摘要：本研究以野生型Solanumsitiens为转化受体材料，首先构建外泌型重组人IFN-α基因的转化载体，采用叶盘法转化获得转基因阳性植株，通过生物胁迫和非生物胁迫，验证人IFN-α基因的生物学活性与功能。利用实验室自建的瓶内互作检测体系，对同瓶内的野生型和转基因植株，分别接种植物病原菌（大豆核盘菌、茄病镰刀菌和小麦赤霉孢菌）进行生物胁迫验证；同时，采用非生物胁迫的方法（高盐、极限pH、干旱和极限温度），即野生型和转基因植株分别置于1.2%NaCl、pH10.0、pH4.0、15%甘露醇、高温65℃和低温-20℃条件下进行非生物胁迫处理，并分析其表型和耐受能力差异。结果表明，转基因植株较野生型更易感病，而在高盐和pH10.0条件下转基因植株长势明显优于野生型，对于极限高温和低温以及干旱胁迫，野生型和转基因植株表型相似。

关键词：IFN-α；生物胁迫；非生物胁迫；功能分析

The Functional Analysis of Recombinant Human IFN-α in transgenic Solanum sitiens

Abstract: The wild type Solanum sitiens was used as the transformation receptor material in this study. Firstly, we constructed the extracellular secretory transformation vector of recombinant IFN-α, and obtain transgenic positive plants through leaf-disk transformation method, the biological activities and functions of IFN-α in S. sitiens were verified by biotic stress and abiotic stress systems. The WT and transgenic plants were simultaneously inoculated with plant pathogens in one bottle, such as Sclerotinia sclerotiorum, Fusarium solani and F. graminearum, and the biological stress verifications were made, under our innovative detection system in bottle. At the same time, we carried out abiotic stress (high salt, extreme pH values, drought, and extreme temperatures), that is, the WT and transgenic plants were treated respectively under 1.2 % NaCl, pH 10.0, pH 4.0, 15 % mannitol, high temperature of 65℃, and the low temperature of -20℃ for abiotic stress, and the phenotype and tolerance differences were analyzed. The results showed that transgenic plants compared to WT plants were more susceptible to plant pathogens, the growth of transgenic plants was significantly superior to the WT plants under the high salt and pH 10.0 stresses, the phenotype of WT plants and transgenic plants was similar in extreme high and low temperature and drought stresses.

Key words: IFN-α; Biotic stress; Abiotic stress; Functional analysis

目录

摘要 1

Abstract 1

引言 2

1材料与方法 3

1.1材料 3

1.2培养准备 3

1.2.1无菌苗获取 3

1.2.2培养基制备 4

1.2.3GUS染液配制 4

1.3方法 4

1.3.1酶切与连接 4

1.3.2菌种转化及鉴定 5

1.3.3叶盘法转化及转基因植株鉴定 5

1.3.4生物胁迫 6

1.3.5干旱与盐胁迫 7

1.3.6极限pH胁迫 7

1.3.7极限温度胁迫 7

2结果与分析 8

2.1大豆核盘菌互作 8

2.2茄病镰刀菌互作 9

2.3小麦赤霉孢菌互作 9

2.4干旱胁迫 10

2.5NaCl胁迫 11

2.6极限pH胁迫 12

2.6.1低pH胁迫 12

2.6.2高pH胁迫 13

2.7极限温度胁迫 14

2.7.1极限高温胁迫 14

2.7.2极限低温胁迫 15

3讨论 15

参考文献 17

致谢 19

重组人IFN-α基因转化Solanumsitiens功能分析

引言

人干扰素（Interferon，IFN）是人机体内产生的一类具有免疫能力的细胞因子，它们具有相似的结构、类似的功能[1]，同时具有广谱抗病毒作用，它的抗病毒机制主要是通过靶细胞表面干扰素受体与干扰素结合，诱导靶细胞产生MX蛋白、蛋白激酶PKR、2-5(A)合成酶等多种蛋白质来抵御病毒，改变某些必须酶的活性[2]，阻止病毒合成所需的蛋白质、抑制机体内病毒的核酸复制和转录[3]。干扰素具有免疫调节功能，可提高淋巴细胞对靶细胞的特异性，维护和促进机体的免疫防护、监视、自稳等功能[4]。按照人干扰素的来源和结构不同，可将其分为α、β和γ三类。上世纪80年代，美国FDA就批准了αⅡa型和αⅡb型干扰素投放进入医疗和商业市场，截止目前，世界上已有50多个国家批准了IFN上市，用于治疗多种疾病。但是，上述干扰素产业多采用动物源方式进行工业生产，然而一直以来，该技术系统存在动物病原菌潜在污染、培养成本昂贵、产率低下等技术瓶颈，至今国际上并无有成熟的以植物（植物细胞）为生产载体进行IFN的工业化生产技术。