Molecular Structure Identification,Key Functional Genes And Transcription Factors Selection For Biosynthesis Of Natural Rubber In Lactarius Volemus

As an important strategic resource and industrial raw material,the natural rubber is mainly produced from Hevea brasiliensis Muell.Arg.But it is a typical resource-constrained industry as it can only grow in tropical areas and cannot be grown in many countries and regions.As a result,research on alternative resources to natural rubber is being carried out around the world,mostly focusing on plant resources with high rubber content,such as Parthenium argentatum Gray,Taraxacum koksaghyz Rodin,etc.In recent years,many macrofungi,mainly of the genus Lactarius,have been found to contain natural rubber in their fruit bodies,such as Lactarius volemus(Fr.)Fr.Its natural rubber content is higher than other types of mushrooms,and its molecular weight is very close to the natural rubber of H.brasiliensis.Therefore,the L.volemus has great potential as a natural rubber alternative resource for research and development.This study aims to provide the theoretical basis and technical support for the better development and utilization of this new natural rubber resource through methods establishment of extraction and determination for natural rubber from L.volemus,molecular structure identification,genome and transcriptome sequencing,and the key functional genes and transcription factors selection for natural rubber biosynthesis.The study results are as follows:1.Based on the previous method by Soxhlet extractor,the extraction and analysis of polyisoprene from sample(less than 0.5 g)in 2 mL microtube was further discussed.The standard curve established by cis-polyisoprene standards was used to correct the extraction yield,and the molecular weight distribution was analyzed by gel permeation chromatography(GPC)to test the integrity of the extraction,so as to provide a technical method for the small and accurate extraction,separation and determination of natural rubber in the mvcelium and fruit bodies of L.volemus.2.By means of nuclear magnetic resonance(NMR)spectroscopy.1 H NMR and 13C NMR were used to detect the natural rubber extracted from L.volemus,and the main chain and branch chain structure of the main component polyisoprene polymer were characterized and analyzed.The main chain was determined to be cis-1,4-polyisoprene structure;For the first time,it was found that its molecular chain also have some branched chains composed of side groups,including epoxy groups,hydroxyl groups,and 3,4-isoprene structures.Among them,the content of epoxy groups is 0.19%(w/w),the content of hydroxyl groups is 0.18%(w/w),and the content of 3,4-isoprene structures is 0.39%(w/w).3.Using the third generation sequencing and Hi-C chromosome conformation capture technology,combined with a variety of advanced splicing software and processes,the whole genome of L.volemus was sequenced,assisted assembled,and functional annotated.In addition,full-length transcriptome sequencing was conducted on samples from different developmental stages,such as mycelium,juvenile,and aged fruit bodies of L.volemus,to study the natural rubber biosynthesis related genes and their transcriptional expression,providing data support for the subsequent mining of natural rubber biosynthesis related genes in L.volemus.The biosynthesis pathway of natural rubber from L.volemus is found to be basically similar to those of most rubber-producing plants,with the upstream pathway of Mevalonate(MVA)biosynthesis pathway,but there is no DXP/MEP(1-deoxy-D-xylose-5-phosphate/2C-methy l-D-erythritol 4-hosphate)biosynthesis pathway that like yeast and most of fungi,and the downstream pathway is polyisoprene biosynthesis pathway.4.Based on the genome and transcriptome annotation results,the relevant functional genes involved in natural rubber biosynthesis were screened,and their transcription factors were predicted;The gene expression levels at different developmental stages such as mycelium,juvenile fruit body,and aged fruit body of L.volemus were analyzed using a weighted gene co-expression network analysis(WGCNA)to obtain gene modules that were more relevant to the changes in natural rubber biosynthesis;Combined with differential gene expression data from the two stages with the greatest differences in natural rubber content,the mycelium and the juvenile fruit body of L.volemus,Venn analysis was used for selection of the key functional genes and transcription factors related to the variety of natural rubber content,as a result including 7 functional genes belonged to 4 enzymes,such as 3-hydroxy-3-methylglutaral-CoA(coenzyme A)reductase(HMGR),methylvalproate 5-phosphate kinase(PMK),geranylgeranyl(-)diphosphate synthase(GGPPS),and hexaprenyl diphosphate synthase(HexPPS),and 22 transcription factors were selected in total.