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研究以Ty1-copia类反转录转座子反转录酶区域的简并引物,对100μmol·L-1脱落酸(ABA)处理的铁皮石斛进行反转录PCR(RT-PCR)扩增,得到260 bp左右的目的条带,说明100μmol·L-1的ABA能诱导Ty1-copia类反转录转座子转录活性的表达。将目的条带回收、克隆和测序后分析得到42条Ty1-copia类反转录转座子反转录酶的保守序列,该序列变异性较大,核苷酸序列长度247~266 bp,同源性为46.3%~98.9%。经plantCARE软件分析发现与铁皮石斛基因组所分离的Ty1-copia类反转录转座子一样,存在多个受胁迫条件作用的调控元件、多个启动子的特征结构TATA box和CAAT box的保守序列及其他一些调控元件;但有转录活性的铁皮石斛Tyl-copia RT序列中与胁迫条件作用相关的调控元件明显增加。翻译成氨基酸后,有15条序列出现不同程度的终止密码子突变,19条序列出现移码突变,保守序列“SLYGKQ”全部发生不同程度的突变。其氨基酸序列经过系统聚类后可分为5类;且与基因组Tyl-copia RT序列聚类后发现多数发生转录的Tyl组反转录转座子RT序列与基因组Tyl组反转录转座子RT序列亲缘关系较远,说明经ABA诱导后发生转录的Tyl组反转录转座子RT序列与基因组Tyl组反转录转座子RT序列有很大的差异。
In this study, degenerate primers of Ty1-copia retrotransposon reverse transcriptase region were amplified by RT-PCR from 100 μmol·L -1 abscisic acid (ABA) The result showed that the ABA of 100μmol·L-1 induced the transcriptional activity of Ty1-copia retrotransposon. The target bands were recovered, cloned and sequenced to obtain the conserved sequences of 42 Ty1-copia retrotransposon reverse transcriptases. The nucleotide sequence of the Ty1-copia transcriptase transcriptase was 247-266 bp in length, The source was 46.3% ~ 98.9%. The results of plantCARE software showed that there were many regulatory elements under stress conditions, like the Ty1-copia retrotransposon isolated from Dendrobium officinale genome, the conserved sequences of the TATA box and CAAT box of multiple promoters And other regulatory elements; however, transcriptional activity of Tyl-copia RT sequences in response to stress conditions significantly increased regulatory elements. After translated into amino acids, 15 sequences showed varying degrees of stop codon mutations, 19 sequences showed frameshift mutations, and the conserved sequence “SLYGKQ” all had different degrees of mutations. After clustering with the Tyl-copia RT sequence, most of the transcripts of the Tyl group RT transcripts and the Tyl group RT transposons RT sequence is far genetic relationship, indicating that there is a great difference between the Tyl group RT transposon RT transcripts induced by ABA and the Tyl group RT transposon RT sequences.