【作者】 王璐；

【导师】 刘迪；

【作者基本信息】 延边大学 ， 农艺与种业（专业学位）， 2021， 硕士

【摘要】 桑黄(Sanghuangporus spp)在其生长过程中必不可少的两个因素为营养因素与环境因素,环境因素是桑黄生长发育过程中重要因子,其中光照是环境因素中主要的影响因素之一。菌丝的长势情况、外部形态以及菌落的色泽变化等都会因光照的影响而发生改变。本研究以杨树桑黄(Sanghuangporus vaninii)菌丝为试验对象,采用冷光源光质培养箱设置红光、蓝光两种光质处理,设黑暗为对照,观测桑黄菌丝生长状态、生物量与抗氧化酶活性及多糖的含量变化,并从转录组学水平上分析桑黄菌丝各光质处理与其酶活性相关的差异表达基因。通过对抗氧化酶活性以及多糖含量变化过程中关键转录因子进行表达分析,初步分析光质影响桑黄菌丝酶活性与多糖含量变化的分子机制,为以后更好的开发桑黄药用价值以及经济价值提供理论依据。主要研究结果如下:1.桑黄菌丝釆用LED冷光源光照培养箱,分别设置光质为红光R(625～740nm)、蓝光B(440～475nm)及暗培养CK三个处理,设置培养温度为28℃,湿度35%,光照强度为600 Lux。R处理条件桑黄菌丝生长较快,桑黄菌落中心呈深黄色边缘趋向白色,且生物量累积较多;对照条件下桑黄菌丝长势次于R处理,菌落颜色呈淡黄色,生物量累积次于红光处理;B处理下桑黄菌丝长势最慢,菌落明显小于R处理及对照,但菌落生长浓密,菌落颜色较深呈黄褐色,生物量累积较少。综合分析来看,红色光较适宜桑黄菌丝体生长。2.在相同的环境条件及培养条件下,不同光质处理桑黄菌丝在生长阶段结束后各处理对超氧化物歧化酶(Peroxidase,POD)、过氧化物酶(Superoxide dismutase,SOD)、过氧化氢酶(Catalase,CAT)活性的影响。R处理的桑黄菌丝POD、SOD酶活性显著高于B处理及对照,但对照处理与B处理无明显差异;B处理桑黄菌丝CAT酶活性相较R处理及对照下的CAT酶活性显著,且三个处理下的CAT酶活性均有明显差异,R处理的CAT酶活性表现最低。多糖含量在R处理的桑黄菌丝显著高于B处理及对照,B处理与对照处理无显著差异。3.不同光质处理下的桑黄菌丝转录组测序,最终共获得60.63 Gb的Clean Data,组装后共获得36091条Unigenes,对得到的全部Unigenes进行功能注释,共获得有注释结果的Unigenes19128条。各样本的有效数据量分布在6.25～7.35 G,Q30碱基分布在94.75～95.73%,平均GC含量为51.29%。基于比对结果,进行蛋白编码基因表达量分析。根据蛋白编码基因在不同样本中的表达量,进行差异筛选,共设有3个差异分组,其检测到的差异基因数量分别为:517、2364、3639。将不同光质处理的桑黄菌丝进行差异表达分析,共筛选出8520个差异表达基因;差异表达基因GO功能注释到64个功能分类。4.本研究中,KEGG数据库中注释到2条糖代谢相关通路,其中共筛选了与糖代谢相关的16个差异基因。转录组测序分析结果表明,合成糖分有关的基因表达水平显著下调是R处理桑黄菌丝多糖含量显著增高的关键,这说明与前期试验测得结果相同;多个基因显著下调,该基因可能直接或者间接参与了多糖的合成与代谢。这些与桑黄菌丝多糖合成的相关基因包括磷酸烯醇丙酮酸羧激酶、肌醇磷酸酶、1,6-二磷酸-D-果糖醛缩酶、葡萄糖-6-磷酸异构酶、己糖激酶、三糖磷酸异构酶、琥珀酸-半醛脱氢酶、3-磷酸甘油醛脱氢酶等。这些Unigenes及其注释信息,为今后深入研究多糖代谢途径提供了依据,也为桑黄经济价值的开发提供依据。更多还原

