Mechanism On Degrading Lignocellulose In Seed Coat Of Cremastra Appendiculata By Coprinellus Disseminatus

Cremastra appendiculata（D.Don）Makino is a rare perennial medicinal herb of the genus Cremastra in the orchid family.It is used in medicine with dried pseudobulbs.Under natural conditions,the seed germination rate of C.appendiculata was very low,and the germination period was long.Based on the previous research subject,a fungus of Coprinellus disseminatus that promotes germination of C.appendiculata seeds,was screened by our team.The fungus can promote germination of C.appendiculata seeds well,and the germination rate reached about 71%,which may be because the seed coat inhibited the development of embryo and seriously affected the permeability and absorption of nutrients of embryo.However,C.disseminatus significantly promoted germination of C.appendiculata seeds,which may be due to the fact that the fungi can secrete lignocellulose enzymes to degrade lignocellulose in seed coat,thus breaking the physical barrier of seed germination,and enhancing the water absorption and air permeability of germination of C.appendiculata seeds,which is conducive to the absorption of water and nutrients,This study laid a foundation for the development of seed embryos during the symbiosis between C.appendiculata seeds and C.disseminatus.The results of this experiment are as follows:（1）First of all,the experiment of in vitro degradation of exogenous lignin was carried out.With the longer culture time,lignin content has been reduced,and the C.disseminatus secreted related enzymes to degrade exogenous lignin.The contents of hemicellulose and lignin in seeds cocultured at 0 day,6 days and 12 days were determined.Compared with the seeds at 0 day,The hemicellulose of cocultured seeds decreased for 6 days without significant difference,and the lignin content of the seeds at 12 days of co-culture decreased faster,which reached a significant difference.Then,seeds were made resin section on 0 day to 15 days,and it was found that the seed coat of the 12 days seeds almost disappeared,and a large number of mycelia invaded the seed cells.After 13 days co-culture,the seed coat disappeared and the seed embryo cells proliferate.The activity of laccase,cellulase and xylanase was higher at 12 days after co-culture.In conclusion,12 days of co-culture was the best time for the effect of C.disseminatus on lignocellulose in seed coat of C.appendiculata seeds.（2）Guaiacol method and sodium carboxymethyl cellulose method were used to qualitatively verify the abilities of laccase,cellulase and xylanase of C.disseminatus.The lignin degrading enzyme conditions were optimized by co-culture with C.appendiculata seeds for 12days.The laccase produced by the white parasol can discolor guaiacol medium,while aniline blue medium does not discolor,so the white parasol may not produce lignin peroxidase.Through the enzyme activity determination of extracellular medium for 12 days co-cultured with the seeds of C.appendiculata seeds and C.disseminatus,the suitable medium for enzyme production with single factor was first used.Finally,on the basis of the single factor test,the orthogonal test of nutritional factors was carried out.Finally,the suitable conditions for the production of enzymes were screened:30 g·L^-1 maltose,6 g·L^-1 peptone,2 g·L^-1 Mg²⁺and medium p H=6,1 L dd H₂O.（3）In this study,according to the above orthogonal experiment to optimize the suitable conditions for enzyme production,the C.appendiculata seeds and C.disseminatus were co-cultured for 12 days.Based on the transcriptome data,major lignocellulose degrading enzyme genes of C.disseminatus were screened.Under the conditions suitable for lignin degrading enzyme production,real-time fluorescence quantitative PCR was performed on the selected lignocellulose degrading enzyme genes and lignin and hemicellulose content was determined.Compared with the control,The results showed that compared with the control group,the expressions of 1 glyoxal oxidase gene,2 cellulase genes,1 dye decolorization oxidase gene,1catalase gene,1 glucose oxidase gene and 5 laccase genes were up-regulated.Compared with the control,the lignin content and hemicellulose content of C.appendiculata seeds were significantly decreased,and the expression of lignin degrading enzyme genes was significantly up-regulated,reaching a significant difference,which was consistent with the results of physiological measurement.The difference of xylanase gene expression was not significant,and the hemicellulose content was also decreased,which was not statistically significant,which was consistent with the results of physiological measurement.The expression of two cellulase genes was up-regulated,which indicated that the C.disseminatus could secrete related enzymes to degrade lignocellulose of the seed coat during the symbiosis of C.appendiculata seeds.