【作者】 张昕；

【导师】 林景卫；

【作者基本信息】 沈阳农业大学 ， 细胞生物学， 2019， 硕士

【摘要】 真菌免疫调节蛋白（FIPs）是一种提取于高等真菌的小分子蛋白质,其具有与免疫球蛋白重链区相似的结构且生物学活性与植物凝集素相似。已发现的多种真菌免疫调节蛋白均具有凝集血细胞、抗肿瘤、抗过敏、促淋巴细胞增殖等生物学活性,使其具有良好的临床应用前景。目前FIP的功能研究主要集中在对人体和动物的生物学作用,而其对真菌本身的生物学功能尚不清楚。本研究以金针菇为研究对象,构建FIP-fve和LZ-8超表达载体,通过农杆菌介导法转入金针菇,得到阳性转化子;将FIP-fve基因超表达、FIP-fve基因沉默和野生型金针菇进行栽培,对比和分析表型变化,提取蛋白并进行蛋白质检测分析,为研究FIP-fve对金针菇本体的生物学功能奠定基础。首先,本论文克隆了FIP-fve基因和LZ-8基因,并构建相应的表达载体,其中构建了两种FIP-fve超表达载体,一种是pCAMBIA1301-FIP-fve,是在pCAMBIA1301载体的35S启动子后连入目的基因FIP-fve,另一种是pCAMBIA1301-GPD-FIP-fve,是将金针菇内源启动子GPD基因同FIP-fve基因插入pCAMBIA1301多克隆位点,随后构建LZ-8基因超表达载体pCAMBIA1303-GPD-LZ-8,同样将GPD启动子基因和LZ-8基因插入多克隆位点。其次,选用农杆菌介导转化的方法,建立并完善超表达载体的金针菇遗传转化体系,侵染材料为1 cm²的金针菇菌丝块,侵染条件为农杆菌菌液浓度OD₆₀₀为0.5,农杆菌菌液中加入AS至终浓度为200μmol·L^-1,侵染时间为30 min。25℃下,在含有200μmol·L^-1AS的共培养基中培养3天,用含有9μg·mL^-11 Hyg的YPG培养基进行筛选,最终获得了FIP-fve超表达的金针菇转化子,其中,pCAMBIA1301-FIP-fve转金针菇的农杆菌转化效率为17.5%,pCAMBIA1301-GPD-FIP-fve转金针菇的转化效率为22.5%,而pCAMBIA1303-GPD-LZ-8转金针菇的转化效率为10%。最后,将筛选出的FIP-fve基因超表达的金针菇进行菌丝培养,同FIP-fve基因沉默金针菇和野生型金针菇共同进行实验室内栽培,至子实体成熟,观察金针菇表型变化,并提取蛋白进行蛋白质检测分析。野生型金针菇中菇原基数为46,pCAMBIA1301-GPD-FIP-fve转金针菇的三株FIP-fve超表达金针菇的菇原基数分别为69,55和71;pCAMBIA1301-FIP-fve转金针菇的三株FIP-fve超表达金针菇的菇原基数分别为92,116和51。采用Bradford法测试金针菇子实体总蛋白含量,结果表明FIP-fve基因超表达的金针菇蛋白浓度与野生型金针菇蛋白浓度相近,而FIP-fve基因沉默的金针菇总蛋白浓度相对较低。另外,通过SDS-PAGE分析目的蛋白质FIP-fve的含量,结果表明野生型金针菇中FIP-fve的含量为0.73 mg·g^-1,pCAMBIA1301-GPD-FIP-fve转金针菇的三株FIP-fve超表达金针菇的FIP-fve含量分别为0.75 mg·g^-1,0.76 mg·g^-1和0.75 mg·g^-1;pCAMBIA1301-FIP-fve转金针菇的三株FIP-fve超表达金针菇的FIP-fve含量分别为0.80 mg·g^-1,0.71 mg·g^-1和0.76 mg·g^-1;FIP-fve基因沉默的金针菇中FIP-fve含量为0.71 mg·g^-1和0.69 mg·g^-1,进一步进行Western Blot检测,结果表明,FIP-fve超表达的金针菇中FIP-fve表达量升高,FIP-fve基因沉默的金针菇中FIP-fve表达量下降。更多还原

