华山新麦草特异重复序列的筛选、克隆及Southern杂交分析

为了寻找华山新麦草特异重复序列,以华山新麦草、栽培一粒小麦、栽培二粒小麦、野生一粒小麦、野生二粒小麦、圆锥小麦、阿拉拉特小麦、茹科夫斯基小麦、斯卑尔脱小麦、普通小麦"中国春"(CS)为材料,利用200条RAPD引物筛选出11条华山新麦草特异条带(命名为RHS1~RHS11),并对这些条带进行回收、克隆、测序。将序列在NCBI数据库中进行比对,其中RHS1、RHS2、RHS3、RHS4、RHS6、RHS8、RHS9在NCBI数据库中未发现与其同源的序列。RHS5、RHS7、RHS10、RHS11在NCBI数据库中有相似序列,其最高覆盖度和最高相似性分别为:83%和100%、99%和85%、49%和100%、19%和83%。通过Southern blotting进行序列特异性检测,RHS1、RHS2、RHS4、RHS6和RHS9等5个克隆在10种材料中都没有杂交信号;RHS7、RHS10和RHS11在华山新麦草及其他9种材料中都有弥散分布的杂交信号;RHS3、RHS5和RHS8只在华山新麦草中有杂交信号的序列。这些结果说明RHS3、RHS5、RHS8为华山新麦草基因组特异重复序列。

Screening, Cloning and Southern Blotting Analysis of Specific Repetitive DNA Sequences of Psathyrostachys Huashanica

In order to screen specific repetitive sequences of Psathyrostachys huashanica, 11 specific repetitive DNA fragments of Psathyrostachys huashanica were screened by using 200 RAPD random primers and Triticum dicoccum, Triticum amyleum, Triticum dicoccoides , Triticum turgidum, Triticum araraticum, Triticum zhukovskyi, Triticum spelta, Chinese Spring and Psathyrostachys huashanica as materials. Those fragments were cloned, sequenced, and designed as RHS1～RHS11. Blast analysis of those sequences with those in nucleic acid database of NCBI website revealed that there were no homological sequences of RHS1, RHS2, RHS3, RHS4, RHS6, RHS8, RHS9, and there were homological sequenes for RHS5, RHS7, RHS10 and RHS11 with the coverage and maximum identity of 83% and 100%, 99% and 85%, 49% and 100%, 19% and 83%, respectively. The 11 sequences was further analyzed by Southern Blotting, and the results showed that there were no signals for RHS1, RHS2, RHS4, RHS6 and RHS9 in all 10 materials;And dispersive signals for RHS7, RHS10 and RHS11 were observed in Psathyrostachys huashanica, as well as other 9 materials, only RHS3, RHS5 and RHS8 had specific signals in Psathyrostachys huashanica. Therefore, RHS3, RHS5 and RHS8 were the specific repetitive sequences of genome of Psathyrostachys huashanica.