Cloning And Application Of Antifreeze Protein Gene Of Tenebrio Molitor

It has been discovered that many creatures have antifreeze or its related gene. Antifreeze proteins (AFPs) lower the non-equilibrium freezing point of water (in the presence of ice) below the melting point, thereby producing a difference between the freezing and melting points that has been termed thermal hysteresis. Different kinds of antifreeze protein has been discovered in a kinds of organisms include polar fish, plant, insect and microbe. In order to insure themselves survive under the cold stress, many plants has been synthesized a lot of novel proteins to protect themselves form cold damage which named cold-induced proteins. Such proteins include cryoprotective proteins, antifreeze proteins, dehydrin and membrane related proteins.As a lower temperature impressible plant, sweetpotato can not resist the damage come form cold stress when the temperature come to under 10℃. When storage at 2℃for 2 months, coat will be change color, decompose and pathological softening. If the temperature has been droped in the progress of growth of the young seedling suddenly, sweetpotato will be damaged enven be killed by the cold stress.It will bring a large loss to farmer. So, it's very important to construct new antifreeze sweetpotato materials.Nowadays, 8 Tenebrio molitor antifreeze proteins (TmAFPs) have been identified, coding 84, 96 and 102 amino acids polypeptide respectively. The latest research has proveed TmAFPs have the strongest thermal hysteresis activity to reduce freezing point about 5.58℃.In our paper, a complete coding sequence of TmAFP which cotent 339bp has been cloned from the cold induced larva of Tenebrio molitor by RT-PCR. Such fragment coding 112 amino acid residues, which consists of 7 right-handed b-helixs with 12(CTXSXXCXXAXT) residues per coil. A multiple sequence alignment with known TmAFP revealed that the sequence we gained have sequence identity with 99％similarity in nucleotide acid sequence to the well known AFP gene Tmafp4-9 and 97.3％similarity in AA sequence at protein level. Four bases (G5→C5, C44→T44,G229→A229 and A319→T319) has been substituted in Tmafp by comparing with Tmafp4-9 resulted in the change of four AA residues (Gly2→Ala2,Thr15→Ile15, Va177→Met77 and Thr107→Ser).TmAFP gene has bee expressed in E. coli (BL21), recombinant TmAFP has been purified and applied in the preparation for E. coli competent cells. The effect of freezing and thawing on E. coil viability, transformation efficiency was observed after the addition of TmAFP at different concentrations, temperatures, and storage time. TmAFP was shown a dual effect in low-temperature storage: lower concentration TmAFP can protect cells from the damage of freeze, but higher concentration TmAFP may has a deleterious effect on cells. It was also suggested that different storage time has feeble effect on the competent cell but storage temperatures were a dramatic effect on it.In order to protective sweetpotato from cold damage in the progress of root-strorage and seedling growth, a series of plasmids have been constructed by the use of SP promoter (the promoter of storage protein of sweetpotato), CaMV35S promoter, Tmafp and the agrobacterium plasmids, and were transformed to sweetpotato by agrobacterium EHA105.S-adenosyl-L-methionin (SAM) is the important metabolic-intermediate far-ranging presence in creature such as animal, plant and even microbe, which take part in 40 kinds of biochemistry interaction. Recently, it has been discovered that S-Adenosyl-L-Methionine synthase (SAMS) can be induced expression under the stress of lower temperature and desiccation, it suggest such SAMS maybe a very useful protein in resistance the stress condition of lower temperature and desiccatio. Suppression subtractive hybridization (SSH) method was used to identify novel genes regulated by cold stressed in sweetpotato. A full length cDNA coding region of S-Adenosyl-L-Methionine synthase (SAMS) which contains 1182bp was isolated by this way combined with RT-PCR technology. Sequence analysis revealed that this gene hared 85.48％similarity in gene sequence and 93.6％similarity in protein sequence with the other SAMS gene in plant. Fused expression plasmid vector pET-SAMS were constructed with sams and pET32a. A protein about 63KDa was expressed after induced by IPTG at different temperature in BL21. The works to clone the promoter of SAMS are on doing.