豆梨中谷胱甘肽还原酶基因的分离、表达特点及酶活性分析

Isolation,expression of glutathione reductase genes and analysis of the enzyme activity in Pyrus calleryana

【目的】测定谷胱甘肽还原酶活性变化,结合其催化产物含量、编码基因表达特点分析,明确该酶是否参与豆梨应对镉胁迫的调控过程。【方法】采用紫外/可见分光光度法检测谷胱甘肽还原酶(glutathione reductase,GR)活性、谷胱甘肽池组成及过氧化氢(hydrogen peroxide,H_2O_2)含量,RT-PCR和PCR克隆PcGRchl和PcGRcyt的cDNA和DNA序列,利用生物信息学方法进行序列比较分析,荧光定量PCR检测它们在镉胁迫下转录水平变化。【结果】镉胁迫情况下,豆梨叶片GR活性上升,谷胱甘肽池中总谷胱甘肽(total glutathione,T-GSH)和还原型谷胱甘肽(reduced glutathione,GSH)减少、氧化型谷胱甘肽(oxidized glutathione,GSSG)上升,H_2O_2积累增加。豆梨叶绿体PcGRchl和胞质PcGRcyt的序列长度、基因结构及所编码蛋白特征各不相同,它们在豆梨叶片中的表达量上调以响应镉胁迫信号,以PcGRchl的转录占主导。外源GSH预处理有助于豆梨预先储存GSH,减缓镉胁迫下H_2O_2的积累,与Cd(2 mmol·L^(-1)CdCl2·2.5H_2O+Hoagland营养液)组相比较,GC(2 mmol·L^(-1),GSH预处理12 h转入2 mmol·L^(-1)CdCl2·2.5H_2O+Hoagland营养液)组PcGRchl和PcGRcyt的表达水平没有太大变化,但GR活性部分受抑制;外源BSO预处理抑制植株叶片GSH合成,镉胁迫下BC(2 mmol·L^(-1)丁硫氨酸-亚砜亚胺预处理12 h转入2 mmol·L^(-1)CdCl2·2.5H_2O+Hoagland营养液)组H_2O_2产生加剧,GR活性上升幅度变大,但PcGRchl和PcGRcyt的转录不受影响。上述结果表明,GSH预处理或BSO预处理,可改变豆梨叶片GSH池的组成,从而在蛋白质水平上反馈调节镉胁迫情况下GR活性。【结论】豆梨GR参与应对镉胁迫的调控过程。镉处理后,豆梨叶片中GSH减少,促使GR酶活性上升,以补充植株应对逆境所需GSH,这一过程主要通过叶绿体PcGRchl的转录上调来实现;GSH或BSO预处理,改变植株GSH含量,从而影响GR活性,减缓或加剧镉胁迫下H_2O_2的产生,这一过程主要是在GR基因翻译后的蛋白水平上进行反馈调节。

