农杆菌介导黄曲霉菌遗传转化体系的初步建立

发布时间：2018-05-15 11:43

  本文选题：黄曲霉菌 + 农杆菌介导的遗传转化　； 参考：《安徽农业大学》2015年硕士论文

【摘要】：黄曲霉菌(Aspergillus flavus)是一种腐生型植物病原真菌,其次级代谢物黄曲霉毒素(Aflatoxin,AFB1)是现如今人类研究发现的具有强致癌性的真菌毒素。黄曲霉菌可侵染玉米、花生、大米等农作物种子并导致相关农产品霉变,严重危害人和动物的健康。了解黄曲霉菌致病的分子机理对预防作物种子霉变具有重要意义。农杆菌介导的遗传转化(ATMT)已成为真菌功能基因组研究的有力工具。本研究采用ATMT技术对黄曲霉菌进行遗传转化,主要结果如下:1.通过黄曲霉原始菌株对不同抗生素的敏感性实验,确定了农杆菌介导转化过程中以博来霉素为抗性筛选剂,100μg/mL博来霉素可完全抑制1×106个/mL浓度的黄曲霉孢子。经过农杆菌抑菌实验,确定了在筛选过程中头孢噻肟钠(Cef)的最适浓度为300μg/mL。2.为构建适合于黄曲霉菌遗传转化的双元表达载体pDHBle,首先对pAN7-1载体进行改造,将受控于gpdA启动子和trpC终止子的潮霉素抗性基因替换以博来霉素抗性基因(ble),构建成pANBle载体;再将pANBle载体中含ble基因的表达盒PgpdA-ble-TtrpC插入pDHt载体,构建成双元表达载体pDHBle。3.本研究以黄曲霉孢子为材料制备原生质体并对影响原生质体制备的酶浓度、酶解时间、酶解温度等条件进行了优化,其最优条件为:黄曲霉孢子在酶液浓度为纤维素酶∶蜗牛酶∶溶壁酶=1.5%∶1.5%∶1.5%,30℃酶解3h,原生质体制备率高达97.3%,再生率达89.2%。为农杆菌介导黄曲霉原生质体提供了可靠的实验材料。4.初步建立了农杆菌介导黄曲霉菌转化体系。探讨了不同共培养温度、时间、农杆菌浓度和不同的受体材料对农杆菌介导黄曲霉菌遗传转化体系的影响,结果表明,当农杆菌OD600值在0.5,孢子浓度为1×105个/mL,共培养温度为22℃,共培养2d时,获得的转化效率较高。以原生质体为受体材料的遗传转化正在进行中。5.通过PCR对获得的转化子进行验证。根据转化子上已知ble基因的上下游引物进行PCR,将检测结果为阳性的转化子接种于含100μg/mL博来霉素的SM培养基上,30℃连续培养6代后进行转化子的遗传稳定性检测,结果表明,PCR检测结果为阳性的转化子,继代培养后仍可检测到ble基因,遗传稳定性良好。综上所述,本文构建了以ble为抗性筛选标记基因的双元表达载体,通过ATMT法首次对黄曲霉菌进行遗传转化,获得了阳性转化子,初步建立了农杆菌介导黄曲霉菌遗传转化体系,为后续的T-DNA插入突变体库的构建及黄曲霉菌致病相关功能基因的研究奠定了基础。
[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，

本文编号：1892331