农杆菌介导转化黑曲霉分生孢子及产孢缺陷突变子T-DNA插入位点分析

Agrobacterium tumefaciens mediated transformation of Aspergillus niger conidiospores and sequence analysis of T-DNA insertion site in conidiation-defective mutant

摘要:

摘要： 【目的】利用农杆菌（Agrobacterium tumefaciens）T-DNA系统，建立转化黑曲霉（Aspergillus niger）分生孢子的方法，构建T-DNA插入突变子文库，为黑曲霉基因组功能注释研究打下基础。【方法】采用携带二元质粒载体pCAMBIA1301的农杆菌EHA105，诱导转化黑曲霉分生孢子，筛选具有潮霉素抗性的突变子。分析抗性稳定突变子菌株的表型，采用反向PCR方法分析T-DNA插入位点相邻位置的序列，并推测突变基因可能具有的功能。【结果】实验获得具有稳定潮霉素抗性转化子193株，转化率为5.6?102转化子/108分生孢子。部分转化子表型出现较为明显改变，其中一株不能产孢，对其T-DNA插入位点序列分析比对结果显示，突变基因属于超级转运家族（major facilitator superfamily, MFS）。【结论】本研究建立的农杆菌转化黑曲霉分生孢子平台，结合T-DNA插入突变位点分析，可以为黑曲霉基因组功能注释研究提供一种简便有效的途径。

Abstract:

Abstract: [Objective] Development of a method of transforming Aspergillus niger conidiospores with Agrobacterium tumefaciens T-DNA system and using it as a tool for genome annotation via construction a T-DNA insertion mutant library of A. niger. [Methods] A. tumefaciens EHA105 carrying the binary vector pCAMBIA1301 was used to transform A. niger conidiospores under induction conditions. Hygromycin resistant transformants were screened on the selected medium. Sequence analysis was performed usimg inverse PCR method in stable mutants. [Results] In total, 193 hygromycin resistant stable transformants were obtained and the transformation rate was about 5.6?102 transformants /108 conidiospores. Obvious morphological changes were observed in some mutants and sequence analysis of T-DNA inserted site was performed in one conidiation-defective mutant. The result showed that the interrupted gene belonged to major facilitator superfamily (MFS). [Conclusion] Our results indicated that the transformation system of A. niger conidiospores set up in this study, combing with sequence analysis of T-DNA insertion site, might be an effective way for genome annotation in A. niger.