【Abstract】 Fungal immunoregulatory proteins（FIPs）are small molecule proteins extracted from higher fungi that have a similar structure to the immunoglobulin re-ligation region and are biologically similar to plant lectins.A variety of fungal immunomodulatory proteins have been found to have biological activities such as agglutinating blood cells,anti-tumor,anti-allergy,and lymphocyte proliferation,which have good clinical application prospects.At present,the functional research of FIP mainly focuses on the biological effects on humans and animals,and its biological function on the fungus itself is still unclear.In this study,Flammulina velutipes was used as the research object to construct FIP-fve and LZ-8overexpression vectors,and transformed into Flammulina velutipes by Agrobacterium-mediated transformation to obtain positive transformants;FIP-fve gene overexpression,FIP-fve gene silencing and wild Flammulina velutipes was cultivated,contrast and analysis of phenotypic changes,protein extraction and protein detection and analysis,to lay the foundation for the study of FIP-fve on the biological function of Flammulina velutipes.Firstly,the FIP-fve gene and LZ-8 gene were cloned and the corresponding expression vector was constructed.Two FIP-fve overexpression vectors were constructed,one was pCAMBIA1301-FIP-fve,which was 35S in pCAMBIA1301 vector.The promoter is ligated into the target gene FIP-fve,and the other is pCAMBIA1301-GPD-FIP-fve.The endogenous promoter GPD gene of the Flammulina velutipes and the FIP-fve gene are inserted into the multiple cloning site of pCAMBIA1301,followed by construction of the LZ-8 gene.The overexpression vector pCAMBIA1303-GPD-LZ-8 was also inserted into the multiple cloning site of the GPD promoter gene and the LZ-8 gene.Secondly,Agrobacterium tumefaciens-mediated transformation was used to establish and improve the over-expression vector of Flammulina velutipes genetic transformation system.The infecting material was 1 cm² of Flammulina velutipes mycelium,and the infective condition was Agrobacterium liquid concentration OD₆₀₀00 of 0.5,Agrobacterium AS was added to the bacterial solution to a final concentration of 200μmol·L^-1,and the infestation time was30 min.The hyphae were cultured for 3 days at 25℃in a co-culture medium containing 200μmol·L^-11 AS,and screened with YPG medium containing 9μg·mL^-11 Hyg,and finally FIP-fve overexpressed Flammulina velutipes transformants were obtained.The transformation efficiency of pCAMBIA1301-FIP-fve to Flammulina velutipes was 17.5%,the transformation efficiency of pCAMBIA1301-GPD-FIP-fve to Flammulina velutipes was 22.5%,and the transformation efficiency of pCAMBIA1303-GPD-LZ-8 to Flammulina velutipes was 10%.Finally,the selected FIP-fve gene overexpressing Flammulina velutipes hyphae cultured,and the FIP-fve gene was silenced Flammulina velutipes and Flammulina velutipes was cultured in laboratory,until the fruiting bodies matured,and the phenotypic changes of Flammulina velutipes were observed and extracted.The protein is subjected to protein detection analysis.The number of mushroom primordium of wild type Flammulina velutipes was 46,and the number of mushroom primordium of three strains of FIP-fve overexpressing Flammulina velutipes of pCAMBIA1301-GPD-FIP-fve were 69,55 and 71;the number of mushroom primordium of three strains of FIP-fve overexpressing Flammulina velutipes of pCAMBIA1301-FIP-fve were 92,116 and 51 mushrooms.The total protein content of Flammulina velutipes fruit body was tested by Bradford method.The results showed that the protein concentration of Flammulina velutipes overexpressed by FIP-fve gene was similar to that of wild type Flammulina velutipes,while the total protein concentration of Flammulina velutipes with FIP-fve gene silencing was relatively low.In addition,the content of FIP-fve of the target protein was analyzed by SDS-PAGE.The results showed that the content of FIP-fve in wild-type Flammulina velutipes was 0.73 mg·g^-1,and the FIP-fve content of the three FIP-fve over-expressing Flammulina velutipes of pCAMBIA1301-GPD-FIP-fve was 0.75mg·g^-1,0.76 mg·g^-11 and 0.75 mg·g^-1;the FIP-fve content of three FIP-fve overexpressing Flammulina velutipes of pCAMBIA1301-FIP-fve was 0.80 mg·g^-1,0.71 mg·g^-11 and 0.76mg·g^-1;the FIP-fve content of Flammulina velutipes of FIP-fve gene silencing was 0.71mg·g^-11 and 0.69 mg·g^-1,further tested by Western Blot,and the results showed that the expression of FIP-fve in Flammulina velutipes of FIP-fve overexpressed was increased,and the expression of FIP-fve in Flammulina velutipes of FIP-fve gene silencing was decreased.更多还原