一种不依赖载体构建的质体转化方法与应用

Plastid transformation has been widely used in various fields such as plastid functional genomics study, molecular farming, metabolic engineering and genetic improvement in crops. However, the construction of plastid transformation vectors is very tedious and time-consuming, it is difficult to clone exogenous genes that are toxic to bacteria or more likely to recombinate in E. coli. At the same time, the vectors from E.coli can take only up to 15 kbp of DNA, more than 20 kbp metabolic pathways that be incorporated into plastids is not easy. In this study, self-assembly of multiple linearized DNA fragments containing overlapped sequence through homologous recombination system existed in the plastid was utilized to transform plastid. Previously, it had been tested that the linear single DNA fragment could transform plastid. Next, we will continue to verify the possibility of two fragments, three fragments and more than three fragments. Furthermore, the toxic protein plyGBS will be expressed in the plastid by the method of no vectors, and the polysaccharide metabolic pathway which contains 25 kbp nucleotides from Streptococcus agalactiae will be introduced into the plastid. The establishment of this technology will simplify plastid transformation and provide a new strategy for bacterial toxic proteins, easily recombinant proteins and complex metabolic engineering.

质体生物技术在质体功能基因组学、分子农场、代谢工程以及作物遗传改良等多个方面有广泛应用，但质体转化载体的构建过程繁琐，难于克隆细菌有毒或易诱导重组的外源基因，不易通过基因枪导入大片段的DNA。本研究将利用质体内存在的同源重组系统，通过含有重叠序列线性化的DNA多片段在质体内自组装达到转化质体的目的，建立不依赖克隆过程的质体转化技术。本项目内容首先建立单线性化DNA片段、两片段及多片段转化质体的过程，然后通过无载体技术在质体内表达有毒蛋白PlyGBS和导入含有16个基因的基因簇的多聚糖代谢通路。此方法的建立将简化质体转化的技术流程，为细菌有毒蛋白、易诱导重组蛋白和复杂代谢工程提供了新策略。

质体基因工程具有外源基因高表达和生物安全性等独特优势，在分子农业、代谢工程和基因改良等领域具有广阔的应用前景。迄今为止大部分质体转化技术均依赖于质粒载体的构建，然而质体转化载体并没有通用载体且构建过程十分繁琐，不易构建含有长操纵子的载体，难于克隆对细菌有毒或易诱导重组的外源基因。在本研究中，我们利用质体内源丰富的同源重组使用具有同源序列的线性DNA片段开发了一种简易的无克隆质体转化方法。我们研究发现，两个线性的DNA片段之间重叠200 bp或以上便足以以传统的质体转化效率整合到质体基因组中。两个线性的DNA片段之间重叠50 bp也可以成功整合到质体基因组中，但是其质体转化的效率远远低于于传统的质体转化方法的效率。同时，我们还对通过线性DNA片段和传统质体转化方法得到的质体转化植株进行了蛋白水平的检测，不论是GFP蛋白的表达量还是蛋白功能都没有显著性差异。而且我们还对两种方法得到质体转化植株在遗传特性进行了相关检测。在T1代种子测试和T1代植物整个生长过程中都没有显著性差异。此外，我们成功地利用此方法将来源于噬菌体的裂解酶基因plyGBS(其表达产物对大肠杆菌有毒)和一个长操纵子导入烟草质体基因组。该技术的建立简化了质体转化过程，为在质体中表达细菌毒性蛋白和长操纵子提供了一种新的策略。

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