Functional Verification Of Saccharopine Dehydrogenase Gene In Flammulina Filiformis

Flammulina filiformis is rich in lysine,and lysine has a good effect on children's height and intellectual development.Therefore,F.filiformis is known as a mushroom that enhances intelligence.Based on the whole genome sequence of F.filiformis,key genes in the lysine biosynthesis pathway of F.filiformis were screened,and it was confirmed that lysine was synthesized through the ?-aminoadipate pathway(AAA pathway).Based on the AAA pathway and the key genes in the pathway as the core,studying its molecular and regulatory mechanism will help develop the edible and commercial value and lay a theoretical foundation for the development of new products and the cultivation of new varieties of F.filiformis.In this study,two exogenous influencing factors,temperature and methyl jasmonate,were used to induce the lysine synthesis pathway of F.filiformis,to explore its molecular mechanism and regulatory mechanism,and to identify the target genes that play an important role in the synthesis process,and further verify the purpose the function of genes.It helps to deepen the understanding of the lysine biosynthesis pathway of F.filiformis.This is also the first time that low temperature stress and methyl jasmonate induction have been performed on the lysine synthesis pathway of F.filiformis.The main research works and conclusions are as follows:1.Low-temperature stimulation was performed on the mononuclear strain Dan3 mycelia of F.filiformis to explore the regulation mechanism of SDH gene expression and lysine synthesis by cold treatment.After culturing F.filiformis Dan3 liquid mycelia at different temperatures(10,15,20,25 ?)for 7-10 days,the mycelia were collected for quantitative PCR and lysine detection.The liquid mycelia of F.filiformis Dan3 was cultured for 7-10 days(25 °C)and treated at an ambient temperature of 15 °C for 0,2,4,6,8,10,and 12 h,respectively.The mycelia were collected for quantitative PCR and lysine detection.The results showed that after treatment at different temperatures,the expression of HCS and HIDH genes increased at 15,20 ?;the expression of AAT1 gene increased at 10,15,20 ?,and the expression was higher at 20 ?;gene expression increased at 20 ?;SDH gene expression increased at 15 ?.After the mycelium was treated at 15 ?,except for 4 h treatment,the expression of SDH gene increased,and the lysine content increased.The results show that low temperature affects the expression of lysine synthesis genes and lysine synthesis in Flammulina velutipes mycelium,providing a reference for exploring the mechanism of lysine synthesis.2.Me JA induction was performed on the mononuclear strain Dan3 mycelia of F.filiformis to explore the mechanism of Me JA induction on SDH gene expression and lysine synthesis.Me JA solution was prepared using absolute ethanol as a co-solvent,and Me JA concentration(0 ?M,5 ?M,10 ?M,50 ?M,100 ?M,150 ?M,200 ?M,250 ?M,300 ?M)were used to induce the liquid mycelia of F.filiformis Dan3.The mycelia were collected for quantitative PCR and lysine detection.The results showed that after induction with different Me JA concentrations,the expression of SDH gene was upregulated and down-regulated,and the lysine content was up-regulated.It is speculated that it may be the synergistic effect of multiple enzymes during the lysine biosynthesis process.Me JA may affect other enzymes in the pathway while affecting SDH,and then affect the final lysine synthesis.Therefore,it is necessary to study the changes in the transcription levels of other genes in the pathway by Me JA to provide a strong theoretical information for a comprehensive understanding of Me JA's regulation of SDH,so as to improve the understanding of the role of SDH in the lysine biosynthetic pathway.3.Bioinformatics analysis of F.filiformis SDH gene includes sequence analysis,homology analysis,promoter analysis,and phylogenetic tree construction.To verify SDH overexpression function,an overexpression vector of SDH for F.filiformis(named p YN6981-SDH)was constructed,and the SDH gene was integrated into the chromosomes of F.filiformis by Agrobacterium-mediated genetic transformation to verify its overexpression function.The specific method is as follows: the mononuclear strain Dan3 mycelia of F.filiformis was selected as the recipient and two kinds of millets(foxtail millet and proso millet)were selected for the first time as the mycelium culture and the infection medium for comparison.In the transformation process of Agrobacterium strain LBA4404,the co-culture duration was seted for 3,4,5,6 days as the optimization condition,and the first and second screening were performed on PDA plates containing hygromycin and cefotaxime.The transformants were tested by PCR and e GFP green fluorescence,and the positive transformants were subjected to the detection of real-time PCR and lysine content.Agrobacterium-mediated transformation efficiency of F.filiformis mycelium is relatively high,and the average transformation efficiency is 41.0 % and 30.1 % in the foxtail millet and proso millet respectively,both of which reached the highest transformation efficiency on the 6th day of co-cultivation,57.5% and 47.5% respectively.The expression of SDH gene and the content of lysine are both upregulated,which verified the overexpression function of SDH gene,and further explained that changes in SDH gene will affect the synthesis of lysine,which is speculated to be a key gene in the pathway.This study mainly verified the function of SDH gene from two aspects: Agrobacterium-mediated genetic transformation and regulation of environmental factors.It was speculated that SDH is a key gene in the lysine biosynthesis pathway of F.filiformis.The results of this study provide ideas for exploring the regulatory mechanisms of other key genes in the pathway,have a further understanding of the lysine biosynthesis pathway and its key genes at the molecular level and provide theoretical basis for breeding F.filiformis with high lysine content.