Construction Of Prokaryotic Expression Vector For Mulberry Melatonin Synthesis And Functional Analysis Of Its Key Genes

Melatonin(Melatonin,MT)was first isolated from the pineal gland of cattle in 1958.In 1995,Dubbels and Hattori et al identified melatonin humans in higher plants.The biosynthesis of melatonin has been analyzed successively in animals and model plants.With the in-depth study of melatonin,its physiological function has been paid more and more attention by scientists.Melatonin plays an important role in chronobiology in animals,regulating circadian rhythm,maintaining rhythm stability,improving sleep,treating neurasthenia and improving immunity.In plants,melatonin can participate in all stages of plant growth and development,regulate plant physiological activities,and more importantly,melatonin can scavenge reactive oxygen species in plants to improve their tolerance to adverse environment.Plants rich in melatonin will show strong stress resistance.at the same time,environmental stress can also affect the level of endogenous melatonin in plants,which can enhance the tolerance of plants to external stresses by increasing the level of melatonin.Abiotic stress caused by extreme temperature,drought,salt stress,radiation and chemical stress,and biological stress caused by bacteria and pests.In plants,melatonin is synthesized by substrate L-tryptophan via Tryptophan decarboxylase(TDC),Tryptamine-5-hydroxylase(T5H),N-acetylserotonin methyltransferase(SNAT),N-acetylserotonin methyltransferase(ASMT)or Caffeic acid-O-methyltransferase(COMT).Melatonin is produced by continuous enzymatic reaction.We are concerned that the first two enzymes TDC and T5 H,which play a role in plants and animals,act in the opposite order.Among them,TDC enzyme is considered to be the first rate-limiting enzyme in melatonin biosynthesis pathway.The expression and regulation of melatonin gene is affected by species,environment and other factors,and plays different physiological roles in plants,such as regulating plant growth and development,resisting external stress and so on.However,there are few studies on T5 H enzyme.Some studies have shown that T5 H enzyme plays an irreplaceable role in the synthesis of melatonin,and plants without T5 H enzyme can hardly synthesize melatonin.Mulberry originated in China and was originally planted as silkworm feed.Now mulberry has been developed and utilized as a tree species with important edible,medicinal and ecological functions.Studies have shown that mulberry is a plant with high melatonin content.And some researchers have found that the expression level of TDC gene is closely related to the content of melatonin in mulberry,and T5 H enzyme is an indispensable key enzyme in the process of melatonin synthesis,but there is almost no research on the function of endogenous expression of these two genes in plants.In 2017,we identified the presence of melatonin in Sichuan mulberry and compared with several mulberry varieties,we found that the content of melatonin in Chuan mulberry was the highest.37 candidate genes related to melatonin biosynthesis were identified in the whole genome database of Chuan mulberry by bioinformatics method and all of them were successfully cloned.Among them,there is one TDC(MnTDC)gene and seven T5H(MnT5H1-7)genes.Combined with phylogenetic tree and sequence alignment,and the expression level of each gene was analyzed,it was found that the expression level of MnTDC gene was higher in Chuan mulberry leaves,and the expression level of MnT5H2 gene in Sichuan mulberry tissues was higher than that of other T5 Hs genes.Through the enzyme activity experiment in vitro,it is determined that the enzymes encoded by these two genes can synthesize the corresponding intermediates in the melatonin biosynthesis pathway,which indicates that the thorough study of TDC and T5 H genes is of positive significance for further exploring the physiological function of TDC and T5 H enzymes and regulating the content of melatonin in plants..This study is based on the previously completed informatics analysis of MnTDC and MnT5H2 genes.By constructing plant overexpression vectors of MnTDC and MnT5H2 to improve Agrobacterium-mediated transfer into tobacco,3 MnTDC overexpression transgenic lines and 5MnT5H2 overexpression transgenic lines were successfully obtained.Three strains with significant differences in expression levels were selected and treated with salt stress.By measuring chlorophyll content,proline content,malondialdehyde content,peroxidase activity and catalase activity,and stress related genes Expression