摘要
目前随着化学农药的过度施用,给环境和人类健康带来极大的危害,因此开展生物防治的研究和应用,正受到世界各国的广泛重视。在开发的众多生防微生物中,生防真菌在生物防治,特别是植物病原物防治方面,起着十分重要的作用。生防真菌因其对环境和人、畜无毒等优点,有着化学农药不可取代的优势。淡紫拟青霉不仅是一类具有极大生防潜力的线虫寄生真菌,同时也是一种有效的昆虫病原真菌。为了充分的利用淡紫拟青霉这一生防资源,本实验通过遗传工程,对淡紫拟青霉E2-4进行了改良,获得了毒力效果有显著提高的工程菌株。具体的实验内容如下:
以本实验室筛选和保存的淡紫拟青霉E2-4为原始出发菌株,首先对其进行了一系列的毒力试验。本文通过测定不同浓度的淡紫拟青霉E2-4孢子悬浮液对线虫卵囊孵化的影响发现,低浓度的孢子悬浮液(10~4个/mL)处理线虫卵囊后,线虫的孵出数还高于对照在清水的条件下所孵出的线虫数;随着孢子悬浮液浓度的增加,线虫卵囊孵化的线虫数开始有明显的减少趋势,孢子悬浮液浓度为10~5、10~6、10~7、10~8个/mL时对线虫卵囊的相对抑制率分别为21.72%、42.21%、49.19%和54.95%;孢子浓度为10~6、10~7、10~8个/mL时对线虫卵囊孵化的抑制作用与对照处理相比存在极显著差异。在体式显微镜下观察10~7个/mL孢子液处理的卵囊,可见在卵囊表面萌发出大量菌丝,将卵囊层层裹住,抑制了线虫的孵化。
本研究采用液固两级发酵法,以大豆粉为液体培养基,以大米为主要固体基质制备了淡紫拟青霉E2-4的固体菌剂;并在此固体发酵的基础上研究了加入诱导物后对E2-4产孢量的影响;结果显示在大米中加入几丁质和壳聚糖这两种诱导物后孢子产量分别可增至4.15×10~9和2.37×10~9个/mL,均有利于提高E2-4的产孢量,使用制备的E2-4固体菌剂对番茄根结线虫病进行小量盆栽试验,发现E2-4对番茄根结线虫防治效果较好,与对照相比:长势明显优于对照,茎和叶变褐、枯萎等症状较少,且根部根结极少。
同时,本实验还测试了E2-4对蚜虫的毒力,结果表明:不同孢子浓度对蚜虫的致死率存在一定的差异;孢子悬浮液浓度为10~4个/mL时对蚜虫的毒力不明显,与对照差别不大;孢子悬浮液浓度达到10~5、10~6、10~7和10~8个/mL后,毒力效果明显增强,尤其是高于10~5个/mL后的浓度,不但致死率高,而且死虫褐化程度也明显高于对照;从体式显微镜下观察死亡的虫体,可见用孢子悬浮液处理过的蚜虫体表长着一层绒毛状的菌丝。从光学显微镜下观察,触角等结节处可见有孢子和菌丝粘附在上面。
通过研究试验表明:淡紫拟青霉E2-4对根结线虫和蚜虫均具有一定的生防能力。为进一步提高E2-4的毒力,使其更有效的应用于生防工作中,对其进行了农杆菌介导的遗传转化的改良。首先构建了含几丁质酶基因(chi)和GFP的淡紫拟青霉的表达载体。载体构建的方法为:在表达载体pCAMBIA 1302的基础上构建适合于淡紫拟青霉遗传转化的表达载体,先对pCAMBIA 1302的选择标记基因及其启动子进行第一步改造,构建pTBAR载体,然后再在这个载体的基础上插入外源基因(绿僵菌几丁质酶基因),构成pTBTCHI表达载体。构建的结果如下:通过PCR方法从pC30RG载体中扩增得到Trpc启动子,将适用于真菌表达的启动子Trpc替换pCAMBIA 1302中靠近左边界位于选择标记潮霉素基因上游的Camv 35S启动子;再根据设计的保守引物通过PCR方法从pBHt2载体中扩增获得bar基因替换选择标记潮霉素基因,构成载体pTBAR;然后从绿僵菌HN1中扩增chi基因的开放阅读框,利用套叠PCR的方法将Trpc启动子与chi基因连接起来,组成一表达元件trpc-chi;利用BamH ?和HindШ酶切位点插入载体pTBAR中,构建成功pTBTCHI表达载体。
通过电击转化将pTBTCHI表达载体导入根癌农杆菌GV3103中,并成功转化了淡紫拟青霉,对转化效率进行了优化。结果表明:在AS加入量为200μg/mL,淡紫拟青霉E2-4孢子浓度为106个/mL的转化条件下,在根癌农杆菌生长浓度OD_(600)为0.2-0.3左右时,在抗性筛选培养基上所出现的抗性转化子个数最多。根癌农杆菌浓度过低(OD_(600)≤0.1)和过高(OD_(600)≥0.3)时,转化出的转化子数较少;将共培养条件设定为乙酰丁香酮(AS) 200μg/mL,根癌农杆菌生长浓度OD_(600)值为0.2;孢子浓度除10~6个/mL浓度外其它的浓度均没有得到抗性转化子。浓度为10~4个/mL和10~5个/mL的条件下,共培养6天后仍没有抗性转化子长出;而浓度达10~7个/mL和10~8个/mL时,抗性选择培养基上的卫星菌落多,生长背景杂;将根癌农杆菌生长浓度OD600值定为0.2;孢子浓度定为10~6个/mL条件下,加了AS的处理在选择培养平皿上长出的转化子个数较为稳定,平均在40个左右。而在没有加AS的处理中,只有少量的转化子长出或没有转化子,且PCR检测均为假阳性。
本实验共获得含chi基因的抗性转化子53个,转化子中绝大部分菌落型态与原始菌相似,只有少数与之有稍微的差别。从中随机选取抗性转化子11个进行bar基因的PCR检测,其中有6个扩增得到目的片段。通过荧光显微镜检测,阳性转化子可发出绿色荧光。对抗性转化子进行的Southern blotting检测也证明含chi基因的T-DNA已插入淡紫拟青霉中。
随机选取10个阳性转化子,分析其几丁质酶活力。经Duncan多重比较分析:阳性转化子tchi001、tchi002、tchi007、tchi009、tchi021和tchi030的几丁质酶活表达水平与E2-4存在差异极显著;此6个阳性转化子测得的总蛋白含量与E2-4之间的差异也很显著(a=0.05)。
再对其中两个几丁质酶表达活力相对较高的抗性转化子进行毒力测试,结果表明: tchi001抑制线虫卵孵化的速度明显快于E2-4与tchi007,抑制线虫卵孵化的能力也强于E2-4和tchi007,相对抑制率可达62.11%;而tchi007抑制线虫卵囊孵化的能力虽高于原始菌株,但不存在显著差异。
