| Pleurotus pulmonarius is one of many gourmet cooking materials in modern society for its good-looking appearance and delicious tastes.As P.pulmonarius contains much protein and different varieties of microelement,it meets people’s demands for eating more green food and living a health life.LuoYuan County in FuJian Province is a special place to cultivating and selling P.pulmonarius.But the main variety there is merely Xiu-57 introduced from TaiWan and the quality of it deteriorates after a few year’s planting.Therefore,screening a new variety which is suitable for growing under the local climate conditions is the major course of this study.This study lays a solid foundation for the follow-up research through a series of studies from many different aspects.And the the primary theme of my study is following:We chose 6 strain in total:1 wild strain Xiu-1(sampling from the NanPing wild field)and 5 cultivated strain:ZhongXiu-618、XiuJin、TaiXiu 57-66,Xiu-57 and LuoXiu as original strains in study,and generated 9 protoplast monokaryon strains:Xiu-1-1、Xiu-1-15、ZhongXiu-618-21、ZhongXiu-618-31、XiuJin-1、TaiXiu-3、TaiXiu-7 and Xiu-57-6.There are also 24 hybrid strains available through both of Mon-Mon crossing breeding and Di-Mon crossing breeding.After conducting an experiment to the 24 hybrid strains and parts of original strains and testing the growth rate of hypha and determination of cellulose,hemicellulose and the laccase enzyme activity,the results shows that:in PDA medium,hybrid S5 cultivated grown faster than original strains.In cultivation medium materials,hybrid Z5 grown faster than original strains.We discovered that no matter in whatever substrate there was no correlation between mycelial growth rate and enzyme activity.The growth rate of the same strain in PDA was the same as that in the cultivation medium,the active of cellulose and hemicellulose also has significant positive correlation by analyzing the dependency between the growth rate of mycelia and enzyme activities.According to the yield data of the first time,hybrid Z2L derived from Xiujin and ZhongXiu products more than the original strains.In the process of Mon-Mon crossing breeding,we encountered an issue that monokaryon strains hybridized with low rate.We studied them in molecular aspects by using genomic sequencing technology to make it clear that whether it caused by small amount of mating-type factors.The genome of two monokaryons from ZhongXiu-618,two monokaryons from wild Xiu-1 and one monokaryon from Xiu-57 were sequenced.After assembly,genome sequences were compared with the existing mating-type gene by BLAST and predicted gene structure with AUGUSUT.With the NCBI BLATp and interpro prediect the protein domain proved that these genes were the mating-type A factor and B factor genes in monokaryon strains.Among them,the HD gene of Xiu-1-1、ZhongXiu-618-31 and Xiu 57-6 have 100%similar degree in nucleotide sequence and protein sequence.It suggests they have the same mating-type A factor.Furthermore Xiu-1-1、ZhongXiu-618-31 and Xiu 57-6 their A factor has composed of a pair of HD1 and HD2 genes.However a special strain Xiu-1-15 that it has 2 HD1 and 3 HD2 genes has been found.Xiu-1-1 and Xiu-1-15 obtained from the protoplast of the wild Xiu-1,with different mating types.The number of HD genes in the mating-type A factor of the Xiu-1-15 and the Xiu-1-1 is different,which is not present in other species.In addition,during the process of finding pheromone receptors of B factor,we observed 5 pheromone receptors in each of Xiu-1-1,ZhongXiu-618-31 and Xiu-57-6.And the similar degree of one of the 5 pheromone factors is 100%.We discovered that the number of pheromone receptors of ZhongXiu-618-21 and Xiu-1-15 is 1.0 and 8 respectively.With the sequence of mating-type A factor,we found that ZhongXiu618-21 has two pair of HD gene and double MIP gene.So we suspect that it may be a dikaryotic strain.We get the results of mating type of some monokaryotic strains:Xiu-1-1:A1B1,Xiu-1-15:A2B2,ZhongXiu-618-31:A1B3,Xiu-57-6:A1B4.The reason that some strains can not cross may be due to differences between numbers of mating-type A factor and B factor.And it is expected to further analysis in subsequent experiments.In Mon-Mon crossing breeding,we sampled from the two sides and the middle of two inoculums,obtained 3 different kinds of alloplasm-homokaryon.That is,to the strain sampled from left,its mitochondria may from in the left parents,to the right strain,its mitochondria may from in the right,but to the middle one,its mitochondria may form in the left or the right parents.In this study,we assembly the mitochondrial genome by using the information of genome sequencing,and got ZhongXiu-618-31 and Xiu-1-15 has diference,and comparing the differences between the two mitochondria and genome,designed primers,After testing PCR,we knew that Mon-Mon crossing breeding generates two different varieties of alloplasm-homokaryon.That is,to the strain sampled from left,its mitochondria rooted in the left parents,to the right strain,its mitochondria rooted in the right parents.To the middle one,its mitochondria rooted in the left or the right parents,but only from one of them.Based upon the results above,we discussed the inheritance of Mon-Mon crossing breeding prelimina rily.The experiment conducted divergence analysis to the two kinds of alloplasm-homokaryon through the data utilization of enzyme activities、growth rate and production.In growth rate respects,different mitochoncrials in cultivation medium shows significant variation but in PDA is not.In enzyme activities respect,different mitochoncrials in cultivation medium materials shows significant variation of all the three enzyme activities.In fermentation,only cellulase activity of different mitochoncrials shows variation while himicellulase activity is no,suggesting that alloplasm-homokaryon strains have variations in aspects of mycelia growth rate、cellulase activity、hemicatalepsy activity as well as the production.Agriculture industry can take advantage of mitochondrial differences to do selective breeding in the future hybrid breeding. |
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