论文标题:生防绿色木霉工程菌的构建及其诱导植物抗病性研究
Construction of Biological Control Strains of Trichoderma Viride and Study of Their Ability to Induce Plant Disease Resistance
论文作者 刘士旺
论文导师 郭泽建,论文学位 博士,论文专业 植物病理与分子生物学
论文单位 浙江大学,点击次数 660,论文页数 124页File Size7760k
2003-05-01论文网 http://www.lw23.com/lunwen_434022/ 绿色木霉;隐地蛋白;原生质体;限制酶介导转化;诱导抗病性;培养基
Trichoderma viride; Cryptogein; Protoplasts; Restriction enzyme-mediated integration; Induction disease resistance; media
植物病害一直是制约农作物丰产的主要因素之一。许多重大病害病原菌的频繁变易，导致抗性作物品种经常丧失抗性，另外化学农药的过量施用使病原菌的抗药性增强，造成防治难度加大。有机农药使用后的残留，造成对生物以及环境的破坏，在一定程度上也造成对人体的危害。因此，随着人们环保意识的增强，生物防治特别是生物农药的使用已成为防治植物病害的趋势，市场对开发新型的生物农药也有着迫切的需求。 植物在同病原物竞争进化过程中，已形成一系列防御机制，过敏反应(HR)和系统获得抗性(SAR)就是这种防御机制的典型表现。某些小分子物质能够诱导植物产生这种防卫反应。这种防卫反应的产生，可以抵抗多种多样的病原物对植物的第二次侵染，类似于人或动物的免疫系统，利用植物的这种诱导抗病性和生物防治菌的特性来开发新型的生物农药应该十分可行。绿色木霉(Trichoderma viride)是一种腐生性生物防治微生物，其对植物病原真菌的拮抗作用包括竞争作用、重寄生作用及抗生作用等方面。如果用木霉来表达诱导植物抗病性蛋白，并利用木霉的腐生特点来传播该蛋白，这将大大增加木霉的应用范围，也为开发可持续利用的生物农药提供一条可行的途径。为了验证这种想法，我们使用了隐地疫霉(Phytophthora cryptogea)分泌的隐地蛋白(Cryptogein, Crypt)作为诱导植物SAR的激发子。通过基因转化的方法获得了含有隐地蛋白基因以及其第13位点突变基因的绿色木霉工程菌，绿色木霉在作为隐地蛋白的传播载体的同时，呈现了诱导烟草抗病的功能。实验研究的主要内容包括以下几个部分： 1．构建了绿色木霉转化的pCSNTCC和pCSNTCCm载体。 为了能使绿色木霉合成的外源激发子蛋白分泌到培养液中，在Crypt和其突变CrypK13V(PCR定点突变，第13位氨基酸由赖氨酸‘K’突变为缬氨酸‘V’)基因前插入信号肽序列，引导外源激发子蛋白分泌到木霉细胞外，与寄主植物直接作用，诱导植物的防卫反应。在合成信号肽基因ThChi和目的基因Crypt和CrypK13V基础上，以真菌表达载体pCSN43以及PBS载体为基本框架，经过系列酶切、连接以及转化等分子生物学技术，构建了crmt基因转化绿色木霉的真菌表达载体PCSNTCC和0丁贝W3厂基因转化绿色木霉真菌表达载体PCSNTCCm。2．建立了绿色木霉的转化体系。 对绿色木霉原生质体制备和再生条件进行了较详细的分析，并在此基础上，利用限制酶介导进行了木霉原生质体转化。结果表明，木霉原生质体形成的合适条件为：培养24 h的菌丝用4 mg加L的Glucanex在磷酸盐缓冲液中（pH 6．98）、30C、振荡（40 r／min）酶解 4 h，原生质体产量达到 4．7X 10’个加g。原生质体在含 0．3 mol／L肌醇和 0．3 mol／L KCI的 CM培养基上再生率为 14．5％。限制酶 ho介导转化绿色木霉原生质体，其转化率为每微克DNA得到1—2个转化子。转化子的PCR鉴定以及特异酶联免疫测定（ELISA）、wes上ern杂交检测表明，外源基因己转入绿色木霉菌并在培养液中有稳定的产物表达。30株原生质体再生株被确定为稳定的转化子，其中＊－1至＊－20为Cr－yP t基因转化子，TV－ZI至＊－30为o地们3V基因转化子。3．转化子诱导植物抗病性 转化子的抗病分析结果表明，转基因木霉菌株TV七和 TV六4等能够分泌CryPt或CryK13V，木霉出发株及转基因木霉抱子处理烟草2个品种和十字花科植物拟南芥u thalfana）根部 10 d后，离体接种和植株整株接种病原菌，实验表明转化子能够提高烟草对黑腔病菌（PhytOPhthom P。rasftfca varnlcotianad、赤星病菌（Altemarfa alte。ata）和烟草花叶病毒（TMV）的抗性，分子杂交检测表明，转化于能够诱导烟草PR基因的表达。转化于处理拟南芥，接种丁香假单胞菌和立枯丝核菌，离体接种和分子水平杂交均表明，拟南芥抗病性基本没有得到提高，表明Crypt的诱导抗病性具有一定的专化性。4．筛选转化子TV－3发酵培养基 转化于TV－3培养基筛选实验表明，合适的利于工业化的固体生产RPA培养基，组成配比为：40％稻草，5％豆饼粉，15 ％鼓皮，1．5％蔗糖，产抱量达到 3．36 X10’cfu／mL；液体MYG培养基，组成配比为麦芽汁30％，酵母提取物0．l％，葡萄糖1．5％，MgSO。7Hzo 0．l％，KHzPO；0．1％，菌丝生物量达到6．74 mg／mL。
Plant disease is one of the serious problems affecting plant growth and output of crops etc. It is difficult to control because pathogens mutated frequently, and this leading to the disease resistant plants broke down their resistance. The excessive application of chemical pesticides is not only leading to the pathogen resistance against the chemicals, but also harmful to the environment, and certainly to the health of human beings. Therefore, biological control agents (BCA) are considered as the possible replacement of chemical pesticides, and as an environmental friendly agent for further plant disease control.Hypersensitive response (HR) and systemic acquired resistance (SAR) are the typical representation of plant defense reactions. Once the establishment of SAR, the plants exhibit a broad-spectrum of disease resistance against pathogen attack. Researchers have identified