The Study On Cold Tolerance Of Insect Antifreeze Protein Gene Transformation To Tobacco

Antifreeze proteins (AFPs) have been isolated from a variety of organisms and play an important role. AFP causes the freezing point of a solution to be lowered without influence on the melting point in a noncolligative manner by adsorption to the ice surface. The difference between the melting point and the nonequilibrium freezing point can be determined and is termed thermal hysteresis activity (THA). THA is widely used as an indicator of AFPs activity, so AFPs are often referred to as thermal hysteresis proteins (THPs). The THAs among species are quite different and insect AFPs are comparatively high (3–6℃), so AFP genes from insects were widely used for improving the cold tolerance of crops.MpAFP149 gene was isolated from Microdera puntipennis dzungarica, a local beetle in Xinjiang desert region. The cDNA of MpAFP149 encoded a polypeptide of 120 amino acid residues with calculated molecular mass of 12.7 kDa. MpAFP149 polypeptide (minus the signal peptide) was 98 residues with calculated molecular mass of 10.2 kDa and comprised of tandem repeats of 12-aa sequence (TCTxSxxCxxAx) with regularly spaced Cys. The two Cys within each repeat form a disulfide bond with the exception of repeats 1 and 2, which are linked by an additional disulfide bond. In the experiments, different plant expression vectors carrying MpAFP149 gene were constructed and transformed the model plant tobacco by using different transformation methods for researching the function of MpAFP149 responded to cold in plants and also looking for the best ways to improve the AFPs expression and thermal hysteresis in plants.Basing on the effective system of nuclear transformation to tobacco, the construct of MpAFP149 gene with the signal peptide sequence under control of a cauliflower mosiaic virus 35S promoter was introduced into tobacco by Agrobacterium tumefaciens–mediated transformation. Two T1 generation transgenic tobacco lines T1-5 and T1-39 were obtained which indicated the high transcripts in mRNA level. The cold tolerance of transgenic tobacco was obviously better than wild-type tobacco with cold treatment for 1 d, 2 d and 3 d at -1℃. At an initial stage of cold treatment, ion outflow was all little increased and no difference between transgenic and wild-type tobaccos on one day. There was a clear difference in ion leakage rate after two days exposure to -1℃: the electrolyte leakage reached 65% for wild-type tobacco, 28% for T1-5 and 27% for T1-39. On day three, the ion outflow was increased in similar tendency as day two, with 71%, 28%, 36% for wild-type tobacco plants, T1-5 and T1-39 lines, respectively. The increase of relative conductivity was regarded as the cause of the rise in MDA. MDA concentration in wild-type and transgenic tobacco plants did not vary much before the cold treatment, but it increased more than three times for wild-type tobaccos and two times for T1 transgenic plants after 2-3 days cold treatment. These result suggested that the wild-type plants were suffered severer oxidative lipid injury than transgenic tobacco ones. The amount of both ion leakage and MDA clearly increased with prolonged times of cold treatment. A significant and high correlation (R2=0.9132) was observed between MDA content and permeability of cell membranes to ions which suggested that permeability of cold damaged cell membrane increased with the increasing peroxidation of fatty acids. In terms of phenotype experiments, ion leakage and MDA content, a relationship between the expression of MpAFP149 and an improvement in cold resistance in transgenic plants was inferred.Citrate was found as the enhancer of antifreeze proteins and can improve the thermal hysterisis activity of AFP in vitro. Citrate synthase gene (tacs) was amplified from Nicotiana tabacum L. by RT-PCR according to sequence published on Genebank. Another recombinant expression vector pCAMBIA1302-MpAFP149-tacs was constructed and then transformed the tobacco by Agrobacterium tumefacines. At -1℃, transgenic tobacco showed the better cold phenotype than that of wild-type tobacco, but there was no difference between transgenic tobacco containing only MpAFP149 gene and binary transgenic tobacco containing MpAFP149 and tacs gene. When returned to room temperature, MpAFP149 expressed plant overcame dehydration and got recovery completely. However, the wild-type tobaccos displayed severe chlorosis and wilting and suffered inreversible damage to some extent. The result showed that both transgenic tobaccos either containing MpAFP149 or MpAFP149-tacs gene displayed better cold tolerance than wild-type tobacco, but there was no cooperated relationship between MpAFP149 and citrate to further improve the cold tolerance in transgenic tobacco.In order to resolve the low expression of heterologous gene in transgenic plants, MpAFP149 gene was constructed into bean chloroplast vector by designing the specific primers and transformed tobacco by gene-gun. We expected increasing the MpAFP149 expression to further improve the plant cold tolerance finally. Four transgenic tobacco lines were obtained by repetitive screen using 500 mg/L spectinomycin. At -1℃, chloroplast transgenic tobaccos displayed the better cold tolerance than wild-type tobacco, but there was no difference with transgenic tobacco containing MpAFP149 gene by nuclear transformation. This result was not consisted with our expected aim and it was possible that homologous degree of interested gene MpAFP149 was relatively low.The result of laser scaning microscope and immunogold localization showed that MpAFP149 protein uniformly accumulated in the outer layers of cell wall of transgenic tobacco.The ultrastructure difference at -1℃between transgenic and wild-type plants for consecutive three days was compared by the observation of organelles morphology changes. The organelle morphological alterations in leaves of transgenic and wild-type plants, before and after cold treatment at -1℃, were examined by TEM. The observations revealed that there were altered appearance inside the cells after the cold treatment and the changes were prominent in membrane of cellular organelles, especially in cell membrane, chloroplasts and mitochondria. This protection might be attributed to an interaction between the antifreeze peptides and cell wall, which resulted in the prevention of the ice growth in the apoplast, so that the whole cell could avoid freezing. These results suggested that the expression of the heterologous protein MpAFP149 have significant effects on the morphological changes in organelles, mitigate the freezing-induced structural damage in the membrane system and eventually lead to enhancement of freezing tolerance in transgenic tobacco cells.According to above experiment results, we can draw a conclusion that transgenic tobaccos containing MpAFP149 display the better cold tolerance than wild-type tobaccos. It suggested that the MpAFP149 gene did not be optimized according to tobacco codon bias and can be used as the candidate gene for the improvement of frost resistance of commercially important crops.