| The straw mushroom, Volvariella volvacea, is one of the most important cultivated edible mushrooms, representing a valuable source both in economy and in nutrition. But the straw mushroom has lower individual yield andunable to stand freeze preservation. Thus how to breed novel strains (highly resistant cold and high-yield strain) is an important problem in straw mushroom production and research. However, straw mushroom is a homothallic fungi. Its mycelium is lack of clamp connections and it has scarcity of selectable markers for hybrid screening. So, using conventional breeding methods to improve straw mushroom strain is difficult. Fortunately, the development of recombinant gene techniques, success in report gene transformation into filamentous fungus, success in antifreeze protein gene cloning and transformation provided a probability on solving straw mushroom problems in cold resistance.Thermal hysteresi proteins (THPs) possess the unique property of lowering the freezing point of water (in the presence of ice crystals) without significantly altering the melting point and play an important role in low temperature survival. A lot of antifreeze protein genes were cloned from fish, insects, plants, fungi and bacteria. However, the most common AFP genes transformated into fish and plants are fish AFP genes. However, the activity of antifreeze protein from insects is much higher than that from fish. The activity of THP from Choristoneura funiferana, for example, is 10-30 folds of activity of AFP fron fish. So, antifreeze proteins from insects are attracting people attention increasingly.In this study, RT-PCR technique was used for amplifying THP gene from a Budworm (a larve of Lepidoptera) in Sweden with primer AFP1 and AFP2. The THP gene was cloned into pGEM T-Vector as plasmid pGTHP4. At the same time, we performed the following studies.(1) The sensitivity assay of straw mushroom to selectable reagents: Four selectable reagents including Kanamycin, Hygromycin, Geneticin-G418, phosphinothricin-PPT were added to PDSA plates with different concentration to detect the antibiotics sensitivity of straw mushroom strain VI308 (VB) and VQ which belong to different genus. The result showed that VI308 and VQ are only sensitive to Hygromycin.(2) The determination of the lowest sensitive concentration of strawmushroom to Hygromycin: Three straw mushroom strain, V1308, V1 and V34 were subjected to be detected with different concentration of hygromycin. The result showed that the lowest sensitive concentrations of V1308, V1 and V34 to Hygromycin were 70 g/mL, 50g/mL, 60g/mL respectively on the Agar plates (PDSA) , and were 50 u g/mL, 35g/mL, 40g/mL respectively in the liquid medium (PDSB).(3) Establishment of transformation system of V1308: The plasmid pCAMBIA1301 which contains 35s promoter, Hygromycin B resistance selectable marker gene , Gus (b-glucuronidase gene) report gene was used to transform straw mushroom strain V1308 by particle bombardment with different parameters on intact mycelia. The putative transformants were gained from three step screening. The transformation efficiency with different parameters was performed variance analysis. The result showed that most effective transformation should be gained by using the parameter of 1100 Psi of Helium Pressure, 26 inches Hg of Vacuum Pressure, and 6 cm of target-shelf distance (from stopping plate to target shelf distance). All putative transformants were detected by histochemical assay and confirmed by southern blot hybridization. The results indicated that exogenous GUS gene has integrated into the V1308 genome and stably expressed in the mycelia and fruiting body.(4) Construction of the expression vector for straw mushroom: The plasmid pCAMBIA1301 was digested with restriction enzyme Bst E II and Nco I . The digestion product was separated with 1% of agarose gel and the longer fragment containing 35s promoter and Hygromycin B resistance gene was purified with the Agarose Gel DNA Extraction Kit. In the same way...
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