elicitor proteins for example elicitins and hatpins, which elicit plant defense reactions. It would be feasible to explore biological control pesticides base on the characteristics of plant SAR.Trichoderma viride is an ubiquitous soil saprophyte biological control microbe having the action modes with pathogens of competition for nutrient resource, antibiosis, and mycoparasitism. If T. viride can be used as a producer and carrier of an elicitor protein, it would be applicable for the development of plant specific BCA. To test this idea, we used cryptogein, a proteinous elicitor secreted from Phytophthora cryptogea for the development of bioengineering T. viride. The plasmid containing Crypt or its mutant was introduced into T. viride by the method of restriction enzyme mediated integration (REMI). Our study indicated that T. viride acts as the producer and carrier of Crypt, and at the same time the transgenic lines enhanced disease resistance when applied on tobacco plants. The summary of our study as follows:1. Construction of pCSNTCC and pCSNTCCm plasmidsFirstly, Crypt gene was mutated by change the K at the position 13 of Crypt into V (the mutant named CryK13V) as described earlier. In order to secrete the produced protein out of T. viride cells, a signal sequence of a chitinase gene from Trichoderma (ThChi) was fused to the 5" of Crypt and CryK13V. The chimeric genes were constructed under the control of trpC promoter in vector pCSN43 respectively. Then, a hygromycin resistant gene was introduced into the vectors, and plasmids of pCSNTCC (for Crypt gene) and pCSNTCCm (CrypK13V) were obtained.2. Establishment of T. viride transformation systemThe conditions for protoplasts isolation and regeneration from T. viride were studied. The optimum condition for protoplast isolation was the 24-hours-cultured hypha of T. viride digested with 4 mg/mL Glucanex in phosphate buffer (pH 6.98) for 4 hours at 30 , the yield of the protoplasts was 4.7 107colony forming unit/ mg. In this study, the maximum regeneration rate (14.5%) was obtained in the CM medium containing osmolutes of 0.3 mol/L KC1 and 0.3 mol/L inositol. Further, plasmids of pCSNTCC and pCSNTCCm were transformed into the protoplasts of T. viride by restriction enzyme Xho I mediated integration, with an efficiency of 1-2 transformants per microgram of DNA. Thirty transformants were obtained, TV-1 to TV-20 for Crypt gene and TV-21 to TV-30 for CrypK13V gene. The hygromycin-resistant transformants were determined by polymerase chain reactions. The elicitor protein was detected in the culture media but not in the cells by western blot analysis. The result indicated that the exogenous gene was expressed in T. viride and the produced protein was secreted into the culture media of transformants as expected.3. Expression of Crypt in T. viride enhanced plant disease resistanceTobacco plants (4-6 week-old) were treated with spores of the transgenic or the wild-type T. viride in the soil. Ten days after the treatment, the plants or detached leaves were inoculated with Phytophthora parasitica var nicotianae, Alternaria lalternata, Pseudomonas syringae tabaci (Pst), and
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