苎麻ACC氧化酶基因(BnACO1)的克隆及表达

Cloning and Characterization of ACC Oxidase Gene (BnACO1) from Ramie (Boehmeria nivea)

【目的】克隆苎麻中的一个ACO基因(BnACO1),并对其进行生物信息学分析、表达分析、胁迫应答分析。【方法】通过RT-PCR结合RACE技术从湘苎3号中克隆该基因的全长cDNA序列,利用生物信息学软件对获得的基因序列及编码的蛋白质序列进行分析。利用实时荧光定量PCR分析BnACO1在苎麻不同部位的表达量以及在ABA、干旱和高盐胁迫下的应答。【结果】BnACO1的cDNA序列全长为1 476 bp,其开放阅读框为981 bp,编码326个氨基酸多肽,预测其分子量和等电点分别为36.81 kD和4.95,基因登录号为JX878855。与无花果(AB307720)、香叶天竺葵(U19856)、柿树(AB073009)、梨(JN807390)、猕猴桃(HQ293208)的ACO基因核苷酸序列相似性分别为83%、81%、79%、79%和79%,氨基酸序列的相似性分别为87%、80%、80%、78%和83%。实时荧光定量PCR分析表明苎麻BnACO1在苎麻根、茎、茎尖、叶片、雌花和雄花中均有表达,其中,在雄花中表达最高,在根、茎中表达最低,且该基因受ABA、干旱和NaCl胁迫诱导表达。【结论】获得的BnACO1是ACO家族成员之一,参与ABA、干旱、NaCl胁迫应答,该基因可能在苎麻性别分化中起到一定作用并且在调控苎麻生长发育和应对环境胁迫时具有独特的表达模式。

An 1-Aminocyclopropane-1-Carboxylic acid Oxidase gene（BnACO1） was cloned from ramie and analyzed by bioinformatics method.The expression pattern was revealed in different organs and under stresses.【Method】The full-length sequence of ACO gene cDNA was cloned from Xiangzhu 3 ramie by RT-PCR and RACE methods.Then the sequence of BnACO1 gene and protein sequences were analyzed through bioinformatics means.The relative expression level of BnACO1gene in various organs of ramie and under ABA,drought,NaCl stresses was examined by real-time fluorescence PCR.【Result】The full length sequence and the ORF of the BnACO1 gene were 1 476 bp and 981 bp,respectively,which encoded 326 amino acids.The molecular weight is 36.81 kD and the pI is 4.95.The similarity comparison revealed that the gene nucleotide sequence shared 83%,81%,79%,79%,and 79% of homology with the Ficus carica（AB307720）,Pelargonium（U19856）,Diospyros kaki（AB073009）,Pyrus pyrifolia（JN807390）,Actinidia chinensis（HQ293208） ACO gene,and the similarity of the amino acid sequences with that of these species was 87%,80%,80%,78%,and 83%,respectively.The results of semi-quantitative RT-PCR showed that the BnACO1 expressed in root,stem,shoot tip and blade,female flowers and male flowers,with the higher expression level in male flowers,while the lowest expression level in root and stem.The results of real-time PCR showed that the BnACO1 was induced by ABA,dehydration and NaCl.【Conclusion】BnACO1 belongs to ACO gene family,which involved in abiotic stress responses to ABA,drought and NaCl.It may play a role in gender differences of ramie and have distinctive expression pattern in the regulation of development and stress responses.