Objective Telomere genes play an important role in the process of plant tissue senescence and differentiation. In this study, the telomere genes CcTBP1 and CcPOT1 of ancient Cinnamomum camphora were cloned and analyzed by bioinformatics, subcellular localization and detection of their expression level in ancient C. camphora and its rejuvenating cuttings, to preliminarily reveal the function of telomere genes in the process of adventitious root formation of ancient C. camphora rejuvenating cuttings, and lay a foundation for exploring the telomere regulation mechanism of ancient C. camphora rejuvenating cuttings.
Method In this study, leaf RNA was extracted from 1200 years old ancient C. camphora. The telomere genes CcTBP1 and CcPOT1 were cloned by PCR and homologous cloning, as well the bioinformatics analysis was conducted to their sequences. The over-expression vector was constructed for subcellular localization analysis. The CcTBP1 and CcPOT1 expression level of different tissues and adventitious root formation process was analyzed by qRT-PCR in ancient C. camphora and its rejuvenation cutting seedlings.
Result (1) Two telomere genes were cloned, named CcTBP1 and CcPOT1, respectively. The CDS sequence of CcTBP1 gene was 2 163 bp and encoded 720 amino acids. The CDS sequence of CcPOT1 gene was 1 407 bp and encoded 468 amino acids. (2) The results of sequence similarity and phylogenetic tree analysis showed that the sequence similarity of CcTBP1 protein and Cinnamomum kanehirae RWR76720.1 was 98%, and their genetic relationship was the closest; the sequence similarity of CcPOT1 protein and Cinnamomum kanehirae RWR96964.1 was as high as 99%, and their genetic relationship was the closest. (3) Subcellular localization analysis showed that CcTBP1 protein was located in the nucleus, and CcPOT1 protein was located in the cytoplasm and nucleus. (4) The qRT-PCR expression analysis in root, stem and leaf showed that the expression level of CcTBP1 gene was highest in root of ancient C. camphora, the expression level of CcPOT1 gene was highest in root of rejuvenation cutting seedlings. Expression analysis showed CcTBP1 was down-regulated tendency during adventitious root formation process, and the expression level of ancient C. camphora was higher than rejuvenation cutting seedlings. And CcPOT1 gene was up-regulated tendency during adventitious root formation process, and the expression level of ancient C. camphora was lower than rejuvenation cutting seedlings.
Conclusion The telomere genes CcTBP1 and CcPOT1 play an important regulation function in the adventitous root formation process of ancient C. camphora rejuvenation.