草莓miR156靶基因SPL9的克隆与表达分析

Cloning and Expression Analysis of miR156-Targeted SPL9 Gene from Strawberry

【目的】从草莓中克隆SPL(SQUAMOSA promoter binding protein-like)转录因子基因SPL9,并分析其在草莓植株不同生长时期的表达水平及与miR156的表达关系,探讨SPL9在草莓植株生长发育中的作用。【方法】根据苹果SPL基因家族的保守序列设计简并引物,以草莓叶片为试材,利用RT-PCR克隆草莓SPL9基因片段,在此基础上利用RACE方法克隆SPL9的cDNA全长。利用实时定量RT-PCR技术分析草莓叶片中SPL9及miR156的表达水平。【结果】从草莓(Fragaria×ananassa)品种‘全明星’中克隆出SPL9基因的cDNA全长序列,命名为FaSPL9。其CDS长度为1 143 bp,编码381个氨基酸,与拟南芥AtSPL9、葡萄VvSPL9、枳PtSPL9、苹果MdSPL9以及玉米ZmSPL9的氨基酸序列同源性分别为:40.30%、64.57%、56.09%、70.33%,38.35%。FaSPL9有着高度保守的SBP结构域和一个双向核定位信号KRXXXRRRK。FaSPL9基因序列中包含miR156的靶位点,实时定量RT-PCR结果表明,在草莓植株的不同生长时期FaSPL9的表达量不同,且与miR156呈现相反的表达模式。【结论】从草莓中分离出FaSPL9基因,其作为miR156的靶基因,推测FaSPL9对草莓植株的生长发育具有重要的调控作用。

【Objective】The aim of this study was to clone the full length cDNA of the gene encoding SQUAMOSA promoter binding protein-like 9（SPL9） from strawberry,to analyze its expression level at different growth stages of strawberry and the expression relationship between SPL9 and miR156,and preliminarily to elucidate the role of SPL9 gene in strawberry plant development.【Method】The fragment of SPL9 gene was amplified from strawberry leaves by RT-PCR with the degenerate primers designed according to the sequences of Malus SPL genes.The full-length cDNA of SPL9 gene was obtained with RACE.Real time RT-PCR was used to analyze the expression levels of SPL9 and miR156 in strawberry leaves.【Result】The full-length cDNA sequence of SPL9 was cloned from strawberry（Fragaria×ananassa） cultivar ＇Allstar＇ and the gene product was designated as Fragaria×ananassa SPL9（FaSPL9）.The CDS length of FaSPL9 is 1143bp that encoded a predicted protein of 381 amino acid residues.The amino acid identity compared with Arabidopsis thaliana AtSPL9,Vitis vinifera VvSPL9,Poncirus trifoliate PtSPL9,Malus domestica MdSPL9 and Zea mays ZmSPL9 was 40.30%,64.57%,56.09%,70.33%,and 38.35%,respectively.There are highly conserved SBP structure domain and two-way nuclear localization signal KRXXXRRRK in FaSPL9.The nucleotide sequence of FaSPL9 includes the target site of miR156.The real time RT-PCR result showed that the expression levels of FaSPL9 changed at the different development stages of strawberry plants,and the expression of FaSPL9 was strikingly complementary with the expression of miR156.【Conclusion】FaSPL9 gene was cloned from strawberry.As the target gene of miR156,FaSPL9 may play an important role in regulating the growth and development of strawberry plants.