对线虫的致死率试验结果显示:这三个菌株与对照处理间存在极显著差异。分析还表明阳性转化子tchi001和tchi007之间的毒力分析没有显著差异。但是阳性转化子tchi001和tchi007的总体毒力均明显高于原E2-4菌株,它们对线虫的校正致死率都超过了60%,分别为75.67%和65.67%。
The excessive application of chemical pesticides has left the agro-ecosystem collapse, environmental contamination and human health concerns. So the biological strategy for biological control is receving much attention worldwide. Among the biocontrol microorganism, pathogenic fungi have very important contribution to biological control, especially for plant etiological agent. Compared with chemical pesticide, pathogenic fungi were no toxic to environment, human and animals. Therefore , mycoinsecticides are being considered as alternative and supplement to chemical pesticides. Paecilomyces lilacinus is well known not only one of nematodes egg-pathogenic fungi, but also a kind of entomopathogenic fungi ,which currently receiving attention for their potential in biological control agent. However, the acceptance of fungal products for pest control is very limited .Because the fungal insecticides are not as fast acting as chemical products and sensitive to adverse environment and lose their effectiveness more rapidly. So it is necessary to improvement P.lilacinus strain .With the basis of gene engineering ,chitinase gene from Metarhizium anisopliae was cloned and transformated to P.lilacinus E2-4 by Agrobacterium tumefaciens-mediated procedure.Transformants of P.lilacinus E2-4 were obtained and the biocontrol ability of transformants were significant enhanced.The main results are as following:
1) Firstly ,in order to sure the biocontrol potential of P.lilacinus E2-4 which was screened and stored by our lab, some virulence test were done . The effection of P.lilacinus E2-4 sporium suspension to the hatching of nematode egg masses showed that the number of nematode hatching from nematode egg masses which were treated by low concentration of sporium suspension(10~4conidia/mL)were more exceded than control treated by water.When the concentration of sporium suspension was increased to 10~5conidia/mL,numbers of nematode were significantly reduced and the fractional inhibition of 10~5, 10~6,10~7,10~8coidia/mL to nematode egg masses were 21.72%,42.21%, 49.19 % ,54.95 % respectively.The effection of sporium suspension10~6、10~7 and 10~8conidia/mL to the hatching of nematode egg masses existed significant deviation between control . The nematode egg masses treated by sporium suspension were observed by Olympus stereomicroscopy, found that many mycelial of P.lilacinus on the surface of egg masses and wrapped around it.