【Abstract】 There are two essential factors in the growth of Sanghuangporus spp,which are nutrition and environment,environmental factors are important factors in the growth and development of Sanghuangporus vaninii,which light is one of the main factors.The growth of mycelium,the external morphology and the color change of colony will change with the influence of light.In this study,the mycelium of S.vaninii was used as the experimental object.The growth state,biomass,antioxidant enzyme activity and polysaccharide content of S.vaninii mycelium were observed in the cold light quality incubator with red light and blue light treatment and dark as the control.The growth status,biomass,antioxidant enzyme activity and polysaccharide content of S.vaninii mycelia were observed in a cold light quality incubator with red light and blue light,and dark as control.The differentially expressed genes related to enzyme activity of S.vaninii mycelia were analyzed by transcriptome analysis.Through the expression analysis of key transcription factors in the process of antioxidant enzyme activity and polysaccharide content change,the molecular mechanism of light quality affecting the enzyme activity and polysaccharide content change of S.vaninii mycelia was deeply analyzed,which provided theoretical basis for better development of S.vaninii medicinal value and economic value in the future.The main research contents and results are as follows:1.The mycelia of S.vaninii were cultured in the incubator with LED cold light source.The light quality was red light R(625-740 nm),blue light B(440-475 nm)and dark culture CK.The culture temperature was 28℃,the humidity was 35%,and the light intensity was 600 lux.Under the condition of red light treatment,the mycelium of S.vaninii grew faster,the center of S.vaninii colony was dark yellow,the edge was white,and the biomass accumulated more;under the control condition,the mycelial growth of S.vaninii was inferior to that of R treatment,the colony color was light yellow,and the biomass accumulation was inferior to that of red light treatment;the mycelial growth of S.vaninii in treatment B was the slowest,and the colony was significantly smaller than that in treatment R and the control,but the colony grew densely,the color of the colony was dark and yellowish brown,and the biomass accumulation was less.Comprehensive analysis showed that red light was more suitable for mycelium growth of S.vaninii.2.The effects of different light quality treatments on the activities of(Peroxidase,POD),(Superoxide dismutase,SOD)and(catalase,CAT)were studied.The activities of POD and SOD in R treatment were significantly higher than B treatment and control,but there was no significant difference between control treatment and B treatment The CAT activity of B treatment was significantly higher than that of R treatment and control,and there were significant differences among the three treatments.The CAT activity of R treatment was the lowest.The polysaccharide content of S.vaninii in R treatment was significantly higher than that in B treatment and control,but there was no significant difference between B treatment and control.3.The transcriptome of S.vaninii treated with different light quality was sequenced,and a total of 60.63 Gb of clean data was obtained.After recombination,36091 unigenes were obtained,all unigenes are annotated,and 19128 unigenes with annotation results are obtained.The distribution of effective data was 6.25～7.35 G,the distribution of Q30 basic group was 94.75～95.73%,and the average GC content was51.29%.Based on the comparison results,the protein coding gene expression was analyzed.According to the expression levels of protein coding genes in different samples,there were three differential groups,and the number of differential genes detected were 2517,2364 and 3639.A total of 8520 differentially expressed genes were screened from the mycelia of S.vaninii treated with different light quality;Go function of differentially expressed genes was annotated into 64 functional categories.4.In this study,two pathways related to glucose metabolism were annotat-ed in KEGG database,and a total of 16 differential genes related to Carbohy d-rate metabolism were screened.The results of transcriptome sequencing analysi s showed that the significant down-regulation of gene expression level related t o polysaccharide synthesis was the key to the significant increase of polysacch ari-de content of S.vaninii mycelia in R treatment,which indicated that the res ults were the same as those in previous experiments;Multiple genes were signi fica-ntly down regulated,which may be due to the different treatment conditio ns h-ave a certain impact on the polysaccharide synthesis of S.vaninii mycelia.Thes-e genes are directly or indirectly involved in the synthesis and metabolis m of polysaccharides.These genes related to the biosynthesis of S.vaninii myce lial pol-ysaccharides include phosphoenolpyruvate carboxykinase,inositol phosph atase,fr-uctose 1,6-bisphosphate aldolase,glucose-6-phosphate isomerase,hexoki nase,trio-sephosphate isomerase,succinate-semialdehyde dehydrogenase,glyceral dehyde 3-phosphate dehydrogenase.These unigenes and their annotation inform ation provide the basis for the further study of polysaccharide metabolism path way in the future,and also provide the basis for the development of economic value of S.vaninii.更多还原