摘要
【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�
[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.Th
作者
李慧
阚家亮
王影
蔺经
杨青松
常有宏
LI Hui;KAN Jialiang;WANG Ying;L1N Jing;YANG Qingsong;CHANG Youhong(Institute of Pomology,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,Jiangsu,China;Jiangsu Key Laboratory for Horti- cultural Crop Genetic Improvement,Nanjing 210014,Jiangsu,China;College of Horticulture,Nanjing Agricultural University,Nanjing 210095,Jiangsu,China)
出处
《果树学报》
CAS
CSCD
北大核心
2019年第1期11-20,共10页
Journal of Fruit Science
基金
江苏省自然科学基金(BK20151361)
关键词
豆梨
镉胁迫
谷胱甘肽还原酶
活性调节
表达特点
Pyrus calleryana
Cadmium stress
Glutathione reductase
Activity regulation
Expression characteristic