P.lilacinus E2-4 solid granula were made by liquid-solid diphasic fermentation. Soybean meals were used as the substrate of liquid fermentation and the substrate of solid fermentation were grains of rice .Studied on the effect of inducer on conidia yield.When chitin and chitosan were added to grains of rice as the inducer, the conidia yield of P.lilacinus E2-4 added to 4.15×10~9 and 2.37×10~9 conidia/mL.A pot experiment was conducted to test the effect of P.lilacinus E2-4 solid granula on tomato root-knot nematodes.The results proved that the occurrence of tomato root-knot nematodes was inhibited and plant growth was promoted by P.lilacinus E2-4 solid granula. And CK which were not treated by P.lilacinus E2-4 solid granula occurred diseases ,for example the leaves become brown or yellow and even to withered , the root had many root knots. The disease symptoms of tomato stalks、leaves and root were less by P.lilacinus E2-4 compared with CK meanwhile.
A experiment was conducted to test the effection of P.lilacinus E2-4 sporium suspension to the death rating of chinese cabbage aphids.The result showed that the death rating of aphids treated with different concentration of sporium suspension have some difference.Treated with 10~4 conidia/mL sporium suspension,the virulence to aphids was not distinct difference compared with control which treated by water. And then the death rating of aphids were increased obviously,when the concentration of sporium suspension was increased to 10~5、10~6、10~7 and 10~8conidia/mL.The mycelial threads could be observed on the surface of aphids by microscope.
2)According to the above experiments proved that P.lilacinus E2-4 have the biocontrol ability to root-knot nematodes and aphids.Therefore ,in order to improve the virulence of E2-4 strain for more effective biocontrol application, a simple,highly efficient and reliable transformation system of P.lilacinus mediated by Agrobacterium tumefaciens was developmented ,cloning of chitinase gene from M.anisopliae and then enhancing virulence of P.lilacinus by genetic transformation.
First of all , a expression vector including chi gene and gfp gene was constructed. Using pCAMBIA1302 ,a commonly used vector for plant transformation,as the backbone,the binary vector pTBAR was constructed .According to the fact that P.lilacinus was not sensitive to hygromycin,the herbicide resistance gene bar which was PCR amplified from vector pBHt2 was used as selectable marker gene for P.lilacinus transformation and bar gene was under the promoter Trpc which was amplified from vector pC30RG in pTBAR. And then a expression element trpc-chi was constructed, through cloning chi gene from M.anisopliae and connecting with Trpc promoter by invaginate PCR.At last , vector pTBAR was inserted trpc-chi expression element by BamH ? and HindШsitus, and expression vector pTBTCHI was constructed successfully. 3) Mediated by A.tumefaciens strain GV310~3,T-DNA of pTBTCHI was successfully integrated into P.lilacinus genome. After being perfected transformation steps were described below. The transformation efficiency of P.lilacinus was highest when the co-cultivation condition was A.tumefaciens OD_(600)=0.2-0.3, P.lilacinus spore 10~6conidia/mL and 200μg/mL AS. When the A.tumefaciens OD_(600) was low to 0.1 or high over 0.3, the transformation efficincy was low. For the concentration of spore , the resistant transformants obtained from transformation were very few when the concentration of spore was 10~4conidia/mL,10~5conidia/mL, 10~7conidia/mL and 10~8conidia/mL,except
10~6conidia/mL.Under the condition of A.tumefaciens OD_(600)=0.2, the concentration of spore was 10~6conidia/mL, AS was added to broth IM ,the transformation efficincy were stable ,and could obtain about 40 transformants/dish. When no AS added to IM co-culture ,no resistant transformants obtained.
A total of 53 resistant transformants were obtained , the most of their morphology were similar with E2-4 strain ,and only few have different. In order to test if the transformants were positive transformants, 11 resistant transformants were random selected and extracted genomic DNA which were used to amplified bar gene by PCR. Six resistant transformants were amplified the bar gene.Because of the T-DNA which integrated into P.lilacinus including gfp gene, the positive transformations could be observed green fluorescence by fluorescence microscope. And Southern blotting analysis on the transformants randomly chosen from resistant transformants revealed that the T-DNA have insertioned to P.lilacinus.
4)To determine if expression of chi can enhance the virulence of P.lilacinus. Four positive transformants were applied to the chitinase expression analysis in the exuxiae of the cicada medium.The result of Duncan multiple range test fo variable showed that the chitinase activity of tchi001、tchi002、tchi007、tchi009、tchi021 and tchi030 compared with E2-4 existed significant deviation. The protein level produced by six transformants were also very different with E2-4.
Virulence test were conducted by two positive transformants tchi001 and tchi007 ,which have higher chitinase activity .The result showed that both of the two transformants and E2-4 strain had inhibitory action to the haching of nematode egg masses. But the inhibitory action speed of tchi001 was obviously faster than E2-4 and tchi007, the ability to inhibit hatching of nematode egg was also higher than E2-4 and tchi007. The death rate test to nematodes revealed that all of the transformations and wild type E2-4 strain had evident virulence to tomato root-knot nematodes. As seen from the analysis of SAS , against E2-4, the correct lethality rate to tomato root-knot nematodes of tchi001 and tchi007 were all over 60%, The result suggesting that expression chitinase can enhance the virulence of P.lilacinus.
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