水稻愈伤组织端粒酶催化特征检测

The Detection of Rice Callus Telomerase Catalytic Characteristics

端粒是位于染色体末端富含鸟嘌呤的双链DNA结构。端粒酶以自身的一段RNA为模板,按照碱基互补配对的方式产生端粒重复序列以延伸端粒长度,维持染色体的稳定。以水稻愈伤组织为实验材料,分离、提取端粒酶,并以不同3’端的寡核苷酸作为前导引物对体外水稻端粒酶催化特征及不同引物对酶活性的影响进行了探究。结果显示:S水稻愈伤组织端粒酶提取液在延伸前导引物时,以催化合成的TAGGGTT末端百分比含量最高,表明在催化延伸寡核苷酸的过程中,倾向于优先催化合成TAGGGTT端粒重复序列。尤其在前导引物3’末端为TT碱基和GT碱基最突出,其次是TA碱基,而AG碱基和GG碱基不明显,推测水稻端粒酶模板RNA与前导引物的结合位点为一个碱基,偏好性为T>A>G。这些特征、特性对尚未克隆的水稻端粒酶RNA,以及对深入研究端粒末端结构及染色体遗传稳定性具有重要意义。

Telomere is the G-rich double strand DNA structure that lies at the end of chromosome. Telomerase elongates telomere length by synthesizing telomeric repeats according to its template RNA and stabilizes chromosome structure. In this research, rice callus telomerase is isolated and extracted. In addition, leading primers, some oligonucleotides which are different in their 3 termini, are used to detect telomerase catalytic characteristics and the efficacy of different oligonucleotides during telomerase elongation. The results show that the highest percentage of synthesized telomeric repeats belongs to TAGGGTT during rice callus telomerase elongating the leading primers in vitro, demonstrating rice callus telomerase preferentially synthesizes TAGGGTT telomeric repeat during elongating leading primers. Telomerase preferentially elongates primers ended with TT and GT in their 3＇ end, then TA followed by primers ended with AG and GG in the 3＇ end. It is speculated that the binding site for telomerase template RNA and the leading primers is one base pair, with a preferentiality of T〉A〉G. This research is of prime significance for further understanding telomere end structure and chromosome genetic stability.