本文選題：膠孢炭疽菌 + 重組PCR技術(shù)��； 參考：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：核桃(Juglans regia L.)是重要的“木本油料”戰(zhàn)略樹種,但隨著核桃集中栽培面積的擴(kuò)大和抗病品種的缺乏,核桃病害日漸嚴(yán)重。核桃炭疽病(anthracnose)是由膠孢炭疽菌(Colletotrichum gloeosporioides)引起的,可致果實(shí)壞疽、葉片焦枯,同時(shí)還危害嫩梢,導(dǎo)致產(chǎn)量銳減,是目前核桃生產(chǎn)中的災(zāi)難性病害。本論文構(gòu)建了適于真菌的綠色熒光蛋白(Green fluorescent protein,gfp)表達(dá)載體,農(nóng)桿菌介導(dǎo)法(Agrobacterium tumefaciens-mediated transformation,ATMT)對膠孢炭疽菌進(jìn)行了遺傳轉(zhuǎn)化得到了帶有綠色熒光蛋白標(biāo)記的膠孢炭疽菌菌株,為研究膠孢炭疽菌對核桃的侵染過程,了解核桃與膠孢炭疽菌的互作機(jī)制提供了技術(shù)支撐。研究結(jié)果如下:1.真菌gfp獨(dú)立表達(dá)載體(DL-gfp)的構(gòu)建。以真菌trp C基因的啟動(dòng)子Ptrp C和終止子Ttrp C作為gfp和潮霉素磷酸轉(zhuǎn)移酶基因(Hygromycin B phosphortransferase gene,hph)的啟動(dòng)子和終止子。通過PCR重組技術(shù)(Overlap PCR)分別構(gòu)建了hph的表達(dá)元件(Ptrp C+hph+Ttrp C)和gfp表達(dá)元件(Ptrp C+gfp+Ttrp C);通過In-fusion酶連接技術(shù),將兩個(gè)表達(dá)元件與帶有T-DNA區(qū)間的線性化Vector(p Green II 62-SK)相連,構(gòu)建獨(dú)立表達(dá)載體DL-gfp。2.真菌gfp融合表達(dá)載體(RH-gfp)的構(gòu)建。以真菌trp C基因的啟動(dòng)子Ptrp C和終止子Ttrp C作為gfp和hph基因的啟動(dòng)子和終止子進(jìn)行構(gòu)建。通過Overlap PCR將啟動(dòng)子Ptrp C和hph基因重組到一起,且hph基因敲除終止密碼;將Overlap PCR獲得片段(Ptrp C+hph)與gfp、終止子Ttrp C和線性化后的Ti質(zhì)粒(p Green II 62-SK)通過In-fusion酶連接重組,構(gòu)建融合表達(dá)載體RH-gfp。3.重新分離純化致病力較強(qiáng)的m9膠孢炭疽菌。實(shí)驗(yàn)室4℃斜面保存的膠孢炭疽菌m9菌株,直接在馬鈴薯葡萄糖瓊脂培養(yǎng)基(PDA)培養(yǎng),菌絲徒長,產(chǎn)孢量少,菌株產(chǎn)孢能力下降,通過將m9菌株重新侵染核桃葉片,獲得大量的m9的分生孢子,在PDA平板上純化后使用。4.ATMT轉(zhuǎn)化膠孢炭疽菌。優(yōu)化轉(zhuǎn)化條件:膠孢炭疽菌分生孢子濃度為107個(gè)/m L,農(nóng)桿菌GV3101(含DL-gfp載體)濃度OD620≈0.5;乙酰丁香酮(AS)誘導(dǎo)濃度為200μM,轉(zhuǎn)化菌株的潮霉素B篩選濃度為100μg/m L,用于農(nóng)桿菌菌株抑制的氨噻肟頭孢霉素100μg/m L;誘導(dǎo)轉(zhuǎn)化溫度為22℃,時(shí)間為2天。5.獲得了帶有熒光蛋白標(biāo)記的膠孢炭疽菌m9轉(zhuǎn)化子。經(jīng)過多次繼代培養(yǎng),篩選出能夠穩(wěn)定表達(dá)gfp標(biāo)記基因和hph篩選基因的轉(zhuǎn)化子,為后續(xù)研究轉(zhuǎn)化子和野生型菌株生物學(xué)特性的差異以及觀察膠孢炭疽菌轉(zhuǎn)化子和核桃之間病原互作的活體動(dòng)態(tài)過程奠定了基礎(chǔ)。
[Abstract]:Juglans regia L.It is an important "woody oil" strategic tree species, but with the expansion of concentrated cultivation area and the lack of disease-resistant varieties, walnut disease is becoming more and more serious.Anthracnoseis is caused by Colletotrichum gloeosporioides. it can cause fruit gangrene, scorched leaves, and damage young shoots, which results in sharp decrease of yield, which is a catastrophic disease in walnut production.In this paper, the expression vector of green fluorescent protein green fluorescent protein (GFP) suitable for fungi was constructed. Agrobacterium tumefaciens-mediated transformation by Agrobacterium tumefaciens was used for genetic transformation of Bacillus anthracis to obtain Bacillus anthracis strain labeled with green fluorescent protein.It provides technical support for studying the infection process of anthrax on walnut and understanding the interaction mechanism between walnut and anthracis.The results are as follows: 1.Construction of fungal gfp independent expression vector DL-gfp.The promoter Ptrp C and the Terminator Ttrp C of trp C gene were used as promoter and Terminator of gfp and hygromycin B phosphortransferase gene.Ptrp C hph Ttrp C) and Ptrp C gfp Ttrp C of hph were constructed by PCR recombination technique, and two expression elements were connected with linearized Vector(p Green II 62-SKK with T-DNA interval by In-fusion enzyme ligation technique.The independent expression vector DL-gfp.2. was constructed.Construction of fungal gfp fusion expression vector RH-gfp.The promoters Ptrp C and Ttrp C of trp C gene were used as promoters and Terminators of gfp and hph genes.The promoter Ptrp C and hph gene were recombined together by Overlap PCR, and the hph gene knockout stop codon was removed, and the Overlap PCR fragment was ligated with GFP, Ttrp C and the linearized Ti plasmid p II 62-SKK were ligated by In-fusion enzyme.Construction of fusion expression vector RH-gfp.3.To reisolate and purify M. 9 anthracis with strong pathogenicity.The strain m9 was cultured directly in the potato glucose Agar medium (PDAs). The mycelium was long, the sporulation quantity was low, and the sporulation ability of the strain was decreased. The strain m9 was infected with the leaves of walnut again.A large number of conidia of m9 were obtained and purified on PDA plate. 4. ATMT was used to transform Bacillus anthracis.The optimum transformation conditions were as follows: the conidial concentration of Bacillus anthracis was 107 / mL, the concentration of Agrobacterium tumefaciens GV3101 (containing DL-gfp carrier) was OD620 鈮，

本文編號：1767372