磁纳米载体介导棉花遗传转化机理研究

With the continuous improvement of big data resources in cotton genome and transcriptome, lots of genes related to stress resistance, fiber development, and yield have been identified and obtained by use of GWAS, QTL and CRISPR/Cas9. These genes were providing abundant genetic resources for cotton molecular breeding. Recently, creating new transformation method to import this gene into cotton has become a hot spot in the research of cotton molecular breeding. These methods could be used to create high-quality, high-yielding and stress-resistant cotton materials for cotton breeding. Our previous work had created a new method which is magnetic nanoparticles mediated cotton genetic transformation. This method could overcome the bottleneck in traditional transformation methods, such as genotype dependence and long-term transformation in plant tissue cultural progress, and low transformation efficiency in pollen tube transformation. Despite our method had successfully used in cotton transformation, there are some mechanisms are still not clear, such as genetic transformation mechanism and regulate mechanism of integrated copy number of the exogenous gene. In this project, after artificial pollination of pollens which be transformed by nanoparticles carrier with GUS-GFP plasmid, the absolute copy number, DNA in situ hybridization and single cell sequencing will be used to resolve the integration mechanism and copy number regulatory mechanism in the nanoparticle-mediated transformation process. The results of will further improve and establish a new method which is simple, high efficiency, clear mechanism, no genotype-dependent of receipt material, suitable for a variety of seed plant. Also, this project will provide a theoretical basis for other crops which were difficult to transform, such as wheat and corn.

随着棉花基因组、转录组等大数据不断完善，借助GWAS、QTL、CRISPR/Cas9等技术鉴定了大量抗逆、纤维发育、产量相关基因，为棉花分子育种提供了丰富的基因资源。发掘新型遗传转化方法将基因资源导入棉花创制优质、高产、抗逆等复合性状新材料，成为棉花分子育种的研究热点。本课题组前期研究工作中建立的纳米载体介导棉花遗传转化新方法，克服了传统方法中受体材料基因型依赖性强、转化周期长、转化效率低等限制因素，但是该方法遗传转化机理、外源基因整合拷贝数调控机制尚不清楚。本项目拟利用DNA原位杂交、单细胞测序、拷贝数绝对定量等技术，分析纳米载体携带GUS-GFP双标记质粒进入胚囊和染色体整合的动态变化过程，解析纳米载体遗传转化方法整合机理及拷贝数调控机制，进一步建立一套操作简单高效、机理明晰、无受体材料基因型限制、普适性好的遗传转化新方法，同时为该方法在小麦、玉米等其他难转化植物应用中提供理论依据。

随着基因组测序技术的飞速发展，棉花、小麦、玉米等大多数作物基因组序列获得解析，同时利用GWAS、QTL等方法使得大量重要农艺性状相关基因得到了完美定位和克隆，从而导致功能基因遗传转化成为验证基因功能和利用功能基因进行分子育种的核心研究工作。此外，随着现代化农业生产对优良品种特性要求的不断提高，单基因、双基因、三基因等少数基因遗传转化已不能满足复杂性状遗传改良的需要，急需要开发一些操作简单、基因数量负载能力高、转化效率高、安全性好、无受体材料基因型限制、转化周期短、适宜于大多数农作物的新型遗传转化系统，实现快速基因功能验证和多基因性状的遗传改良，为加速培育综合性状优良的生物新品种提供技术保障。.本项目通过研究磁性纳米载体介导棉花花粉转染技术及其转基因机理，建立一个操作简单、机理基本明晰、适宜多个物种的转基因技术。最终对该技术的一些操作指标进行了界定：遗传转化用的MNP:DNA复合物的最佳比例是1:2；依据不同质粒DNA的分子量大小，MNP:DNA复合物的最佳稀释倍数为100~1000倍，稀释倍数低于100倍会影响双受精过程导致不孕，稀释倍数高于1000倍导致转基因效率急剧下降；外源DNA在花粉管萌发过程中具有表达活性；外源DNA在双受精过程中通过随机断裂整合于基因组上，且以多拷贝的方式整合；大量田间转基因试验发现利用纳米载体介导的转基因方法无法获得单拷贝的转基因材料。.同时，本项目在已有的实验基础上，积极发掘新纳米材料并应用于花粉遗传转化，初步建立了炭纳米载体、金纳米载体介导花粉转基因技术体系，为后续的纳米载体介导遗传转化研究奠定了理论和实验基础。.此外，在本项目的资助下，发表2篇SCI论文，获得授权专利1项，培养硕士研究生1名。其中授权专利涉及的知识产权为利用基因编辑技术创制抗旱、耐盐碱棉花新种质GhGI-2，申请并获批了中间试验（图6），为后期种质材料的成果转化奠定了基础。