A yeast artificial chromosome-splitting vector designed for precise manipulation of specific plant chromosome region
A yeast artificial chromosome-splitting vector designed for precise manipulation of specific plant chromosome region
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DOI：
10.1263/jbb.99.55
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发表时间：
2005-01-17
影响因子：
2.8
通讯作者：
Harashima, S
中科院分区：
文献类型：
--
作者：
Kim, Y;Kaneko, Y;Harashima, S
构建了酵母人工染色体（YAC）裂解载体pKI 0 1，用于操纵克隆为YAC的植物染色体片段，将片段的特定区域转入植物细胞。载体pKI 01由Km（r）和ADE 2基因（分别用于植物和酵母转化体的选择性标记）、反向端粒重复序列Tr和CEN 4组成。为了证明pKI 01的实用性，将含有来自拟南芥5号染色体的590-kb片段的YAC CIC 9 e2分裂成特异性片段。将位于距离590 UP片段右端100 kb处的1-kb靶区域克隆到pKI 01中。将得到的质粒pKY 03导入到含有YAC CIC 9 e2的酿酒酵母中。发现Ade（+）转化体含有490和100 kb的两个新的YAC，并且缺乏原始的590 kb YAC，这与预期的分裂事件一致。为了释放所需的YAC CIC 9 e2的中间区域，构建了两个额外的分裂载体pKY 11和pKY 14。通过进行两轮分裂，即，第一轮用pKY 11从YACCIC 9 e2的右端100 kb处产生490和100 kb YAC，第二轮从新的490 kb YAC的右端50 kb处产生440和50 kb YAC，携带Km（r）的植物染色体片段的中间50 kb区域作为分裂的YAC被成功释放。这些结果表明，本研究中构建的YAC分裂载体可用于产生任何所需的植物染色体片段作为YAC，以最终重新引入植物细胞。
A yeast artificial chromosome (YAC) splitting vector, pKI01, was constructed for manipulating plant chromosome fragments cloned as YACs in order to transfer specific regions of the fragments into plant cells. Vector pKI01 consists of Km(r) and ADE2 genes (selective markers for plant and yeast transformants, respectively), inverted telomeric repeats Tr and CEN4. To demonstrate the utility of pKI01, YAC CIC9e2 harboring a 590-kb fragment from Arabidopsis thaliana chromosome 5 was split into specific fragments. A 1-kb target region positioned 100 kb from the right end of the 590 UP fragment was cloned into pKI01. The resultant plasmid, pKY03, was introduced into Saccharomyces cerevisiae harboring YAC CIC9e2. The Ade(+) transformants were found to contain two new YACs of 490 and 100 kb, and to lack the original 590 kb YAC, consistent with the expected splitting event. To release the desired middle region of YAC CIC9e2, two additional splitting vectors were constructed, pKY11 and pKY14. By conducting two rounds of splitting, i.e., the first round 100 kb from the right end of YAC CIC9e2 with pKY11 to generate 490 and 100 kb YACs and a second round 50 kb from the right end of the new 490 kb YAC to generate 440 and 50 kb YACs, the middle 50 kb region of a plant chromosome fragment harboring Km(r) was successfully released as a split YAC. These results indicate that YAC splitting vectors as constructed in this study are useful for generating any desired plant chromosome fragment as a YAC for eventual re-introduction into plant cells.