水稻Xa21基因在水稻和玉米中的比较物理定位

Comparative Physical Location of Rice Xa21 Gene in Rice and Maize Using the BAC FISH Approach 

比较基因组分析证明，禾本科不同种基因组间存在广泛的同线性和共线性。对水稻（ＯｒｙｚａｓａｔｉｖａＬ．）这一模式植物与其它禾本科植物基因的原位杂交比较定位可以揭示禾本科植物基因组结构的共同特点和进化规律。利用含Ｘａ２１基因的ｐＢ８２２作探针筛选水稻的细菌人工染色体（ＢＡＣ）文库，建立了一个包含３个ＢＡＣ克隆的重叠群。用生物素标记其中一个ＢＡＣ克隆，对水稻“广陆矮４号”和玉米自交系黄早四进行了染色体荧光原位杂交。同时，用ｐＢ８２２也作了原位杂交检测。在水稻第１１染色体长臂中间检出了杂交信号，信号与着丝粒的百分距离约为２４。在玉米的第１、３和第８染色体长臂观察到杂交信号，表明玉米基因组中具有三个Ｘａ２１的同源序列座位。ＢＡＣＦＩＳＨ的信号检出率达在４０％以上，大大高于质粒探针ｐＢ８２２的检出率（１５％），而且可在同源染色体和姊妹染色单体上同时检出杂交信号的比例较高，证明了利用ＢＡＣ克隆荧光原位杂交进行比较物理定位的可行性和优越性。在ＢＡＣＦＩＳＨ中必须用相应基因组的ＣｏｔⅠＤＮＡ封阻，以排除重复序列的干扰。

In grasses, comparative genome analyses have demonstrated that there were widespread syntenies and colinearities of the genes among different species within a family. As a model genome, analysis of the rice (Oryza sativa L.) genome has allowed us to reveal the basic evolutionary units of the cereal genomes. The authors used a rice genomic clone of the gene Xa21 inserted in the plasmid pBluescript as a probe to screen rice bacterial artificial chromosome (BAC) library. A contig of three BAC clones was constructed. One of the BAC clones was adopted as the tested probe. The tested plants were O.sativa subsp. indica cv. “Guang Lu Ai 4” and the inbred line of Zea mays cv. Huang Zhao 4. The biotin labeled BAC clone was hybridized onto the rice and maize metaphase chromosomes by fluorescence in situ hybridization (FISH). The Xa21 cloned in pBluescript was also hybridized onto the maize chromosomes. The results obtained from the BAC and plasmid clones were the same. In rice, the Xa21 was located in the long arm of the chromosome 11, the percent distance from the centromere to the signal was about 24. This result was in accordance with the rice genetic map. In maize, three hybridized sites were detected ant located on the long arms of chromosomes 1, 3, and 8 respectively. It suggested that the homologous sequences of Xa21 were triplicated in maize. By BAC FISH technique, the detection rate was over 40%, much higher than that by FISH with plasmid clones, for which it was only about 15%. Moreover, the rate of signals which were detected simultaneously on two members of a homologous chromosome pair and sister chromatids was higher by BAC FISH technique than that by FISH with plasmid clones. It demonstrated the feasibility and advantage of BAC FISH technique in gene comparative physical location. C 0tⅠ DNA was necessary for blocking the nonspecific DNA because the tested results were interfered seriously by repeated sequences. The signals could be found on many chromosomes without blocking.