本文選題：Pci-laeA + Penicillium��； 參考：《西南大學(xué)》2012年碩士論文

【摘要】：背景： 橘青霉(Penicillium citrinum)屬于子囊菌亞門,不整囊菌綱,散囊菌目,散囊菌科,青霉屬,是重要的工業(yè)生產(chǎn)菌株。美伐他汀(mevastatin)是橘青霉產(chǎn)生的重要次級代謝產(chǎn)物,這種聚酮類化合物是甲基戊二酸單酰輔酶A (HMG-CoA)還原酶抑制劑,通過競爭性結(jié)合HMG-CoA還原酶有效抑制膽固醇的生物合成。美伐他汀經(jīng)過生物轉(zhuǎn)化后生產(chǎn)的他汀類藥物是臨床治療高膽固醇血癥的首選藥物。研究美伐他汀生物合成機制和調(diào)控網(wǎng)絡(luò)有助于定向改造目的菌株,而這些研究首先需要建立一套穩(wěn)定高效的遺傳轉(zhuǎn)化方法。目前,常用的絲狀真菌遺傳轉(zhuǎn)化方法有：CaCl2-PEG介導(dǎo)的原生質(zhì)體轉(zhuǎn)化法、電擊轉(zhuǎn)化法、醋酸鋰轉(zhuǎn)化法、基因槍轉(zhuǎn)化法和限制性內(nèi)切酶介導(dǎo)的轉(zhuǎn)化法等。由于絲狀真菌的復(fù)雜性,其遺傳轉(zhuǎn)化體系有別于植物細(xì)胞、動物細(xì)胞和原核生物,因此至今仍有許多種類的絲狀真菌未能建立合適的遺傳轉(zhuǎn)化體系,以至無法進行深入研究。根癌農(nóng)桿菌介導(dǎo)的絲狀真菌轉(zhuǎn)化(Agrobactirium tumfacience mediated-transformant)由于具有轉(zhuǎn)化效率高、操作簡便、受體廣泛、單拷貝插入和遺傳穩(wěn)定等優(yōu)勢在很多絲狀真菌中得到應(yīng)用,但在橘青霉中的應(yīng)用尚未有報道。因此我們選擇根癌農(nóng)桿菌介導(dǎo)的方法來構(gòu)建橘青霉遺傳轉(zhuǎn)化體系。 目的： 本文旨在建立一套能夠在橘青霉中實現(xiàn)高效穩(wěn)定運行的遺傳轉(zhuǎn)化系統(tǒng),為實驗室橘青霉的美伐他汀生物合成機制和全局性調(diào)控研究奠定方法基礎(chǔ)。 方法： 1利用重組DNA技術(shù)構(gòu)建含Pci-laeA的重組質(zhì)粒載體,探討抗生素選擇、孢子濃度、根癌農(nóng)桿菌種類、根癌農(nóng)桿菌濃度、乙酰丁香酮濃度、共培養(yǎng)溫度、共培養(yǎng)時間以及共培養(yǎng)方式等因素對根癌農(nóng)桿菌介導(dǎo)的橘青霉遺傳轉(zhuǎn)化的影響,篩選優(yōu)化出合適的轉(zhuǎn)化條件。 2通過PCR技術(shù)擴增橘青霉基因組中增潮霉素B抗性標(biāo)記基因(Hygromycin B resistence gene)篩選驗證陽性轉(zhuǎn)化子,并在無潮霉素B(Hygromycin B)篩選壓力的平板上連續(xù)傳代考察其遺傳穩(wěn)定性。 3對穩(wěn)定遺傳的轉(zhuǎn)化子進行表型觀察,并利用HPLC方法測定發(fā)酵液中美伐他汀生物合成量。 結(jié)果： 1實驗結(jié)果顯示使用50μg/ml的潮霉素B能夠篩選到陽性轉(zhuǎn)化子,500μg/ml的頭孢噻肟鈉可以有效清除殘余根癌農(nóng)桿菌。孢子濃度以107個/ml為佳；農(nóng)桿菌LBA4404的轉(zhuǎn)化效率略高于EHA105,采用濃度為OD600≈0.8左右的根癌農(nóng)桿菌效率較好；乙酰丁香酮的最佳濃度為80μg/ml；24℃下共培養(yǎng)36h-48h時效率最高；液體共培養(yǎng)技術(shù)簡便有效。 2利用PCR技術(shù)成功從轉(zhuǎn)化子中擴增出hyg抗性標(biāo)記基因,陽性轉(zhuǎn)化子篩選成功。在無Hyg篩選壓力的連續(xù)5次傳代下,90%的轉(zhuǎn)化子可以穩(wěn)定遺傳。 3表型觀察的結(jié)果發(fā)現(xiàn),野生型橘青霉與Pci-laeA基因轉(zhuǎn)化株相比其菌絲由淡黃色變成灰白色,孢子和菌絲形態(tài)無顯著改變。 4美伐他汀產(chǎn)量檢測結(jié)果顯示,Pci-laeA基因轉(zhuǎn)化株的美伐他訂生物合成量有顯著提高。 結(jié)論： 本研究成功建立了以根癌農(nóng)桿菌為介導(dǎo)的橘青霉遺傳轉(zhuǎn)化體系,初步探索了全局性調(diào)控因子PCi-laeA的功能。此方法為深入研究橘青霉產(chǎn)美伐他汀的生物合成機制和全局性調(diào)控因子的功能研究奠定了基礎(chǔ)。
[Abstract]:Background :

The present invention has the advantages of high conversion efficiency , simple operation , wide range of receptors , single copy insertion and restriction enzyme - mediated transformation .

Purpose :

The aim of this paper is to establish a genetic transformation system which can realize high - efficiency and stable operation in Penicillium citrinum , which lays a foundation for the study of the biosynthesis mechanism and global regulation of mevastatin of Penicillium citrinum .

Method :

1 . The recombinant plasmid vector containing Pci - laeA was constructed by using recombinant DNA technology . The effects of antibiotic selection , spore concentration , Agrobacterium type , Agrobacterium tumefaciens concentration , acetosyringone concentration , co - culture temperature , co - culture time and co - culture mode on Agrobacterium tumefaciens - mediated genetic transformation of Penicillium citrinum were studied .

2 . The positive transformants were screened by PCR , and the genetic stability of the positive transformants was tested on the plate with the screening pressure of mycin B .

3 . Phenotypic observation was carried out on the stably inherited transformant , and the biosynthesis of mevastatin in the fermentation broth was determined by HPLC .

Results :

1 . The results showed that 50.mu . g / ml of fosfomycin B could be used to screen the positive transformants , and 500 渭g / ml Cefomycin could effectively remove Agrobacterium tumefaciens . The spore concentration was 107 cells / ml .
The transformation efficiency of Agrobacterium LBA4404 was slightly higher than that of EHA105 , and the efficiency of Agrobacterium tumefaciens was better than that of EHA105 .
The optimal concentration of acetosyringone was 80 渭g / ml .
The efficiency was the highest at 24 鈩，

本文編號：1875261