Cloning And Functional Analysis Of A Glutaredoxin Gene CpGRX1from Chimonanthus Praecox(L.) Link

Glutaredoxin (Grx), also known as Thioltransferase, is about12KD in the molecular weight. Grx is a special branch of the large thioredoxin superfamily and the structure of Grx is similar to that of thioredoxin(Trx). The active sites of Grx, which exposed to the surface of protein, are generally conservation.The Grx usually has GSH binding site. Grx is stable to heat. Grx, reduced coenzyme Ⅱ (DADPH), glutathione (GSH), glutathione reductase (GR) compose of the glutaredoxin system, which widely exist in bacteria, plants and mammals. According to sequences of the active sites, Grx can be broadly divided into three subclass in plants, including the CxxC/S (subclass Ⅰ, Namely CPYC type), CGFS (subclass Ⅱ, Namely CGFS type) and CCxC/S/G (subclass Ⅲ, Namely CC type). The active sites of subclass Ⅰ is generally CPYC; the active sites of subclass Ⅱ is CGFS; the active sites of subclass Ⅲ is CCxx and subclass Ⅲ only is exists in paints. The antioxidant mechanism of Grx is the exchange resction of thiol (-SH-) and disulfide (-S-S-) of the cysteine residues on the Grx. It maintains and repairs the active of protein or enzyme and regulates the redox state of cellular. Grx gene palys an important role in plant growth and development, antioxindant, and pathogen resitance and so on.In this study, a glutaredoxin gene has been cloned from cDNA library of Chimonanthus praecox. Using the research methods of bioinformatics analysis, quantitative PCR and the overexpression of model plant to analysis the function of CpGRX1gene. The main results are as follows:1. Structure analysis of CpGRX1sequence and encoded protein To analyze EST of the cDNA library of Chimonanthus praecox, a glutaredoxin gene, named CpGRXl (GenBank:JQ990126), has been randomly cloned. CpGRXl gene has a full length492bp, with an open reading frame of321bp. The molecular weight is about11.18KD, encoding106amino acids. Protein structure analysis showed that the active sites of the protein is CPYC, have the GSH binding sites; the protein is composed of four β-sheet and five a-helical, and the four β-sheet surrounded by five α-helical. It belongs to Thioredoxin-like superfamily. Above analysis showed that the protein is a typical glutaredoxin of subclass Ⅰ (Namely CPYC type).2. The transcriptional level analysis of CpGRX1gene in Chimonanthus praecoxThe Real-time quantitative PCR analysis showed that CpGRXl gene expressing in all tissues of Chimonanthus praecox and has the highest expression in stem. The CpGRXl in expression of the flower-bud period and wither period in flowering stages is ultra-high, with strong time-specific. After the abiotic stress treatment of Chimonanthus seeding, for the expression of CpGRXl gene in leaves, CpGRXl gene hase rapid responses to oxidative stress and salt stress; It showed a regular decline-rising trend to drought stress; CpGRX1gene has no siganificant change to low and temperature stress.The results showed that CpGRX1gene may be associated with antioxidation, drought and salt of the defense in Chimonanthus praecox.3. The overexpression of CpGRXl gene in transgenic tobaccoThe plant expression vector of pCAMBIA2301-CpGRX1was Constructed. Useing agrbacterium tumefaciens-mediated method, the the recombinant genome has been transferred into the wild tobacco. Through the rooting screening, GUS staining and PCR assay of the transgenic tobacco plants,25strains of CpGRX1transgenic tobacco plant have been successfully obtained.