利用CATCH靶向克隆及测序技术获取植原体基因组

Phytoplasma is a kind of prokaryotic microorganism which is strictly parasitic in phloem and transmitted through insect mediators. It can cause many important plant diseases, including jujube witches'-broom disease. It has been difficult to obtain enough pure phytoplasma DNA for sequencing because of the lack of isolation and culture methods, hindering the development of phytoplasma genomics research. The proposed study plans to use CATCH targeted cloning and sequencing technology which was developed by the applicant to obtain phytoplasma genome from host. Taking advantage of the high specificity and editability of CRISPR-Cas system, CATCH can separate tens or hundreds of kb of large target DNA fragments from complex genomic background, and the obtained target fragments can be cloned to vectors by Gibson Assembly. Sanger sequencing or high-throughput sequencing of the specific genome-wide libraries can be used to obtain accurate genome sequences of phytoplasma at much lower sequencing costs. These methods can effectively solve the problem of host DNA contamination and obtain the complete sequence of phytoplasma specifically, and clone the repetitive gene clusters such as PMU in phytoplasma quickly and accurately. This technology also can be potentially applied to other plant pathogenic microorganisms which are strictly parasitic and difficult to isolate and purify.

植原体是一类在植物韧皮部严格寄生并通过昆虫介体传播的原核微生物，能够引起包括枣疯病在内的多种重要植物病害。由于不能分离培养，很难获得足够的高纯度的植原体DNA用于测序，严重阻碍其基因组学研究的发展。本项目计划应用申请人前期开发的CATCH靶向克隆及测序技术从寄主中特异性获取植原体基因组。该技术利用CRISPR-Cas系统特异性高、可编辑性强的特点，可以将几十kb甚至上百kb的目标DNA大片段从复杂背景中分离，得到的目标片段通过Gibson Assembly原理连接到载体上完成克隆。对上述特异性全基因组文库进行Sanger测序或高通量测序，即可利用较低测序成本获得植原体全基因组的准确序列。上述方法可有效解决宿主DNA污染问题，特异性获取植原体完整序列,快速准确克隆植原体中PMU等重复性基因簇。该技术可推广应用于其它严格寄生且难以分离纯化的植物病原微生物。

植原体是一类在植物韧皮部严格寄生并通过昆虫介体传播的原核微生物，能够引起包括枣疯病在内的多种重要植物病害。由于不能分离培养，很难获得足够的高纯度的植原体DNA用于测序，严重阻碍其基因组学研究的发展。本项目基于CRISPR-Cas系统特异性高、可编辑性强的特点，成功构建了利用CATCH技术在溶液中靶向克隆植原体基因组片段的实验体系，同时针对部分无法在大肠杆菌宿主体内表达的有毒基因，构建了以酵母为宿主靶向克隆植原体基因组片段的实验体系，并成功利用这两种体系克隆植原体基因组上的小片段DNA。上述方法可有效解决宿主DNA污染问题，有望特异性获取植原体完整序列，快速准确克隆植原体PMU等重复性基因簇。该技术可推广应用于其它严格寄生的植物病原微生物或难以分离培养的环境微生物。