抗冷冻蛋白基因遗传转化草菇的研究

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 and unable to stand freeze preservation. Thus how to breed novel strains (highly resistant and high-yield strain) is an important problem in straw mushroom production and research. Despite its importance, information related to the breeding of straw mushroom is limited, and where breeding is carried out, the strategies employ conventional sexual processes. Because of its homothallic life circle, lack of clamp connections and scarcity of selectable markers for hybrid screening, using conventional breeding methods to improve straw mushroom strain is difficult. Fortunately, the development of recombinant gene techniques throws a light on solving this problem. The latter approach requires a transformation system and most transformation models described for basidiomycetes thus far are based on nutritional complementation of auxotrophic mutations with equivalent wild-type homologous or heterologous genes. However, isolation and characterization of such mutants are time consuming. So another selection strategy is developed. It uses drug resistance genes as selectable markers. Resistance genes that have been commonly used for transformant selection in edble fungi are antibiotics hygromycin .Thermal hystersis proteins (THPs) possess the unique property of lowering the freezing point of water (in the presence of ice crystals) without significantly altering the the melting point and play an important role in low temperature survival. A lot of antifreeze genes were cloned from fish, insects, plants, fungi and bacteria. Experumentally THPs have been used in the ryopreservation of tissues and cells and to induce cold tolerance in freeze susdeptible organisms. THPs represent a emarkable example of parallel and convergent evolution with different proteins being adapted for an antifreeze role. However, the most common of AFP gene to transformate fish and plants are fish AFP gene. But at presend reported, some of the insects anti-freeze protein is higher activety than fish antifreeze protein. Sush as the THP of Choristoneura funiferana is as 10-30 times activity as fish AFP.RT-PCR technique was used for amplifying THP gene from a Budworm in Sweden with primer AFP1 and AFP2. Then THP gene was ligated to pGEM T-Vector to be the plasmid pGTHP4. The plasmid pCAMBIA1301(contain 35s promoter, 35s poly A terminator, hpt selectable marker gene ,nospolyA terminator, gus)was digested with restriction enzyme BstEⅡand NcoⅠto get a longer fregment containing promoter, and the plasmid pGTHP4 was digested with restriction enzyme BstZⅠand NcoⅠto get a short fragment containing THP gene. The longer fragment and the short fragment were ligated with add at a specific adapter to pCTH823, a expression vector of V. volvacea . Two transformation systems of straw mushroom was established with 35s promoter, gus report gene, hpt selectable marker gene by particle bombardment and Agrobacterium-mediated transformation. Exterior THP gene was transformated to straw mushroom（V1，V34，V1308） by particle bombardment and Agrobacterium-mediated . We selected 4，9，2 transformants of straw mushroom V1，V34 and V1308 by adding appropriate hygromycin to medium. 9 putative transformants were detected by PCR amplifying from genomic DNA, Southern blotting and the mycelia tolerant to cold stress (4℃) text. those result showed that a bright THP gene bend about 400bp was amplified from transformants genomic DNA; and Southern blot analysis confirmed the integration of transforming THP gene into the genome of transformants with different copies and different site; All putative transformants can re-grow well at 28℃ after tolerant to 4℃ low temperature for 10-15days, and the control can't re-grow after tolerant to 4℃ low temperature for 3 days ..In addition, according to analysis of relativity of the growth speed of transformants show that the growth s

利用引进的启动子、选择标记基因、超毒性农杆菌菌株、抗冷冻蛋白目的基因，构建高效表达载体；采用农杆菌介导法和基因枪法转化草菇菌株，建立草菇高效的转化系统，培育抗冷冻的转基因草菇新菌株，解决草菇菌种冷冻贮藏和鲜菇冷冻保鲜的难题，促进草菇生产和流通及出口创汇；同时为应用T-DNA标签法分离草菇基因及其功能研究提供技术保证。