中文摘要：

利用分子生物学技术扩增T-DNA插入突变体中T-DNA侧翼未知序列是克隆突变相关基因、研究T-DNA整合方式的基础，TAIL-PCR是扩增已知序列侧翼未知序列的有效方法，本研究根据哈茨木霉的基因组特点，引用和设计了12条随机AD引物，用这些引物分别与3条右边界嵌套特异引物进行组合对哈茨木霉突变子的T-DNA侧翼未知序列进行扩增，发现不同的随机引物的扩增效率差异很大，以AD5的扩增效率最高，能扩增出80％的T-DNA插入位点的侧翼序列。随机挑取哈茨木霉T-DNA插入突变子52个，选用引物AD5对T-DNA插入位点的侧翼序列进行TAIL-PCR扩增并分析扩增序列发现，在获得的42条侧翼序列中7条只含有质粒序列、33条对应着单一的侧翼序列T-DNA、另外2条序列相同。34条T-DNA侧翼边界序列中13条保存着完整的右边界序列，21条T-DNA边界序列存在一定程度的缺失现象，说明农杆菌转化哈茨木霉的过程中T-DNA右边界存在一定程度的剪切。该研究的进行对明确农杆菌介导的木霉遗传转化过程中T-DNA的整合方式具有一定意义，为研究农杆菌转化木霉的机理奠定了实验基础。研究也精细确定了33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。

英文摘要：

Thermal asymmetric interlaced PCR （TAIL-PCR） is an effective method for cloning the flanking sequence of known sequence. In this paper, we used 12 arbitrary primers to amplify the flanking sequence from randomly chosen mutants and found the arbitrary primer ADS was the optimal primer for T. harziunm T-DNA mutants. Using ADS as arbitrary primer, 42 specific fragments of T. harziunm genomic DNA flanking T-DNA were successfully amplified from 52 mutants randomly selected from the T-DNA insertion mutant library of T. harziunm. These fragments were then sequenced and analyzed. Through the sequence analysis of 42 specifically amplified fragments, it was shown that the 33 sequences were different from each other, only 7 of which con- tained the vector backbone and 2 had identical sequences. Thirteen sequences from the 34 T. harziunm genomic DNA framents had intact identical T-DNA right border sequences. However, the T-DNA right border of the other 21 sequences had different nicks. The spotted positions of T-DNA on the T. harziunm genome laid a foun- dation for further functional genomic research. The spotted positions of T-DNA in the genome of T. harziunm transformants provided a solid basis for further functional genomic research of T. harziunm.