly understoodat the biochemical and molecular level. New methods should be employed to analyze the genefuncations, in order to better understand the induced resistance by BABA and the applicationpotential of BABA induced resistance.cDNA amplified fragment length polymorphism analysis （cDNA-AFLP） was used to revealtranscripts whose expression is rapidly altered during the treatment of soil drench ofβ-Aminobutyric Acid （BABA） to tomato （Lycopersicon esculentum L） roots.The main results are following1. 3 kits were used for RNA extraction from 3 tomato tissues, including leaf, stem and root,were evaluated upon quality、yields of RNA isolated. RNAplant was found to be the most effectiveone among the 3 reagents evaluated. With this kit, higher amount and quality of RNA could besuccessfully extracted from all 3 kinds of tissues tested.2. After treated with 25mmol/L BABA, cDNA-AFLP was used to reveal transcripts whoseexpression is rapidly altered during the treatment of soil drench of BABA to tomato roots, withDDH₂O as control. Among 5,000 transcript-derived fragments （TDFs）, 23 showed increasedabundance and 6 showed decreased abundance. By cloning and sequencing, the sequences of 12TDFs were obtained. A high percentage of the differentially regulated TDFs identified in this studyhas homology to genes commonly regulated during plant defense/stress responses. According to thefunctions of homology genes, these 12 TDFs could be divided into 5 groups.①BIF13 and BIF 14: members of P69, a pathogenesis-related proteins （PR）;②BIF 2, BIF 3、6、17 and BIF 4: high homologies to genes all responsible for transductionacross membrane system of little molecular materials such as H₂O, Ca²⁺ and anti-fungal agents.③BIF5: signal transduction proteins, high homology to Ca²⁺-binding protein which is animportant dement in Ca²⁺ signaling pathway.④BIF1: molecular chaperone protein, keeping activity of certain proteins under stresscondition.⑤BIF10, BIF and BIF 12: no homology to known genes.3. With the RACE and PCR technology, a PM-type （Plasma membrane-type） Ca²⁺-ATPasegene was isolated. The full-length cDNA is 3389 bp, and the open reading frame （ORF） is 3090bp,encoding 1029 amino acid. Both RT-PCR and the analysis of sequence, including Blastn、Blastx and ORF confirmed it is the first time that PM-type Ca²⁺-ATPase was isolated in Solanaceae plant.4. Northern Blotting was performed to reveal transcript level of PM-type Ca²⁺-ATPase indifferent tissues at different times. The result indicated that Ca²⁺-ATPase was abundant in root, lessin leaf and stem; furthermore, the transcript level of target gene was up-regulated and reached itspeak at 6 hours after the treatment, and it nearly could not transcript after 72 hours.5. The deduced amino acid sequence showed high identities with PM-type Ca²⁺-ATPase, buthad low identities with ER-type Ca²⁺-ATPase. Amino acid analysis with TMpred software indicatedthat the polypeptide chain contained 7 motifs、4 domains and 8 transmembrane helixes, which wererelated to the activity of the putative protein. The gene take participate in calcium signaling pathway.6. A full-length cDNA library induced by BABA was constructed by using SMART（switchingmechanism at 5’ end of RNA tanscript） and Primer Extension techniques. The titers of unamplifiedand amplified cDNA library were 4.4×10⁶ pfu/mL and 1.8×10¹⁰ pfu/mL respectively. The rate ofrecombination was 96%. The average size of inserted fragments was 700-1000bp.7. A pair of degenerate primers was designed against the conserved regions that was used forSemi-quantitive RT-PCR to amplify cDNA segment from two citrus genotypes （Citrus unshiu Marcand Poncirus trifoliate） treated with low temperature. The results revealed that Ca²⁺-ATPase wasup-regulated by low temperature in citrus, suggesting this gene participating in adversity signalingconduction.更多还原