园艺学报 ›› 2020, Vol. 47 ›› Issue (2): 355-369.doi: 10.16420/j.issn.0513-353x.2019-0418

• 研究报告 • 上一篇    下一篇

中国樱桃 LTR 类反转录转座子反转录酶序列的克隆和分析

刘厚宇 1,2,3,吴敏芳 1,文晓鹏 1,*   

  1. 1 贵州大学农业生物工程研究院/贵州大学生命科学院,山地植物资源保护与种质创新教育部重点实验室,贵阳550025;2 贵州大学林学院/贵州省森林资源与环境研究中心,贵阳 550025;3 贵州省清镇市农业农村局,贵州清镇551400
  • 出版日期:2020-02-25 发布日期:2020-02-25
  • 基金资助:

Cloning and Analysis of LTR Reverse Transcriptase from Chinese Cherry (Prunus pseudocerasus Lindl.)

LIU Houyu1,2,3,WU Minfang1,and WEN Xiaopeng 1,*   

  1. 1The Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education),Institute of Agro-bioengineering and College of Life Sciences,Guizhou University,Guiyang 550025,China; 2Research Center for Fortest Resources and Environment of Guizhou Province,College of Forestry,Guizhou University,Guiyang 550025,China;3Agricultural and Rural Affairs Bureau of Qingzhen,Qingzhen,Guizhou 551400,China
  • Online:2020-02-25 Published:2020-02-25

摘要: 利用同源序列法根据 Ty1-copia 和 Ty3-gypsy 类反转录转座子反转录酶保守区设计简并引物,从中国樱桃(Prunus pseudocerasus Lindl.)中分别扩增出约 260 和 430 bp 的目标片段,并进行克隆、测序、序列分析及转录活性检测。共获得 44 条 Ty1-copia 类反转录酶序列,13 条 Ty3-gypsy 类反转录酶序列,序列间显示高度异质性,部分序列存在缺失、终止密码子及移码突变。系统发育树显示 Ty1-copia 可以分为 TAR、Ale 两类,Ty3-gypsy 可以分为 Tat、Reina、Tekay 等 3 类。Ty1-copia 和 Ty3-gypsy 类反转录酶 dN/dS值均小于 1,其中 Ty3-gypsy 高于 Ty1-copia。13 条 Ty1-copia 以及 7 条 Ty3-gypsy 反转录酶序列在樱桃正常生长条件下均有转录活性,但不受 8 种非生物胁迫的诱导。PpRT7 在高温胁迫 24 h 时转录活性升高,并且具有组织特异性。

关键词: 樱桃, 中国樱桃, Ty1-copia, Ty3-gypsy, 反转录酶, 序列分析, 转录活性

Abstract: In order to provide fundamental clue for genetic evolution and variation mechanism,the reverse transcriptase gene fragments of Ty1-copia and Ty3-gypsy were isolated and sequenced using degenerate oligonucleotide primers from genomic DNA of Chinese cherry(Prunus pseudocerasus Lindl.). And the transcriptional activity of retrotransposon were detected by reverse transcription polymerase chain reaction(RT-PCR). The results showed that a total of 44 unique clones from Ty1-copia and 13 unique clones from Ty3-gypsy were obtained. Nucleotide sequence of Ty1-copia and Ty3-gypsy ranged in length from 262 to 269 bp and 414 to 435 bp,respectively. Alignment of nucleotide and amino acid sequences showed the high heterogeneity in LTR retrotransposon. Both Ty1-copia and Ty3-gypsy groups displayed mutation including premature stop codons,frameshift mutation and deletion. Phylogenetic analysis showed there were two lineages(TAR and Ale)in Ty1-copia group,and three lineages(Tat,Tekay,Reina)in Ty3-gypsy group. Furthermore,ratios of non-synonymous to synonymous(dN/dS)of Ty1-copia and Ty3-gypsy were less than 1,and Ty3-gypsy were greater than those of Ty1-copia,suggesting that they were evolving under a subfunctionalization model driven by purifying selection. Transcriptional activity was detected in 13 Ty1-copia and 7 Ty3-gypsy RT sequences and didn’t change as exposure to eight types of abiotic stresses. A Ty1-copia RT sequence named PpRT7 was highly transcriptionally activated by high temperature(42 ℃),and showed tissue specific expression.

Key words: Prunus pseudocerasus Lindl., Ty1-copia, Ty3-gypsy, reverse transcriptase, sequence analysis, transcriptional activity