摘要随着基因组高通量测序以及“生物探矿”技术的发展，编码次生代谢产物的大片段 基因簇不断涌现，为新型药物开发提供了丰富的前体化合物。然而，传统的大片段克隆 技术依赖于繁琐的基因文库构建与筛选，无法满足大片段基因簇克隆的需求。本课题基 于对现有 TAR（Tansformation associated recombination）克隆技术的研究，构建出可诱导 线性化的捕捉载体 pSH47-SceI，利用半乳糖诱导捕捉载体上内切酶 I-Sce1 的表达，实现 捕捉载体在酵母细胞内的线性化，再将基因组片段转化入含有线性捕捉载体的酵母中， 有效地提高了原有 TAR 克隆技术中将载体和片段同时转化入酵母的捕捉重组效率，为大 片段基因簇的克隆提供了新思路。74520

毕业论文关键词 TAR 克隆 syringomycin 基因簇 酿酒酵母 同源重组

毕 业 设 计 说 明 书 外 文 摘 要

Title Establishment of an inducible TAR cloning system and the cloning of syringomycin gene cluster

Abstract Natural secondary metabolites are the main sources for novel drug discovery, the genes involved in the biosynthesis of these compounds are usually clustered in the genome。 With the advent of genome sequencing and "bio-prospecting", complete genome sequences are now being determined at a rapidly increasing rate。 Traditional methods for the large genome sequence cloning are  too  dependent  on library construction and  screening。  Inspired  by the  yeast   TAR

（ Transformation  associated   recombination ） cloning,   here   we   developed   an inducible

linearization trapping system, in which the capture vector pSH47-SceI can be linearized at its I-SceI site by induction with galactose, thus enabling the ability to recombine with the target DNA fragments directly。 By using this system, we captured the bleomycin resistance gene as a proof of concept, then we further cloned the syringomycin gene cluster from Pseudomonas synringae HS191。 Compared to the original TAR cloning technology, which simultaneously transformed the vector and the fragments into yeast cells, this system is more convenient and efficient,   opening  a new avenue  for the  cloning  of large  gene  cluster。

Keywords  TAR  cloning,      gene  cluster,      yeast,     homologous  recombination

目 次

1 引言 1

1。1 大片段基因簇克隆 1

1。2 TAR 克隆策略 2

1。3 CRISPR/Cas9 基因组编辑体系 3

2 实验材料 6

2。1 菌株和质粒 6

2。2 培养基 6

2。3 酶类和抗生素 7

2。4 引物 7

2。5 仪器材料 7

3 pSH47-Sce1-DE 诱导表达载体的构建