作 者 ：魏丽娟;张娟*;刘林德;王蕾;赵同欣
期 刊 ：植物研究 2014年 34卷 5期 页码：687-693
Keywords:molecular markers, sex differentiation, RAPD, SCAR,
摘 要 ：南蛇藤（Celastrus orbiculatus Thunb）属卫矛科南蛇藤属落叶藤本植物，是中国传统中药材，雌雄异株，少量雄全同株。由于目前在分子水平上对南蛇藤性别差异的研究较少，极大地限制了对其的开发利用。本研究利用RAPD分子标记对南蛇藤雌株、雄株和雄全同株进行了差异比较。100个引物中有5个引物（S127、S140、S148、S174及S111）在不同性别南蛇藤的基因组DNA中扩增到存在明显差异的条带。根据序列分析的结果将RAPD引物转化成特异性较强的SCAR引物后，仅引物S111扩增出一条雌性特异性条带。序列分析发现，该片段包含两个超过100个氨基酸的开放阅读框，其功能有待进一步的研究。
Abstract:Celastrus orbiculatus Thunb. belongs to Celastrus L. of the family Celastraceae. As a kind of traditional Chinese herbal medicine, it has great ornamental value and important medicinal value. It is a dioecious vine, a small amount of them are andromonoecy plants. However, the morphological differences between the male and female plants are not obvious before the maturation of their sex organs, which restricts the early cultivation and breeding. Currently, molecular markers have been widely used in the sex identification of dioecious plants. But the research on the sex differentiation of C.orbiculatus at the molecular level has not been reported. Due to unknown genome sequence, many studies on C.orbiculatus are limited in the field of chemistry and cultivation technology, which greatly limits its utilization. In this study, differentiation among dioecious plants was compared using random amplified polymorphic DNA (RAPD) markers. It is the first time to use the RAPD markers on the study of C.orbiculatus. Using the young leaves of male and female plants as materials, we extracted the DNA using the improved CTAB method. Then through screening of different factors, we established the optimized PCR system. Based on the above PCR system, we selected 100 RAPD primers to conduct the analysis. Significantly different bands were amplified using primer S127, S140, S148, S174 and S111, respectively. These different bands were cloned into the T-vector, and then sequenced. Based on the sequences, the specific SCAR markers were obtained. Only one female-associated dominant SCAR marker from RAPD marker S111 was verified. Sequence analysis showed that this female specific fragment contained nine open reading frames (ORF) between 93-570 bp in length, among which two have more than 100 amino acids. The results of sequence Blast showed that the polypeptide fragment had a higher similarity with plants reverse transcription factor of the gag-POL protein, which belongs to UBN2 super family. We inferred a possible reason of the gender traits is that the insertion of the reversal seat factor may inhibit some important sex-determining factors. In the future, we need to get their full-length sequences using RACE, and then study their function on sex differentiation.