Isolating the MutatorTransposable Element Insertional Mutant Gene mio16 of Maize Using Double Selected Amplification of Insertion Flanking Fragments （DSAIFF）

• Published in: 农业科学学报：英文版 2012, Vol.11 (10), p.1592-1600
• Main Author: ZHONG Wen-juan ZHANG Mei-dong YANG Liu-qi WANG Ming-chun ZHENG Yong-lian YANG Wen- peng GAO You-jun
• Format: Article
• Language: English
• Subjects: RT-PCR; 侧翼序列; 元素; 全长cDNA; 变体基因; 玉米; 突变体库; 选择性扩增
• ISSN: 2095-3119; 2352-3425

Mutator transposable element （Mu） has been used as an effective tool to clone maize （Zea mays L.） genes. One opaque endosperm mutant （miol6） was identified in a pool of Mu inserted mutants. A modified method, termed the double selected amplification of insertion flanking fragments （DSAIFF）, was employed to isolate the Mu flanking fragments （MFFs） of miol6. The target site duplications （TSDs） isolated from the Msp I and Mse I digested MFFs had a same 9-bp sequence and were confirmed to be the flanking sequence of one identically inserted gene. Co-segregation analysis suggested that the MFFs were associated with the mutant opaque endosperm, and miol6 was mapped in silico onto the physical position ranged from 229 965 021 to 229 965 409 bp of the maize chromosome 4.09 bin. The full-length cDNA of the wild-type gene was obtained by an RT-PCR primer-scanning technique, and Mio16 was found to putatively encode a homolog of the Arabidopsis MAP3K delta-1 protein kinase. RT-PCR result the mRNA expression of miol6 region anchored by primers Mu20 and af276 was not interrupted by Mu insertion. Further researches will be done to elucidate how the expression of miol6 is alternated by Mu insertion.