Identification Of BpSE And BpR2R3-MYB Family Genes And Cloning Analysis Of BpSE4 And BpMYB15 Genes In Betula Platyphylla

Betula platyphylla Suk.is rich in secondary metabolites such as triterpenoids and flavonoids with biocontrol and pharmacological effects.In order to sustainable and large-scale production of secondary metabolites by cell engineering or synthetic biology technology,the analysis of its biosynthesis mechanism is a prerequisite.Therefore,based on the genome and transcriptome data of Betula platyphylla,this study explored the key enzyme squalene cyclooxygenase family genes and R2R3-MYB transcription factors regulating flavonoids synthesis,and analyzed their roles in secondary metabolite synthesis.The main results are as follows:Based on the genome and transcriptome data of Betula platyphylla,four squalene cyclooxygenase(SE)genes were identified.The open reading frame of the family members was 1578～1932 bp,and the number of amino acids encoded was 526～643.Phylogenetic tree analysis showed that the BpSE family genes of Betula platyphylla were closely related to the TwSE family genes of Tripterygium wilfordii Hook.f.The analysis of diurnal variation expression patterns of BpSE family genes showed that BpSE1 and BpSE2,BpSE3,BpSE4 reached their peak values at 24:00,12:00,18:00 and 18:00,respectively.During leaf bud development,the expression trends of BpSEl and BpSE2 were similar,and the expression trends of BpSE3 and BpSE4 were similar.The expression trend of BpSE in roots,stems and leaves of Betula platyphylla was consistent under cadmium treatment,and reached the peak at 1 d,and the expression of BpSEl was the lowest.Therefore,it is speculated that there are temporal and spatial differences in the expression of BpSE family members in Betula platyphylla.The BpSE2 and BpSE4 genes were cloned by PCR,and their yeast expression vectors were constructed and transformed into yeast.It was found that the growth rate of BpSE2-transformed yeast was 3%lower than that of non-transformed yeast on the fifth day,while that of BpSE4-transformed yeast was 2%higher.The contents of triterpenoids and sterols in BpSE2 yeast were 1.57 times and 2.64 times higher than those in untransformed yeast,respectively.The contents of triterpenoids and sterols in BpSE4 yeast were 1.30 times and 1.33 times higher than those in untransformed yeast,respectively.Therefore,it is speculated that BpSE2 and BpSE4 are involved in the synthesis of birch triterpenoids.S-nitrosylation prediction showed that there might be conserved S-nitrosylation sites at position 118 and 276 of BpSE4 amino acid sequence.In order to analyze the role of S-nitrosylation site of BpSE4 in triterpenoids and sterols,the vector was constructed after mutation and transformed into yeast.The amino acid sequence of BpSE4 at position 118 and 276 was 1.41 and 1.31 times higher than that of unmutated triterpenoids,and the sterol content was 1.64 and 1.42 times higher than that of unmutated triterpenoids.Based on the transcriptome data of the second and third generations of Betula platyphylla,44 BpR2R3-MYB genes were identified and distributed on chromosomes except chromosome 12.Phylogenetic tree analysis showed that members of the BpR2R3-MYB family were divided into 27 groups,among which BpMYB15 and BpMYB21 might be related to the synthesis of flavonoids.BpMYB15,BpMYB21 and the key enzyme gene BpF3H for flavonoids synthesis were cloned.Expression pattern analysis showed that the expression of BpMYB15,BpMYB21 and BpF3H genes was different in tissues and temporal and spatial expression after day cycle,leaf bud development,cadmium chloride and N6022 treatment,and the expression trends of BpMYB15 and BpF3H were consistent.Transient transformation of BpMYB15 into Betula platyphylla callus showed that overexpression of BpMYB15 promoted gene expression and flavonoid accumulation of key enzymes in flavonoid biosynthesis,while silencing of BpMYB15 reduced gene expression and flavonoid accumulation of key enzymes in flavonoid biosynthesis.