Cloning, Expression Analysis And Promoter Isolation Of GR?F3H Gene From Caragana Korshinskii Kom.

Adversity stress such as low temperature, high salinity and drought can cause severe influence in growing and development of plants. As a key enzyme in antioxidase system,Glutathione reductase(GR) plays vital effect when plants withstand adversity. Adversity stress can also influence the compounding of anthocyanin in plants, Flavanone 3-hydroxylase(F3H) is a key enzyme in anthocyanin synthesis pathway.Caragana korshinskii Kom. is a kind of shrub belonging to the category of leguminosae caragana, which has strong ability to tolerate adversity, can prevent wind and fix sand, as well as preserve water and soil. In this research, we used eremophyte Caragana korshinskii Kom. as material, cloning GR and F3 H gene sequence, doing bioinformatic analysis, expression analysis,as well as promoter analysis. which providing evidence for researches of plants resistancing adversity stress in desert region. The main conclusions are shown as follows:1. A new GR gene was isolated from Caragana korshinskii Kom. The full-length cDNA of GR was 2122 bp, contained a 1650 bp opening reading frame. The deduced protein was 549 amino acids with molecular weight 59.2 kD and isoelectric point 8.2. Homology analysis results indicated that GR showed high identities with Cicer arietinum GR. Protein sequence analysis showed that GR is a hydrophilic protein without transmembrane helix domain and signal peptide structure.RT-PCR analysis revealed that GR expressed in roots, stems, and leaves with almost no tissue specificity, the expression level is highest in leaves. The transcript level of GR was increased in response to cold, salt and drought stress, and the expression quantity tended to lowering before rising, When cold stress time is 36 h, salt density is 100 mmol/L and PEG quality fraction is 15 %, the expression level of GR reached the highest point. Which indicated that GR was inducted by cold, salt and drought stress, we infered that GR participated in abiotic stress response.The promoter of GR was isolated from Caragana korshinskii Kom. The ATG upstream sequence of GR was 648 bp. Sequence analysis showed that this promoter had basal promoter element CAAT-box and TATA-box, contained homeopathic functional elements related to ABA,high temperature, drought and other stress, indicating that this promoter may be inducted by ABA,high temperature, drought and other stress.2. A new F3 H was isolated from Caragana korshinskii Kom. The full-length cDNA of F3 H was 1437 bp, contained a 1095 bp opening reading frame. The deduced protein was 364 amino acids with molecular weight 41.3 kD and isoelectric point 5.9. Homology analysis results indicated that F3 H showed high identities with Onobrychis viciifolia OvF3 H. Protein sequence analysis showed that F3 H is a hydrophilic protein without transmembrane helix domain and signal peptide structure.RT-PCR analysis revealed that F3 H expressed in roots, stems, and leaves with almost no tissue specificity, the expression level is highest in leaves. The transcript level of F3 H was increased in response to cold, salt, drought and high tempreature stress. During the cold, salt and drought stress, the expression quantity tended to lowering before rising, When cold stress time is36 h, salt density is 150 mmol/L and PEG quality fraction is 15 %, the expression level of F3 H reached the highest point; during the high tempreature stress, the expression quantity of F3 H tended to rise as whole as the temperature rised. When the temperature reached 40 ?, the expression quantity reached the highest point. Which indicated that F3 H was inducted by cold,salt, drought and high tempreature stress, we infered that F3 H gene participates in abiotic stress response.The promoter of F3 H were isolated from Caragana korshinskii Kom. The ATG upstream sequence of F3 H was 468 bp. Sequence analysis showed that this promoter had basal promoter element CAAT-box and TATA-box, contained homeopathic functional elements related to light,ABA, high temperature and other stress, indicating that the promoter may be inducted by light,ABA, high temperature and other stress.