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1、浙江大学博士学位论文生防绿色木霉工程菌的构建及其诱导植物抗病性研究姓名：刘士旺申请学位级别：博士专业：植物病理与分子生物学指导教师：郭泽建2003.5.1生防绿色木霉工程甍的构建及其诱导植物抗瘸性研究摘 要植物病害一点是制约农作物丰产的主雾因素之一。许多重大病害瘸原菌的频繁交易，导致抗性份物懿种经常丧必抗性，另外化学农药的过量旖用使瘸原菌的抗药眭增强，造成防治难度加大。蠢抿农药使用后的残塑，造成对生物以及环境的破坏，京一定程度上也造成封人体浆蔻害。嚣此，隧着人织环保意识的增强t生物痪治特澍是生纺农药静搜焉惩戒为黪溶棱物病害豹趋势，市场对开发耨型静生物农药也鸯麓追镯的需求。植物在同病舔物竞争进化
2、过程中，已形成一系列防御梳带9，过敏反应(HR)和系统获得抗性fSAR)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，利用植物的这种诱鼯抗病性和生物防治菌的特性来开发瓤型的生物农药应该十分可行。绿色木霉(Trjchoderma viride)是一种震生性生物防治微生物，其对植物病服真壤的接摭作用包括竞争作用、重寄生作用及抗生手#援等方甏。懿果耀本霉来表达诱导接物摭痍，陡蛋l皇，并裂鼹本霉的瘸生特点来传播该蛋白，这将大大增嬲本霉翁应耀范围，遣为开发霹持续利溺的生耱农药撼供一条可行的
3、途径。为了验谥这种想法，我们使用了隐缝瘦霉(Phytophthoracryptogea)分泌的隐地蛋白(Cryptogein，Crypt)作为诱母植物SAR的激发予。通过基因转化的方法获得了含有隐地蛋臼基因以及其第13位点突变基因的绿色木霉工程菌，绿色术霉在作为隐地疆白的传播载体的同时，呈现了诱导烟草抗病的功能。实验研究灼主要内容包括以下几个部分：。构建7绿色本霉转化的pCSNTCC和pCSNTCCm载体。必了链傻绿惫本箨会残的终潦激发子爨自分泌到培养液串，奁Crypt秘其突变CrypKl3V(PCR定点突交，第i3位氨基酸崮赣氨羧K突变为缀氨酸V)基戳葡插入信号胶序剜，弓|导矫源激发子蛋白分
4、泌搦木霉细胞外，与寄主植物直接作用，诱导煮物的防卫反应。在合成信号肽基因ThChi和目的基因Crypt和CrypKl3V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Crypt基因转化绿色木霉的真菌表达载体pCSNTCC昶留影缮y基因转化绿色本霉囊壤表达载体pCSNTCCm。2建立7绿色本霉抟转靶体系。对绿色本霉原象痰体制备和再生条俘逡行了较详缨靛分据，并在诧萋秣上，；l用限制酶介导进行了术霉原生成体转纯。结采表明，木霉原生质体形成稳合适条侔为：培养24 h的菌丝用4 mgmL的Glucanex在磷酸箍缓j中液中(pH 698)、
5、30、振荡(40 rmin)酶解4 h，原生质体产量达捌47X 107个珊g。原生质体在含03 molL肌醇和03 molL KCI的CM培养基上再生率为145。限制酶XhoI介黟转化绿色本霉原生质体，其转化率为每微克DNA得到I2个转化予。转化子的PCR鉴定以及特巽酶联免疫测定(ELISA)、western杂交检测表明，外源旗因已转入绿色本霉蘩著在培葵滚孛套稳定熬产物表达。30株骧生度体露生株被确定先稳定的转纯子，其中TV-I至TV-20为Crypt蒸因转纯子，善￥一2l至l￥一30必CrypKl3V基阂转亿予。3转化子诱导檬物抗病性转化子的抗病分析结果表明，转基因木霉菌株TV一3和TV-2
6、4等能够分泌Crypt或CryKl3V，木耀出发株及转基因木霉孢子处理烟草2个品种和十字花科植物拟南芥(Arabidopsis thaliana)根部10 d殿，离体接种和棱株整株接种病原菌，实验表明转化子能够提毫烟擎对黑胫病菌(脚tophthora parasitica varnicotianae)、券星癍蕊(AIternariaalternata)露烟孳芯叶痣谍(TMV)粒抗性，分子杂交检测表明，转化予2够诱导遮革糌基毽黪表达。转纯子处理揿南芬，接晕孛丁香暇单胞菡和立秸丝核蔼，离钵接种和分子水平杂交筠袭明，摈南芥抗瘸性基本没有得到提高，表明Crypt的诱导抗痫性具有一定的专纯性。4筛选转化
7、子TV一3发酵培养基转化子TV一3培养基筛选实验表明，合适的利于工业化的固体生产RPA培养綦，组成配比为：40弼草，5豆饼粉，15麸皮，15蔗糖，产孢照达到336109 cfumL；液体孵G培养綦，组成配比为麦芽汁30，酵母提取物01，龌莓糖5， 鲥gs饶。7Hz0 0，l蒜， K磁飚0，1，麓丝生物星达到874 mg向L。关键诵：绿色术霉；隐璁蛋臼：源垒质体；限制酶介导转亿；诱导抗病性：培养纂Co珏str糕etion ofBiological Control Strain ofTrichoderma virideand Study of their Ability to Induce Pla
8、nt Disease ResistancePiant迸辩粼is one ofthe serious probtems afteethg plant growth and ou每ut ofcropsereIt is difficult tO control because pathogens mutated frequently,and this leading tOthe disease resistant plants broke down their resistance，The excessive application ofchemical pesticides is not only
