Cloning, Expressing In Bacteria And Plant Transgene Of Ryegrass

Antifreeze proteins (AFPs) are a family of proteins capable of protecting organisms from damage in freezing or sub-freezing conditions by controlling the growth of ice and inhibiting the recrystallization between ice granules, which were termed thermal hysteresis (TH) activity and recrystallization inhibition (RI) activity respectively. The characteristic of low TH and high RI of plant AFPs has facilitated their application to the transformation into non-hardy plants and to the low temperature storage of food, cells, tissues, and organs in comparison with fish AFPs and insect AFPs, though it's molecular mechanism is still not understood well at present.Ryegrass (Lolium perenne) is an evergreen grass with strong cold resistance due to high active antifreeze protein (LpAFP).The studying paper is searching about clone lpafp gene,expressing and purify LpAFP,structure analysis and Site-directed mutagenesis of LpAFP and constructing plant excess express vector of LpAFP.The results as following:(1) To clone the cDNA sequences of LpAFP, the total RNA of perennial ryegrass treated with cold stress was extracted. Then, the cDNA of ORF mature LpAFP was obtained by RT-PCR. The sequence analysis result showed that mature ORF of LpAFP contained 357 bp, which encoded 118 amino acids. The result of bioinformatics analysis revealed that ryegrass antifreeze protein has high homology with other plant antifreeze proteins of gramineous, and no homology to fish antifreeze proteins and insect antifreeze proteins.(2) Expressing Lpafp in E. coli. BL21(DE3) by linking Lpafp into expression vector system pET-32a and pET-28a.In order to get a mass of purity LpAFP proteins in short time,it is important to provide better cultive and inducement conditions.In M9 culture medium,E.coli. BL21(DE3)for expression recombinant proteins culture in the conditions as better condition:bacteria beginning density OD600 1.25,IPTG density 0.2 mM and inducement time 6-8h.After expressing recombinant proteins,LpAFP is isolatied and purified by three steps of three methods.In first,total protein 100℃water soaking 12min to remove non-heat resistant proteins,the second step is high affinity Ni-NTA resin to isolate recombinant proteins with His-Tag,the last step is reverse HPLC.the purified LpAFP can be mensurate tretice structure.(3) Theoretical Model of LpAFP present here a theoretical three-dimensional model of this 118-residue plant protein based on aβ-roll domain with eight loops of 14-15 amino acids. The fold is supported by a conserved valine hydrophobic core of "XXNXVXG" and internal asparagines ladders at either end of the roll. Here we have validated the model with a series of nine site-directed steric mutations in which outward-pointing short sidechain residues were replaced by better animo acid for "XXNXVXG"model.Induce the mutations vector to expressing and check the every site-directed mutations(4) Finish construction the plant axceed expressing pCAMBIA2301-Lpafp vector structure by linking upstream promoter element (UPE),lpafp cDNA and terminator structure. The result showed that the second generation of transgenic tobaccos displayed electropositive, the lpafp gene link in genome of tobacco.