Cloning And Expressing Regulation Of Class-1 Nonsymbiotic Hemoglobin (GLB1) From Pingyitiancha (Malus Hupehensis Rehd.) And Researching Of Its Function

GLB1 encodes nonsymbiotic hemoglobins in modle plants,whose functions include : (1)They have a high affinity for oxygen as a result of scavenging O₂, (2) Acts as part of electron transport via interacting with flavoprotein, (3)In plants,upon bingding of CO,NO and O₂,as part of the sensing mechanism which regulation of cell metabolism, (4)Combined with small organic moledules and acted in the transportation of fatty or participate in the synthesis of organic compounds under the hypoxia.The aim of this study was to obtain a better insight into the molecular functions of the GLB1 in response to abiotic stress.Under water culture condition,we have cloned class-1 nonsymbiotic hemoglobin encoding gene from pingyitiancha seedlings.The expression pattern of MhGLB1 in different tissues at different conditions was analyzed by quantitative real-time PCR.The GLB1 gene derived from pingyitiancha was heterologously expressed in tomato to study the biological function of the GLB1.1.In an attempt to isolate MhGLB1 gene,the known apple EST sequences was used to design primer for isolation of MhGLB1.Analysis revealed a 477 bp sequence obtained by PCR technology was the full-length of encoding region(Malus hupehensis Rehd. GLB1, GQ423619). MhGLB1 encoding a protein of 158 amino acids with a mass of 17.8 kDa.The deduced amino acid sequence of MhGLB1 showed 95.57,82.82 and 80.12 percent identity to the pear (Pyrus communis),cotton (Gossypium hirsutum),alfalfa (Medicago sativa) nonsymbiotic hemoglobin encoding gene.The phylogenetic tree showed MhGLB1 has nearest homologous relationship with pear GLB1.It will be as the pingyitiancha class-1 nonsymbiotic hemoglobin encoding gene.2. Quantitative real-time PCR analysis revealed that MhGLB1 gene expressed at different levels in roots, stems and leaves,the relative value in roots was highest ..When NO₃^- was supplied to water cultured seedlings, MhGLB1 transcript in roots rapidly accumulated within 2 h,the accumulation of mRNA in stems and leaves increased slowly.In SNP treated roots,the induction of MhGLB1 transcripts was more rapid and peaked at 2h ,followed by a rapid down-regulation after 2h.Accumulation of MhGLB1 was also induced by the hypoxic stress within 12h,thereafter,the accumulation decreased.Under the treatment of ABA,the level of MhGLB1 mRNA was increased accompanied with the accumulation of NO.3.In order to determine the physiological and biochemical function,a construct containing full-length encoding region of MhGLB1 gene in sense orientation driven by the constitutive caulifower mosaic virus 35S promoter was assembled and introduced into tomato.The tolerance of the transgenic tomato under abiotic stress was also analyzed.Compared with the wild-type,the photosynthetic rate of transgenic tomato decreased slowly.The photosynthetic rate,stomatal conductance and transpiration rate of wild-type was decreased 86,86.8 and 90.7 percent respectively, but the transgenic tomato number 1 and 5 decreased only 40.1,72.5,78.7 and 55.3,70.2,82.8 percent respectively.The level of H2O2 and NO was significantly lower than wild-type at 96h of waterlogging.