Molecular Cloning,Expression Characteristics,and Function Of BpMYB21 And BpMYB61 Genes From Birch (Betula Platyphylla SuK.)

Triterpenoids from Betula platyphlla Suk.has antitumor and anti-HIV activities and so it has become the most promising drug preparation and has important application prospects.Transcription factors can be regulated gene expression and then adjusting the production of secondary metabolites,which are commonly used as a tool to transform metabolic pathways.Birch was experimental materials,the full-length and promoter sequences of BpMYB21 and BpMYB61 genes which was screened in our laboratory previously in response to MeJA and SA signals,and were cloned and analyzed by bioinformatics.The expression characteristics and function of two genes in birch and yeast were analyzed.The results of this study will provide technical support and theoretical basis for genetic transformation and biotechnological utilization of triterpenoids from Birch.The results of this study were as follows:1.The full-length cDNA of birch BpMYB21 and BpMYB61 gene was obtained by cloning and using specific primers combined with RT-PCR.Bioinformatics analysis show that the ORF of the BpMYB21 gene is 1014bp and encodes 337 amino acids,which has a MYB binding site and two SANT Super family,is a hydrophilic protein,and has four two-level structures,and Juglans regia myb30-like has 64%similarity,and Populus euphratica MYB86 similarity is 61%.The ORF of the BpMYB61 gene is 1203bp,encodes 400 amino acids,has two MYB binding site conserved domain,is hydrophilic protein,and the same homology of Betula luminifera myb-like High of 97%.Phylogenetic tree showed that the BpMYB21 transcription factor of Betula Platyphylla was on a branch with Juglans regia,BpMYB61 transcription factor and Betula luminifera on one branch.According to the results showed that the BpMYB21 gene is a new member of the MYB family from Birch.2.The expression patterns of BpMYB21 and BpMYB61 gene were analyzed by real-time PCR technique.The results showed that in the three-year-old birch during the middle of June to September mid-last,the expression of BpMYB21 and BpMYB61 gene showed high expression level in August and July.The expression of BpMYB21 and BpMYB61 genes in tissue culture seedlings was as follows:expression level in leaves and stems was significantly higher than in roots.The response patterns of two genes of BpMYB21 and BpMYB61 in different hormones were significant difference.In leaves,the expression of BpMYB21 gene was highest under treatment ABA and GA,while the other treatments were the highest expression of BpMYB21 gene of 6h.BpMYB21 genes were the highest expression in the processing of 12h under treatment ABA and ethylene.In the leaves,the highest expression of BpMYB61 of 6h under ethylene and injury-treated treatment;in the stems,the treatment of ABA,MeJA and SA reduced the expression of BpMYB61,while GA,ethylene and injury treatments increased the expression of BpMYB61 gene.Subcellular localization experiments showed that the expression of BpMYB21 and BpMYB61 in cell membrane and nucleus.3.The promoter sequences of BpMYB21 and BpMYB61 genes with lengths 1302bp and 850bp were amplified by chromosome step-shift method.The results of the promoter sequence analysis showed that the BpMYB21 and BpMYB61 promoter contain basic components such as TATA-box and CAAT-box.At the same time,several plant hormone response elements such as Gibberellin response element,Abscisic acid response element,Jasmine acid response element and Salicylic Acid response element were obtained.There are also drought responses,heat-stress-related elements,anaerobic-inducible response elements,and,defense and stress elements.In addition,BpMYB21 and BpMYB61 gene promoter sequences have MYB transcription factor binding locus MBS components and bHLH transcription factor binding sites G-box components,suggesting that there may be interactions between the two transcription factors and the bHLH transcription factors.4.The yeast expression vectors of BpMYB21 and BpMYB61 were obtained by pYES3/CT plasmid,and were transformed by recombinant vectors to the INVScl,INVScl-pYES2-SS,INVScl-pYES2-SE yeast strain.The results showed that the contents of squalene and total triterpene in the six recombinant yeasts were increased to different extent compared with control yeast strains of INVScl-pYES3,among which the recombinant yeast INVScl-pYES-MYB21-SS had the highest content squalene compared with the control strain of INVScl-pYES3,increased by 89%,and squalene had the highest content in BpMYB61 recombinant yeast INVScl-pYES3-MYB61,which was 78%higher than the control yeast INVScl-pYES3.In BpMYB21 recombinant yeast INVScl-pYES-MYB21-SS,the total triterpene content was increased by 20%.The total triterpenoids content of INVSc1-pYES3-MYB61 of BpMYB61 recombinant yeast was 23%higher than that of INVScl-pYES3 yeast.5.The leaf,petiole and stem segments of birch were infected by pCAMBIA1303-BpMYB21 and pCAMBIA1303-BpMYB61,respectively,44 strains of pCAMBIA1303-BpMYB21 and 45 strains of pCAMBIA1303-BpMYB61 resistant plants were obtained,4 and 7 of GM birch seedlings were identified by PCR,respectively.Key genes in MVA pathway in transgenic plant MYB21-44 were increased in different degrees,and the most significant increase was the HMGR gene,which was 28.24 times of the control.In transgenic plant MYB61-5,the relative expression amount of FPS and SS was increased by 35%and 13%respectively,while the gene expression of HMGR,BPW,SE and BPY were reduced,and the most obvious effect was BPY gene,84%lower than the control.It is suggested that the function of two genes in regulating triterpene synthesis may be different.6.The cis-acting elements bound by six MYB transcription factors were screened from the genes for the synthesis of key enzymes in the triterpenes of birch,and the bait vector of pHis-elements was successfully constructed.The capture vectors pGADT7-Rec2-MYB21 and pGADT7-Rec2-MYB61 were successfully constructed.The results of interaction experiments showed that the No.1 and No.4 elements existed in the promoter of the SE gene and the promoter of the BPX gene,and the two elements were linked to BpMYB21.There is a weak interaction with BpMYB21,and there is a strong interaction with BpMYB61.It is suggested that BpMYB21 and BpMYB61 have regulatory roles on SE and BPX,while BpMYB61 may have stronger regulatory effect.