稻瘟菌突变体T-DNA插入位点的精细定位和插入模式的研究

Spotting the positions of T-DNA on the genome for Magnaporthe grisea transformants and study on the mode of T-DNA insertion

摘要:

随机挑取已构建的37个稻瘟菌T-DNA突变株,利用TAIL-PCR技术扩增出T-DNA插入位点的侧翼序列,测序并进行比对分析。结果显示:成功获得扩增产物并测序的序列共有39条,T-DNA边界序列为稻瘟菌序列的有19条,其余20条为载体主干序列。在这有效扩增为稻瘟菌序列的19条中,有10条是T-DNA右侧翼序列与稻瘟菌序列,9条为左侧翼序列加稻瘟菌序列。分析T-DNA剪切位点,10条右侧翼序列中有9条的剪切位点相同,这与农杆菌介导T-DNA转化植物一样。而左边界的剪切位点就没有这种规律性。研究也精细确定了17个不同突变株的T-DNA插入位置,为后续的基因功能研究奠定基础。

Abstract:

Using thermal asymmetric interlaced PCR(TAIL-PCR)，39 specifical fragments of Magnaporthe grisea genomic DNA flanked on the T-DNA were successfully amplified from 37 M. grisea transformants randomly selected from the mutants induced by T-DNA insertion in our laboratory. These fragments were sequenced and then compared by BLAST with the sequences of M. grisea genomic DNA published in netwwork. T-DNA insertion positions for 17 transformants were spotted on the genome of M. grisea. Of all the 39 amplified specifical fragments，19 were M. grisea genomic DNA and 20 contained the vector backbone sequences. Of the 19 M. grisea genomic DNA fragments，10 flanked on the right and 9 on the left border of T-DNA inserted. Of the 10 M. grisea genomic DNA fragments flanked on the right border of T-DNA，9 had a 102bp identical sequence. However，the 7 fragments flanked on the left border of T-DNA did not have this regularity. The above results demonstrated that T-DNA right nick positions were relatively fixed on the M. grisea genomic DNA，similar to the ones on plant genomic DNA. The spotted positions of T-DNA on the genome for 17 M. grisea transformants established a solid foundation for further functional genomic research of M. grisea.