果树学报2018，Vol.35Issue(2)：137-146,10.DOI:10.13925/j.cnki.gsxb.20170302
杜梨PbNHX1基因的克隆、表达分析及功能验证
Cloning, expression and functional analysis of PbNHX1 gene in Pyrus betulaefolia
摘要
Abstract
[Objective] Pyrus betulaefolia is one of the main rootstocks for pear P.betulaefolia as a rootstock can effectively reduce Na+ accumulation in the root,inhibit the Na+ transportation to the scion,and improve the salt tolerance of pear varieties.In order to reveal the molecular mechanism of salt tolerance and provide experimental basis for the further research,we researched the Na+/H+ antiporter protein in P.betulaefolia.One PbNHX1 gene was cloned from this species and its sequence characteristics and expression characteristics were analyzed.[Methods] The sodium/hydrogen exchanger 1-like gene (XM_ 018651314.1) in Pyrus×bretschneideri (Chinese white pear) was used as the electronic probe to search the transcriptome database of P.betulaefolia NaCl-treated seedlings.Then,one transcript Pbr017120 was obtained for designing gene-specific primers.Indeed,its cDNA and DNA sequences were isolated using RT-PCR and PCR techniques.And the physical and chemical properties of PbNHX1 protein were analyzed by Protparam online software.The introns and exons of PbNHX1 genes were analyzed using Gene Structure Display Server.The signature motifs were analyzed by MEME software and the phylogenetic tree was built by MEGA 6.0.The expression condition of PbNHX1 under the abiotic stresses,such as 200 mmol· L-1 NaCl,10％ (φ) PEG6000 and 100 μmol·L-1 ABA,were analyzed by quantitative PCR (qRT-PCR).Finally,yeast complementary experiments with a salt sensitive yeast mutant AXT3 were performed to verify the functions of PbNHX1 gene.After solid and liquid cultivation,the growth status of transgenic yeast was detected.Their total Na+ and K+ contents were also tested by a flame-graphite furnace atomic absorption spectrometer.[Results] The cDNAs of PbNHX1 was 1 704 bp,and its DNA was 3 594 bp,which included 13 exons and 12 introns.This gene encoded a protein containing 567 amino acids,its relative molecular weight and isoelectric point (pI) was 62.179 ku and 5.55,respectively.Moreover,the PbN-HX1 elements consisted of C2861H4437N669O805S21.The phylogenetic tree showed that PbNHX1 was located in the branch of vacuolar Na+/H+ antiporter,which was far from the plasma membrane Na+/H+ antiporter gene of Arabidopsis AtNHX7 or rice OsNHX8,and was closely related to poplar vacuolar Na+/H+ antiporter gene PtNHX1.3 or Arabidopsis AtNHX2.The expression level of the PbNHX1 was higher in the leaves than that in its roots under normal growth conditions.After treatment with 200 mmol· L-1 NaCl,PbNHX1 transcriptional level obviously increased both in the roots and in the leaves.For example,PbNHX1 expression level increased firstly and then decreased in the roots once the seedlings were treated with salt.Its expression peak appeared at 6 h.At that time,the amount of PbNHX1 transcription was 2.4 times higher than that of the control.Then,its expression level began to decrease closely to the original level at 24 h.On the one hand,the expression level of PbNHX 1 kept on rising in the leaves after the treatment of salt.In the case of 10％ PEG6000,PbNHX1 expression level increased firstly and then decreased in the roots.Its expression peak appeared at 3 h,which was 1.2 times higher than that of the control.After that time,its expression level turned to fall down and recovered closely to the original level at 24 h.In the leaves,the expression level of PbNHX1 continued to rise when the salt existed and it was 22.6 times higher than that of the control at 24 h.After 100 μmol· L-1 ABA treatment,PbNHX1 expression level increased firstly and then decreased in the roots.Its expression peak appeared at 6 h,which was 1.6 times higher than that of the control.Then its transcription declined closely to the original level at 24 h.In the leaves,the expression level of PbNHX1 increased during the treatment period,which was 8.0 times higher than that of the control at 24 h.These results indicated that PbNHX1 was regulated significantly in the leaves under different abiotic stress.The results of YPD solid culture showed that transform of PbNHX1 could recover the growth inhibition of NaCl,KCl and hygromycin B to the nhx1 mutant yeast strain AXT3 when the cells were treated with 20 mg· L-1 hygromycin B,20-50 mmol· L-1 NaCl or 0.5-1.00 mol· L-1 KCl.Meanwhile,the results of liquid culture showed that transform of PbNHX1 reduced the growth inhibition of the mutant strain AXT3 to NaCl and KCl when the cells were treated with 50-100 mmol· L-1 NaCl or 0.5-1.0 mol· L-1 KCl.Furthermore,the contents of Na+ and K+ significantly increased in PbNHX1 transgenic yeast cells compared with the mutant strain without this gene when 20 mmol· L-1 NaCl presence.[Conclusion] Our results have showed that PbNHX1 gene belong to the NHX gene family of P.betulaefoli,which has the inherent characteristics of plant NHXs family.This gene response to NaCl,osmotic and ABA stresses.Transfer of the PbNHX1 gene can increase the salt tolerance of the nhx1 mutant yeast strain AXT3 and partly recover its ion transport capacity and facilitate the accumulation of the Na+ and K+ ions.关键词
杜梨/PbNHX1基因/序列特征/表达特点/酵母互补Key words
Pyrus betulaefolia/PbNHX1 gene/Sequence characteristics/Expression feature/Yeast complementation分类
农业科技引用本文复制引用
刘威,李慧,蔺经,杨青松,常有宏..杜梨PbNHX1基因的克隆、表达分析及功能验证[J].果树学报,2018,35(2):137-146,10.基金项目
国家自然科学基金(31372051) （31372051）
