本文選題：黃曲霉菌 + 農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化　； 參考：《安徽農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：黃曲霉菌(Aspergillus flavus)是一種腐生型植物病原真菌,其次級代謝物黃曲霉毒素(Aflatoxin,AFB1)是現(xiàn)如今人類研究發(fā)現(xiàn)的具有強(qiáng)致癌性的真菌毒素。黃曲霉菌可侵染玉米、花生、大米等農(nóng)作物種子并導(dǎo)致相關(guān)農(nóng)產(chǎn)品霉變,嚴(yán)重危害人和動物的健康。了解黃曲霉菌致病的分子機(jī)理對預(yù)防作物種子霉變具有重要意義。農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化(ATMT)已成為真菌功能基因組研究的有力工具。本研究采用ATMT技術(shù)對黃曲霉菌進(jìn)行遺傳轉(zhuǎn)化,主要結(jié)果如下:1.通過黃曲霉原始菌株對不同抗生素的敏感性實(shí)驗(yàn),確定了農(nóng)桿菌介導(dǎo)轉(zhuǎn)化過程中以博來霉素為抗性篩選劑,100μg/mL博來霉素可完全抑制1×106個/mL濃度的黃曲霉孢子。經(jīng)過農(nóng)桿菌抑菌實(shí)驗(yàn),確定了在篩選過程中頭孢噻肟鈉(Cef)的最適濃度為300μg/mL。2.為構(gòu)建適合于黃曲霉菌遺傳轉(zhuǎn)化的雙元表達(dá)載體pDHBle,首先對pAN7-1載體進(jìn)行改造,將受控于gpdA啟動子和trpC終止子的潮霉素抗性基因替換以博來霉素抗性基因(ble),構(gòu)建成pANBle載體;再將pANBle載體中含ble基因的表達(dá)盒PgpdA-ble-TtrpC插入pDHt載體,構(gòu)建成雙元表達(dá)載體pDHBle。3.本研究以黃曲霉孢子為材料制備原生質(zhì)體并對影響原生質(zhì)體制備的酶濃度、酶解時間、酶解溫度等條件進(jìn)行了優(yōu)化,其最優(yōu)條件為:黃曲霉孢子在酶液濃度為纖維素酶∶蝸牛酶∶溶壁酶=1.5%∶1.5%∶1.5%,30℃酶解3h,原生質(zhì)體制備率高達(dá)97.3%,再生率達(dá)89.2%。為農(nóng)桿菌介導(dǎo)黃曲霉原生質(zhì)體提供了可靠的實(shí)驗(yàn)材料。4.初步建立了農(nóng)桿菌介導(dǎo)黃曲霉菌轉(zhuǎn)化體系。探討了不同共培養(yǎng)溫度、時間、農(nóng)桿菌濃度和不同的受體材料對農(nóng)桿菌介導(dǎo)黃曲霉菌遺傳轉(zhuǎn)化體系的影響,結(jié)果表明,當(dāng)農(nóng)桿菌OD600值在0.5,孢子濃度為1×105個/mL,共培養(yǎng)溫度為22℃,共培養(yǎng)2d時,獲得的轉(zhuǎn)化效率較高。以原生質(zhì)體為受體材料的遺傳轉(zhuǎn)化正在進(jìn)行中。5.通過PCR對獲得的轉(zhuǎn)化子進(jìn)行驗(yàn)證。根據(jù)轉(zhuǎn)化子上已知ble基因的上下游引物進(jìn)行PCR,將檢測結(jié)果為陽性的轉(zhuǎn)化子接種于含100μg/mL博來霉素的SM培養(yǎng)基上,30℃連續(xù)培養(yǎng)6代后進(jìn)行轉(zhuǎn)化子的遺傳穩(wěn)定性檢測,結(jié)果表明,PCR檢測結(jié)果為陽性的轉(zhuǎn)化子,繼代培養(yǎng)后仍可檢測到ble基因,遺傳穩(wěn)定性良好。綜上所述,本文構(gòu)建了以ble為抗性篩選標(biāo)記基因的雙元表達(dá)載體,通過ATMT法首次對黃曲霉菌進(jìn)行遺傳轉(zhuǎn)化,獲得了陽性轉(zhuǎn)化子,初步建立了農(nóng)桿菌介導(dǎo)黃曲霉菌遺傳轉(zhuǎn)化體系,為后續(xù)的T-DNA插入突變體庫的構(gòu)建及黃曲霉菌致病相關(guān)功能基因的研究奠定了基礎(chǔ)。
[Abstract]:Aspergillus flavus is a saprophytic plant pathogen, followed by aflatoxin (AFB1), a highly carcinogenic mycotoxin found in human studies. Aspergillus flavus can infect corn, peanut, rice and other crop seeds and lead to mildew of related agricultural products, which seriously harms human and animal health. It is important to understand the molecular mechanism of Aspergillus flavus to prevent crop seed mildew. Agrobacterium tumefaciens mediated genetic transformation ATMTs has become a powerful tool for the study of fungal functional genomes. In this study, the genetic transformation of Aspergillus flavus was carried out by ATMT technique. The main results were as follows: 1. Based on the sensitivity of Aspergillus flavus to different antibiotics, it was determined that bleomycin as a resistant screening agent could completely inhibit Aspergillus flavus spores at a concentration of 1 脳 106 / mL during Agrobacterium tumefaciens mediated transformation. The optimum concentration of cefotaxime sodium cef2 was determined to be 300 渭 g / mL ~ (2) during screening by Agrobacterium tumefaciens. In order to construct a binary expression vector pDHBlefor Aspergillus flavus genetic transformation, firstly, the pAN7-1 vector was modified, and the hygromycin resistance gene controlled by gpdA promoter and trpC Terminator was replaced with bleb gene to construct pANBle vector. Then the expression box PgpdA-ble-TtrpC containing ble gene in pANBle vector was inserted into pDHt vector to construct a dual expression vector pDHBle.3. In this study, Aspergillus flavus spores were used as materials to prepare protoplasts and the enzyme concentration, time and temperature of enzymatic hydrolysis were optimized. The optimum conditions were as follows: aspergillus flavus concentration was cellulase: snail enzyme: wall-lysing enzyme in the enzyme solution. The protoplast preparation rate was up to 97.3 and regeneration rate was 89.2cm at 30 鈩，

本文編號：1892331