梨矮化砧木‘中矮1号’生长素氢转运体基因PcAHS及其启动子的克隆与表达分析

Isolation and Analysis of Pc AHS Gene and Its Promoter from Dwarfing Rootstock ‘Zhongai 1' Pear

以梨（Pyrus communis L.）紧凑型矮化砧木‘中矮1号’及其母本‘锦香’新梢韧皮部为试材,根据转录组测序结果设计特异性引物,克隆到1个长1 239 bp的基因序列。该序列在两个品种间不存在差异,均编码412个氨基酸,其氨基酸序列与苹果（np001280772.1）、梅（xp008242099.1）、毛果杨（xp002306421.2）、草莓（xp00428771.1）的生长素氢转运体基因（AHS）编码的氨基酸序列的相似性在67%99%之间,命名为Pc AHS。q RT-PCR分析发现,‘中矮1号’新梢韧皮部中Pc AHS的表达量均低于母本‘锦香’,推测其启动子序列存在差异。‘中矮1号’及其母本‘锦香’Pc AHS基因上游启动子长度分别为828 bp和888 bp,二者相似性89.1%。序列分析发现‘中矮1号’Pc AHS基因启动子有一段58 bp（–496bp–553 bp）的缺失;利用植物顺式作用元件数据库PLACE和PLANTCARE分析表明,‘中矮1号’启动子含有一个‘锦香’没有的BPBF转录因子结合元件P-box。推测‘中矮1号’Pc AHS基因启动子特有的片段缺失和P-box转录因子结合元件可能是导致其表达量低并通过影响生长素的运输最终引起矮化的原因。

‘Zhongai 1'is a pear dwarfing rootstock selected from the open pollinated seedlings of ‘Jinxiang'. One gene with 1 239 bp was obtained from the new shoot phloem of‘Zhongai 1'and ‘Jinxiang'and it was designated as Pc AHS. Pc AHS had no difference in sequence between the two varieties and it encoded 412 amino acids which had a sequence identity from 67% to 99% with the AHS gene in Malus × domestica(np_001280772.1),Prunus mume(XP_008242099. 1),Populous trichocarpa(XP_002306421.2)and Fragaria vesca(xp_00428771.1). The expression of Pc AHS gene in new shoot phloem of‘Zhongai 1'was lower than that in its female parent‘Jinxiang'during the shoot growing stage,so we speculated that there may be difference in their promoter sequences. The promoter sequences of Pc AHS gene isolated from‘Zhongai 1'and‘Jinxiang'had 828 bp and 888 bp respectively and the sequence identity was 89.1%. Sequence analysis showed that there is a deletion fragment of 58 bp(–496 bp to –553 bp)from Pc AHS gene promoter sequence in‘Zhongai 1'. According to PLACE and PLANTCARE online database analysis,Pc AHS gene promoter sequence of‘Zhongai 1'contained a particular BPBF transcription factor binding element P-box which was absent in‘Jinxiang'. So we speculated that fragment deletion and the particular transcription factor binding element P-box may be the cause of low expression of the gene and then influence auxin transport,eventually lead to dwarf.