摘 要：将DNA导入丝状真菌有很多种转化方法，农杆菌介导转化法即为其中之一。本研究即通过根癌农杆菌介导转化法，构建丝状真菌里氏木霉RutC-30的遗传转化表达体系。首先构建含有潮霉素B抗性基因的双元载体，然后将该双元载体转化到根癌农杆菌中，并筛选阳性转化子。将该转化子和里氏木霉RutC-30的分生孢子进行液体共培养，并在含有100μg/mL 潮霉素抗性平板上筛选出里氏木霉RutC-30阳性转化子。最后通过菌落PCR鉴定的方法进行鉴定转化子。结果表明：β-葡萄糖苷酶基因cel3a成功整合到里氏木霉RutC-30的基因组中。93574

毕业论文关键词：根癌农杆菌，丝状真菌，里氏木霉，β-葡萄糖苷酶

Abstract：Many transformation methods have been developed to introduce DNA into filamentous fungi。 One of these methods is Agrobacterium-mediated transformation (AMT)。 In this study, we constructed genetic and transformation system in filamentous fungi Trichoderma reesei RutC-30 through Agrobacterium mediated transformation (AMT)。 First, a binary vector containing the hygromycin B resistance gene was constructed。 Then the binary vector was transformed into Agrobacterium tumefaciens and the positive transformants were screened。 After that, Trichoderma reesei RutC-30 conidia, which were incubated in liquid with positive Agrobacterium transformant, were selected on the plates that contained 100 μg/mL hygromycin B。 Finally, the transformants were identified by colony PCR。 The results showed that the β-glucosidase gene cel3a was integrated into the genome of T。 reesei RutC-30。

Keywords：Agrobacterium tumefaciens, Filamentous fungi, Trichoderma reesei, β-glucosidase

目录

1  前言 4

1。1  里氏木霉 4

1。2  纤维素酶系 5

1。3  β-葡萄糖苷酶cel3a 6

2  农杆菌介导转化原理 7

2。1  大致机理与农杆菌的Ti质粒 7

2。2  农杆菌介导转化真菌体系 8

3  实验材料与仪器 9

3。1  主要试剂 9

3。2  实验仪器 9

3。3  培养基及配方 10

3。4  菌株 10

4  实验方法及步骤 10

4。1  农杆菌介导的里氏木霉转化 11

4。1。1  农杆菌的转化 11

4。1。2  孢子的制备 11

4。1。3  农杆菌的制备 11

4。1。4  共培养 11

4。2  转化子PCR鉴定 11

4。2。2   PCR引物及序列 12

4。2。3  PCR体系及条件 12

5  实验结果与分析 12

5。1  农杆菌介导转化里氏木霉的结果 12

5。2  转化子PCR鉴定结果及分析 15

结论 16

参考文献: 17