SAMDC和PAO基因调控多胺参与棉花体细胞胚胎发生的研究

Agrobacterium mediated transformation was an important method for studying of gene function and molecular breeding in cotton, and genetic transformation achieved by somatic embryogenesis. In present, long period, low efficiency and genotype limitation strongly limited application of genetic transformation in cotton. Polyamine was an important growth regulator, somatic embryogenesis were remarkably promoted by polyamines in some plants. In this study, changes of polyamines contents were detected during somatic embryogenesis. Different treatments of exogenous polyamines, polyamines inhibitor were applied in somatic embryogenesis, which disclosed the suitable treatment promoting somatic embryogenesis. Combine with gene expression analysis of somatic embryogenesis related genes in different treatments, revealed physiological mechanism of somatic embryogenesis regulated by polyamines in cotton. 2-3 key genes were selected by gene expression analysis and genetic transformation performances of SAMDC and PAO genes. Compare of histological analysis, polyamines contents, genes expression analysis of polyamines and somatic embryogenesis related genes in transgenic and control plants, which might revealed the molecular mechanism of somatic embryogenesis regulated by polyamine in cotton. These results provided theoretical basis for somatic embryogenesis regulated by polyamines, and might provided a new insight into embryo development in plant.

农杆菌介导的遗传转化是棉花基因功能研究和分子育种的重要手段，需要借助于体细胞胚胎发生实现。目前，棉花体细胞胚胎发生依然存在再生周期长、效率低、受基因型限制等诸多难题，限制了中国特别是新疆棉花品种的遗传改良。多胺是一类重要的生物调节物质，大量研究表明它可以显著地促进多种植物的体细胞胚胎发生。本项目拟通过对新疆棉花体细胞胚胎发生过程中多胺含量测定，外施多胺、多胺抑制剂等研究不同处理对棉花体细胞胚胎发生的影响，结合不同处理对棉花体细胞胚胎发生相关基因表达的影响，揭示多胺调控棉花体细胞胚胎发生的可能生理机制；通过参与多胺合成代谢基因SAMDC和PAO在体细胞胚胎发生过程中的表达分析和转化过程中的表现，选择2-3个关键基因，对转基因稳定后代与对照的体细胞胚胎发生过程中组织学、多胺含量、多胺和体细胞胚胎发生相关基因表达的比较分析，解析SAMDC和PAO调控多胺参与棉花体细胞胚胎发生的可能机制。

为提高棉花遗传转化效率，本项目对棉花体细胞胚胎发生过程不同时期组织的多胺含量测定，利用多胺和多胺抑制剂进行互补实验，探明了多胺在棉花体细胞胚胎发生过程中的变化规律，发现精胺促进胚性愈伤组织向胚状体分化方面发挥重要作用。结合多胺相关基因的表达分析和酶活分析，揭示了多胺在体细胞胚胎发生过程中的生理机制。此外，克隆获得14个GhSAMDC和12个GhPAO基因全长cDNA序列，并对基因表达模式进行了表达分析；选择了4个GhSAMDC和3个GhPAO基因构建了植物表达载体转化模式植物拟南芥、烟草和棉花，获得了转基因材料20余份，在此基础上重点分析了部分基因在体细胞胚胎发生、抗盐和生殖生长方面的功能。此外，通过对棉花体细胞胚胎发生过程中的3个关键时期组织进行了转录组和蛋白组分析，从生理和分子水平解析了多胺及相关基因GhSAMDC和GhPAO在棉花体细胞胚胎发生过程中的作用，为利用多胺调控棉花体细胞胚胎发生进而提高遗传转化效率提供了参考。