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| Isolating the Mutator Transposable Element Insertional Mutant Gene mio16 ofMaize UsingDoubleSelectedAmplification of Insertion Flanking Fragments (DSAIFF) |
| ZHONG Wen-juan, ZHANG Mei-dong, YANG Liu-qi, WANG Ming-chun, ZHENG Yong-lian, YANG Wenpeng GAO You-jun |
1.National Key Laboratory of Crop Genetic Improvement/Huazhong Agricultural University, Wuhan 430070, P.R.China
2.Institute of Upland Food Crops, Guizhou Academy of Agricultural Sciences/Guizhou Center of Maize Engineering Techniques, Guizhou 550006, P.R.China |
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摘要 Mutator transposable element (Mu) has been used as an effective tool to clone maize (Zea mays L.) genes. One opaque endosperm mutant (mio16) 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 mio16. 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 mio16 was mapped in silico onto the physical position ranged from 229 965021 to 229 965409 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 mio16 region anchored by primers Mu20 and af276 was not interrupted by Mu insertion. Further researches will be done to elucidate how the expression of mio16 is alternated by Mu insertion.
Abstract Mutator transposable element (Mu) has been used as an effective tool to clone maize (Zea mays L.) genes. One opaque endosperm mutant (mio16) 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 mio16. 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 mio16 was mapped in silico onto the physical position ranged from 229 965021 to 229 965409 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 mio16 region anchored by primers Mu20 and af276 was not interrupted by Mu insertion. Further researches will be done to elucidate how the expression of mio16 is alternated by Mu insertion.
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Received: 02 June 2011
Accepted:
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| Fund: This research was supported by the High-Tech R&D Program of China (2006AA10A106), the open funds of the National Key Laboratory of Crop Genetic Improvement and China National Fundamental Fund of Personnel Training (J0730649). |
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Corresponding Authors:
Correspondence GAO You-jun, Tel: +86-27-87282689, E-mail: yjgao@mail.hzau.edu.cn; YANG Wen-peng, Tel: +86-851-3760096, E-mail: ywpmaize@126.com
E-mail: yjgao@mail.hzau.edu.cn
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Cite this article:
ZHONG Wen-juan, ZHANG Mei-dong, YANG Liu-qi, WANG Ming-chun, ZHENG Yong-lian, YANG Wenpeng GAO You-jun.
2012.
Isolating the Mutator Transposable Element Insertional Mutant Gene mio16 ofMaize UsingDoubleSelectedAmplification of Insertion Flanking Fragments (DSAIFF). Journal of Integrative Agriculture, 12(10): 1592-1600.
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