High throughput T-DNA insertion mutagenesis in rice:: a first step towards in silico reverse genetics
High throughput T-DNA insertion mutagenesis in rice:: a first step towards in silico reverse genetics
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DOI：
10.1111/j.1365-313x.2004.02145.x
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发表时间：
2004-08-01
期刊：
影响因子：
7.2
通讯作者：
Guiderdoni, E
中科院分区：
文献类型：
--
作者：
Sallaud, C;Gay, C;Guiderdoni, E
在水稻栽培种中构建了一个包含29482个T-DNA增强子捕获系的文库。日本晴通过衔接子锚PCR系统地分离来自前12 707个初级转化体的T-DNA左边界侧翼区域并测序。对7480个大于30 bp（平均长度为250 bp）的基因组序列进行了调查，占总可读序列的56.4%，并与水稻细菌人工染色体/噬菌体人工染色体（BAC/PAC）假分子组装序列进行了匹配，从而将6645个（88.8%）T-DNA插入位点分配到水稻基因组的至少一个位置上。日本晴T-DNA插入似乎相当随机地分布在水稻的12条染色体上，其中第1、2、3和6号染色体的插入频率略高。723个独立的T-DNA插入沿着1号染色体假分子的分布与预测的编码序列没有显著差异，在着丝粒区域周围显示出较低的插入密度，而在基因密度较高的亚端粒区域显示出较高的插入密度。进一步建立的T-DNA插入物沿着最近释放的12个水稻假分子的密度图证实了这种不均匀的染色体分布。T-DNA似乎不太倾向于热点和冷点的整合相比，所揭示的同时分配的Tos 17反转录转座子侧翼序列存放在美国国家生物技术信息中心（NCBI）。T-DNA插入很少整合到重复序列中。基于预测的1号染色体的基因注释，已观察到从推定的ATG起始密码子的前250 bp内的优先插入。使用插入点周围的4kb序列，62%的序列显示出与编码已知蛋白质的基因的显著相似性（E值
A library of 29 482 T-DNA enhancer trap lines has been generated in rice cv. Nipponbare. The regions flanking the T-DNA left border from the first 12 707 primary transformants were systematically isolated by adapter anchor PCR and sequenced. A survey of the 7480 genomic sequences larger than 30 bp (average length 250 bp), representing 56.4% of the total readable sequences and matching the rice bacterial artificial chromosome/phage artificial chromosome (BAC/PAC) sequences assembled in pseudomolecules allowed the assigning of 6645 (88.8%) T-DNA insertion sites to at least one position in the rice genome of cv. Nipponbare. T-DNA insertions appear to be rather randomly distributed over the 12 rice chromosomes, with a slightly higher insertion frequency in chromosomes 1, 2, 3 and 6. The distribution of 723 independent T-DNA insertions along the chromosome 1 pseudomolecule did not differ significantly from that of the predicted coding sequences in exhibiting a lower insertion density around the centromere region and a higher density in the subtelomeric regions where the gene density is higher. Further establishment of density graphs of T-DNA inserts along the recently released 12 rice pseudomolecules confirmed this non-uniform chromosome distribution. T-DNA appeared less prone to hot spots and cold spots of integration when compared with those revealed by a concurrent assignment of the Tos17 retrotransposon flanking sequences deposited in the National Center for Biotechnology Information (NCBI). T-DNA inserts rarely integrated into repetitive sequences. Based on the predicted gene annotation of chromosome 1, preferential insertion within the first 250 bp from the putative ATG start codon has been observed. Using 4 kb of sequences surrounding the insertion points, 62% of the sequences showed significant similarity to gene encoding known proteins (E-value