9、 leading to the pathogen resistance against the chemicals，but also harmful to the environment，and ceaainty to the health of human beings、Therefore,biological control agents(BCA)ale considered as the possible replacementof chemieat pesticidesand as all environmental friendly agent for further plant d
10、iseasecontr01Hypersensitive response f秘袋)and systemic acquired resistance(SAg)are the typicalrepresentation of plant defense reaetinns。Once the estabtishmem of SAR,the plantsexhibit a broad-spectrum of disease resistance against pathogen attack。Researchershave identified elicitor proteins for exampl
11、e elicifins and rarpins，which elicit plantdefense reactions。It woud be feasible to explore biological control pesticides base o摊the characteristics ofplant SARTrichoderma viride is an ubiquitous soil saprophyte bologcat control microbe havfiagthe action modes with pathogens of competition for nutrie
12、nt resource，antibiosis,andmyc昭parasRism，If T viride鼹建be used as a producer and e-翅,rier ofan elicitor protdn、囊would be applicable for the development of plant specific BCATo test this idea，weused eryptogein，鑫proteinous elicitor secreted from Phytophthora cryptogea for thedevelopment of bioengineerin
13、g r vrdeThe plasmld containing Crypt or its mutant嘲introduced into Z viride by the method of restriction enzyme mediated integration(REMI)。Our study indicated that Z viride acts as the producer and carrier of crypt,and谯t the same time the transgenic lines enhanced disease resistance when applied ont
14、obacco plantsThe summary龉et轻stay as follows：1Construetion of pCSNTCC and pCSNTCCm plasmidsFirstly,Crypt gene WaS mutated by change the K at the position 1 3 of Crypt into V(th。mutant named CryKl3 V)as described earlierIn order to secrete the produced proteinout of Z v加胁cells，a signal sequence of a c
15、hitinase gene from Trichoderma(ThChi)was fused to the 5ofCrypt and CryKl3VThe chimeric genes were constructed underthe control of trpC promoter in vector pCSN43 respectively,Then，a hygromycinresistant gene was introduced into the vectors，and plasmids of pCSNTCC(for Cryptgene)and pCSNTCCm(CrypKt3玲wer
16、e obtained2Establishment of Z viride transformation systemThe conditions for protoplasts isolation and regeneration from?=viride were studiedThe optimum condition for protoplast isolation was the 24-hours-cultured hypha of Tviride digested with 4 m咖匕Glucanex in phosphate burr(pH 6。98)for 4 hours at 30the yield ofthe protoplasts was 4，7107colony forming unitmgIn this study,themaximum regeneration rate(145
