基于TILLING技术的变性淀粉木薯种质创新研究

Cassava is an important cash crop; its starch and novel modified starch have huge market with broad prospects. The development and utilization of the novel modified starch cassava is an important way to produce natural novel modified starch. But lack of novel modified starch cassava is the problem. Based on the prelimilary studies, it created cassava mutant library by the combination of chemical mutagenesis and tissue culture; designed the specific primers for TILLING (Targeting Induced Local Lesions in Genomes), based on the available sequences of both GBSS I（Genbank: X74160）and SBE（Genbank: X77012）gene, to screen out the mutant library and identify the mutants; analysed the mutant alleles, and phenotyped the mutants in vitro and in vivo, so as to create novel modified starch cassava. This work, to create novel modified starch cassava germplasms by exploring the alternative breeding strategy via TILLING, will play a significant role in the innovation of cassava germplasms, the discovery of gene functions, as well as the development of cassava and starch industry.

木薯是重要的经济作物，木薯淀粉及变性淀粉市场巨大和前景广阔，开发利用变性淀粉种质是生产天然变性淀粉的重要途径，但变性淀粉木薯种质资源严重缺乏。本项目在前期研究基础上，结合化学诱变和组织培养技术构建木薯突变体库，根据已克隆木薯淀粉合成相关基因GBSS I（Genbank: X74160）和SBE（Genbank: X77012）设计特异引物，利用TILLING（Targeting induced Local Lesions in Genomes，定向诱导基因组局部突变）技术筛选突变体库，检测突变位点，对突变等位基因进行分析和功能预测，并对突变株系进行离体和田间表型鉴定，筛选获得变性淀粉木薯创新种质。本研究采用结合TILLING技术的高效诱变育种方法，探索新的木薯育种策略，创建变性淀粉种质材料，对木薯种质资源创新和功能基因挖掘，以及我国木薯产业、淀粉产业的发展都具有重要的意义。

通过本项目的实施，建立了成熟的体细胞胚再生技术和探讨出木薯组培苗壮苗生根的适宜配方1/2 MS + 0.02 mg/L NAA+ 0.05 mg/L PP333 + 40.0 g/L蔗糖 + 6.0 g/L琼脂（pH，5.8）；通过开展比较重复试验，建立一套高效的木薯突变体创制方法，从中获得突变体材料200余份；利用TILLING 技术分子检测突变体，对突变等位基因进行分析与功能预测，并对突变株系进行表型鉴定，建立了基于TILLING 技术的木薯分子诱变育种技术方法，筛选获得变性淀粉木薯创新种质材料8份，初步建立了木薯分子诱变育种研究平台；获得《一种木薯组培苗壮苗生根的方法》、《一种木薯试管薯的诱导方法》发明专利2项；发表研究论文7篇；联合培养硕士研究生1 名。