[Objective]Pyrus calleryana Decne.is widely used as a pear rootstocks in Asia.Our preliminary experiment proved that glutathione played a vital role in protecting the plant from cadmium(Cd)stress.It is well known that glutathione reductase(GR)is an essential enzyme that recycles oxidized glutathione back to the reduced form.However,the GR function during the above process remians unknown.In this paper,the changes of GR activity,the content of its catalytic products and the expression characteristics of its encoded genes were analyzed in order to understand the regulation process of the GR against Cd stress in P.calleryana.[Methods]90-day-old seedlings of P.calleryana were chosen asthe test materials for physiological and molecular detection.Firstly,252 plantlets were randomly classified into four groups.Then,one group grew in the Hoagland solution as the control,and one group grew in the same solution plus 2 mmol·L^-1 CdCl2·2.5H2O as the Cd treatment.The other two groups grew in the Hoagland solution plus 2 mmol·L^-1 L-buthionine sulfoximine(BSO)or 2 mmol·L^-1 reduced glutathione(GSH)for 12 hours,and then they were transferred to the new nutrient solution containing 2 mmol·L^-1 CdCl2· 2.5 H2O as the BC and GC groups,respectively.The plantlets were incubated for 0 h,1 h,3 h,6 h,9 h,12 h and 24 h respectively,the third and fourth leaves from the top of the plantlets were collected and used for analysis.The GR activity,the composition of glutathione pool,and H2O2 content were determined by UV/Vis spectrophotometry.To isolate the GR encoded sequences,chloroplast GR(GRchl)(XM009376341)and cytosolic GR(GRcyt)(XM009356603)genes in the Pyrus×bretschneideri(Chinese white pear)genome database were used as the probes to search the transcriptome database of P.calleryana Cd-treated seedlings.Two transcripts(Pbr009065 and Pbr030956)were identified as their analogue genes.Then,two pairs of specific gene primers were designed for RT-PCR and PCR amplification,and confirmation of the aforementioned was done by sequencing.The compositions of introns and exons in PcGRchl and PcGRcyt genes were analyzed using the Gene Structure Display Server.Moreover,their translated protein sequences were obtained through BioXM 2.6,and then signature motifs from different GR proteins were found out by MEME software.A quantitative PCR(qRT-PCR)assay was performed to analyze the relative mRNA expression levels of PcGRchl and PcGRcyt when the seedlings were furnished with or without GSH/BSO before 2 mmol·L^-1 CdCl2·2.5H2O treatment,respectively.[Results]Under the condition of Cd stress,the activity of GR increased and the GSH pool changed in leaves of P.calleryana.The reduced glutathione(GSH)content decreased,and the oxidized glutathione(GSSG)content increased.The total glutathione(T-GSH)content decreased.In addition,H2O2 was rapidly accumulated.At the same time,two GR genes were isolated from Cd-treated leaves of P.calleryana,which were named chloroplast PcGRchl and cytoplasmic PcGRcyt,respectively.They were different in sequence lengths,gene structures,and encoded protein characteristics.The cDNAs of PcGRchl was 1 680 bp,and the DNA sequence was 3 069 bp,which included 10 exons and 9 introns,encoding a protein containing559 amino acids.Meanwhile,the cDNAs of PcGRcyt was 1 491 bp,and the DNA sequence was 6 538 bp,which included 16 exons and 15 introns,encoding a protein containing 496 amino acids.MEME software analysis showed that PcGRchl and PcGRcyt encoded proteins had all the specific motifs of chloroplast GR and cytoplasmic GR,respectively.Both of them contained the typical pyridine nucleotide-disulfide redox reductase class I activity site,NADP-binding site and GSSG binding site.Compared with PcGRcyt,PcGRchl contained a chloroplast signal peptide(N-terminal amino acid residues at position 1-65).Meanwhile,there was a specific cytosolic GR domain in PcGRcyt(L155 D157 G158 T159 K160).PcGRchl and PcGRcyt expression levels were up-regulated in the leaves once the seedlings suffered from the Cd stress.Moreover,the transcription of PcGRchl was dominant.Exogenous GSH pretreatment facilitated the prestorage of GSH in leaves of P.calleryana and helped to effectively inhibit the accumulation of H2O2 once the plantlets were exposed to 2 mmol·L^-1 CdCl2·2.5H2O.Compared with Cd group,the expression levels of PcGRchl and PcGRcyt did not change much,but GR activity was partially inhibited in leaves of GC group.Exogenous BSO pretreatment inhibited the GSH synthesis in leaves of P.calleryana.After Cd treatment,H2O2 production exacerbated,and the rising range of GR activity increased,but the transcriptions of PcGRchl and PcGRcyt were not affected in leaves of BC group.The above results indicated that GSH pretreatment or BSO pretreatment could regulate GR activity at the protein level through changing the composition of the GSH pool in the leaves of P.calleryana under Cd stress.[Conclusion]GR may positively take part in the regulation process in the leaves of P.calleryana against Cd stress.After Cd treatment,the GSH content in the leaves of P.calleryana decreased,which compensated for the need of the plants to cope with adversity via promoting GR activity.This process was mainly achieved through the transcriptional up-regulation of chloroplast PcGRch1.Pretreatment with GSH or BSO changed the GSH content in the leaves of the plant,thereby affecting GR activity,slowing or exacerbating H2O2 production under Cd stress.This process was mainly regulated at the protein level after GR gene translation.