| Lentinula edodes is a kind of widely cultivated edible fungi in the world, but in winter, the yield of Lentinula edodes (high temperature type)is too low and unable to stand freeze preservation. Thus how to breed novel strains, which it has high resistance to cold and high yield, is an importan problem in Lentinula edodes cultivation and production. With the development of genetic engineering techniques, success in cold resist gene cloning and transformation provided a probability on solving Lentinula edodes problems in cold resistance.COR (Cold-regulated) genes are inductive ones inducing hypothermia induced protein synthesis, and only under certain conditions (mainly low temperatures and short-day) they were able to be started and expressed. Therefore, mushrooms growing in relatively high temperature could acquire the antifreeze property and could be supplied continuously during winters if effective antifreeze genes were isolated from yellow vine white by molecular biology methods and were cloned into mushrooms that grows in relatively high temperature by recombinant DNA technology, transgenic and clone technology etc. In this study, the full-length of COR gene first obtained from Brassica campestris ssp. Pekinensis, then, construced Expression Vector in Lentinula edodes which includes COR gene, through the Agrobacterium tumefaciens-mediated transformation, got the transformants. Here are the main results.(1) Construced Expression Vector in Lentinula edodes, COR genes were isolated from mRNA of Chinese cabbage using CORR and CORF as primers by RT-PCR techniques, then gene npt Ⅱ of plasmid pcDNA3.1(+)-npt II was replaced by COR gene; secondly, COR gene expression cassette PgpdA-COR-TtrpC, which is suitable to be expressed in edible fungi, was constructed; later on, by using plasmid pDHt/sk-hyg as a template, the cloned COR gene expression cassette was inserted into the plasmid, which constructs a hygromycin resistance binary vector pDHt/sk-hyg-COR; finally, the transformation of antifreeze gene COR was achieved by Agrobacterium-mediated genetic transformation method using hyg resistance gene as a selectable marker. PCR and southern blot analysis confirmed that COR gene was successfully integrated into the genome of mushrooms. Experimental results show that transformant of mushrooms has good genetic stability.(2)Established A.tumefaciens-mediated L. edodes genetic transformation system, Using Atumefaciens-mediated transformation mediate. Successfully implemented genetic transformation of Ledodes Wl by transforming the expression vector pDHt/sk-/hyg-COR into A. tumefaciens and co-cultivating with the Ledodes mycelium. Ledodes Wl is sensitive to hygromycin B, and50μg/ml can totally inhibit its growth. The genetic transformation efficiency is very low, only less than7%. The transformant can stable genetic by PCR and southern blot test. This indicates that the binary vector pDHt/sk-hyg-COR can be used for A.tumefaciens-mediated genetic transformation, and COR gene can also be used to study the genetic transformation of other edible fungi.(3) At the same time, this study optimizes the co-cultured conditions from four aspects, Agrobacterium infection time, co-cultured temperature, co-cultured time, and whether to add AS in the process of co-cultured. The results show that the optimum infection time is20min; the optimum co-cultured temperature is28℃; screening efficiency under hygromycin B between the co-cultured time for1days and2days has no significant difference; adding AS during the co-cultured is necessary for the growth of transformants. |
PDF Full Text Request