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植物研究 ›› 2020, Vol. 40 ›› Issue (5): 648-658.doi: 10.7525/j.issn.1673-5102.2020.05.002

• 研究报告 • 上一篇    

NBA1/MERIT40在3种沙棘中的适应性分化研究——兼论二代测序技术在杂种鉴定中的应用

孙坤, 丁雪洋, 张辉, 李雪丽, 汪颖, 王娟, 刘本立   

  1. 西北师范大学生命科学学院, 兰州 730070
  • 收稿日期:2019-11-20 发布日期:2020-07-10
  • 通讯作者: 张辉,E-mail:zhanghui@nwnu.edu.cn E-mail:zhanghui@nwnu.edu.cn
  • 作者简介:孙坤(1965-),男,博士生导师,主要从事系统与进化植物学方面的研究。
  • 基金资助:
    国家自然科学基金项目(30660060)

Adaptive Differentiation of NBA1/MERIT40 of Three Species of Hippophae L.——Discussion on the Application of Next-generation Sequencing Technology in Hybrid Identification

SUN Kun, DING Xue-Yang, ZHANG Hui, LI Xue-Li, WANG Ying, WANG Juan, LIU Ben-Li   

  1. College of Life Science, Northwest Normal University, Lanzhou 730070
  • Received:2019-11-20 Published:2020-07-10
  • Supported by:
    Under the auspices of the National Natural Science Foundation of China(30660060)

摘要: 随着高通量测序技术的发展,从"组学"水平研究适应性进化分子机制成为进化生物学的研究热点。本研究以沙棘属的中国沙棘、肋果沙棘及二者的同倍化杂交物种棱果沙棘为材料,通过转录组测序,筛选出了受到正选择(w>1)的BRCA1-A&BRISC复合体亚基NBA1基因。生物信息学分析表明,NBA1基因在3种沙棘中编码区全长为771 bp,共编码256个氨基酸,为核定位的亲水性蛋白。中国沙棘与肋果沙棘中NBA1的氨基酸序列在218与236位存在差异,中国沙棘中靠近结合位点的218位突变为甲硫氨酸,不同于肋果沙棘及其他陆生植物中NBA1亚基VWA结构域中保守的亮氨酸,导致二者的蛋白质三级结构发生明显变化,且使中国沙棘与肋果沙棘中该蛋白结合位点的空间排布产生显著差异。综上,推测二者的NBA1亚基在结合其他亚基方面有所不同,主要导致了BRCA1-A复合体修复紫外辐射引起的DNA损伤修复功能方面出现差异,这可能与中国沙棘与肋果沙棘对不同海拔环境的适应有关。此外,Sanger测序验证了二代测序结果的准确性,证明该基因在亲本种中国沙棘与肋果沙棘中是纯合的单拷贝,在杂种棱果沙棘中以共显性的杂合方式存在。进一步分析发现第二代测序数据不适用于在个体水平鉴定杂种,但群体内多个个体的平行测序一定程度上可随机反映杂种的亲本来源。本研究为进一步揭示沙棘属植物对不同海拔的适应分子机制提供了参考数据,同时为利用转录组测序方法鉴定杂交物种提出了建议。

关键词: NBA1, 中国沙棘, 肋果沙棘, 棱果沙棘, 适应性分化, 杂种鉴定

Abstract: With the development of high-throughput sequencing technology, the study of molecular mechanisms of adaptive evolution in the "Omics" level has become a hotspot of evolutionary research. In this study, transcriptome sequencing was carried out using materials of Hippophae rhamnoides ssp. sinensis, H.neurocarpa and H.goniocarpa that is a hybrid species from homoploid hybridization between the former two species, and the gene of subunit NBA1 of BRA1-A&BRISC complex was selected to deeply study on the basis of its positive selection(w>1). Bioinformatics analysis found that the coding region of NBA1 gene was 771 bp in three sea buckthorns in length, which was a nuclear localized hydrophilic protein that encoding a total of 256 amino acids. The amino acid sequence of NBA1 of H.rhamnoides ssp. sinensis and H.neurocarpa have different loci which is loci of 218 and 236. The site of 218 which close to ligand binding sites in H.rhamnoides ssp. sinensis is different from the VWA domain of H.neurocarpa and terrestrial plants, and it has been mutated from conserved leucine to methionine. This resulted in a significant change in the protein tertiary structure of H.rhamnoides ssp. sinensis and H.neurocarpa, and makes the protein binding sites have significant difference in spatial structure. Above all, we speculated that the NBA1 subunits differ in binding to another subunit. Mainly led to the difference in the BRCA1-A complex repair DNA damage repair function that caused by UV radiation, and it may be related to the adaptation of H.rhamnoides ssp. sinensis and H.neurocarpa to different elevations. In addition, Sanger sequencing has verified the accuracy of the next generation sequencing results, and has proved that is a homozygous gene of single copy in two parental species H.rhamnoides ssp. sinensis and H.neurocarpa, and exists in codominant allele way in H.goniocarpa. Further analysis found that the next generation sequencing data is not suitable for identifying hybrids at the level of individual, but it can reflect the parental source of the hybrid randomly to some extent if we parallel sequencing in multiple individuals within the populations. This study provides a reference data for further revealing the molecular mechanism of adaptation of Hippophae L. to different altitudes, and proposes advice in choose transcriptome sequencing methods to identify hybrid species.

Key words: NBA1, Hippophae rhamnoides ssp. sinensis, H.neurocarpa, H.goniocarpa, adaptive differentiation, hybrid identification

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