and other indicators were used to explore the mechanism of MnTDC and MnT5H2 genes in stress response,and to extract and identify melatonin-related compounds in tobacco,and then to verify the function of transgenic tobacco against aging.In addition,we hope to rebuild the biosynthetic pathway of melatonin in Sichuan mulberry in Escherichia coli and increase the biosynthesis of melatonin through prokaryotic expression.Therefore,we have constructed four key enzymes that connect the melatonin synthesis pathway.The prokaryotic expression recombinant vector of the coding gene is expected to lay the foundation for the development and utilization of melatonin.The research results are as follows:1.Construction of plant overexpression vector of MnTDC and MnT5H2 genesThe MnTDC and MnT5H2 gene fragments were successfully cloned from the c DNA of Chuan mulberry.The full length of MnTDC gene is 1521 bp,encoding 506 amino acids,and the protein molecular weight is 56.87 KD.MnT5H2 gene is 1530 bp,encoding 509 amino acids,and the protein molecular weight is 57.49 KDa.The two genes were ligated to the plant overexpression vector p LGNL respectively,and the plant overexpression vectors p LGNL-MnTDC and p LGNL-MnT5H2 were constructed.The constructed plasmids were successfully transferred into Agrobacterium tumefaciens receptive cells,and the strains identified as positive by PCR were preserved.2.Transgenic plants obtained by genetic transformation of tobacco mediated by Agrobacterium tumefaciensSeveral overexpressed transgenic lines(over-expression,OE)were obtained from tobacco transformed by positive Agrobacterium tumefaciens leaf disc method.Among them,Three transgenic lines with overexpression of MnTDC and five transgenic lines with overexpression of MnT5H2 were identified as transgenic positive lines by GUS staining and genomic PCR identification of transgenic tobacco.According to the quantitative detection of target gene expression in transgenic tobacco,q RT-PCR results showed that in MnTDC transgenic tobacco,the expression of OE3 was the highest and that of OE1 was the lowest,and there were significant differences among transgenic lines.OE1,OE2 and OE3 were used as follow-up experimental materials.In MnT5H2 transgenic tobacco,the expression of OE1 was the highest and that of OE5 was the lowest.In all transgenic lines,there was no significant difference between OE1 and OE3,and there was no significant difference between OE4 and OE5.As a result,OE1,OE2 and OE5 transgenic lines with high expression and significant differences were selected as experimental materials in the later experiment.Under natural growth conditions,we determined the substances in melatonin biosynthesis pathway in MnTDC and MnT5H2 transgenic tobacco.the results showed that the transfer of MnTDC and MnT5H2 genes increased the biosynthesis of melatonin,and the plant height of transgenic tobacco was significantly higher than that of wild-type tobacco.This proves that the melatonin content in genetically modified tobacco is increased,which promotes plant growth.3.Functional verification of MnTDC and MnT5H2 transgenic tobaccoUnder salt stress,compared with WT tobacco,transgenic tobacco grew better and could effectively alleviate the plant wilt caused by salt stress.The substances related to melatonin synthesis in tobacco were extracted and detected by UPLC-MS / MS.The results showed that the content of melatonin in transgenic tobacco was higher than that in WT tobacco.The results showed that CAT gene expression and enzyme activity,SOD expression and POD enzyme activity in transgenic tobacco were significantly higher than those in WT tobacco,the contents of osmotic substances such as Pro and genes regulating osmotic pressure such as ERD10 C in transgenic tobacco were significantly higher than those in WT tobacco,and the content of MDA in transgenic tobacco was lower than that in WT tobacco.The results showed that tobacco plants overexpressing MnTDC and MnT5H2 genes could increase the gene expression of Nt CAT and other antioxidant enzymes and the activities of CAT and other antioxidant enzymes under salt stress,so as to eliminate the excessive reactive oxygen species produced in the cells under salt stress,reduce the amount of malondialdehyde produced by plasma membrane lipid peroxidation,maintain the stability of cell membrane structure,and alleviate the chlorophyll degradation caused by salt stress.And up-regulate the expression of osmotic pressure-related gene ERD10 C and increase the content of osmotic regulator proline,so as to enhance the tolerance of plants to salt stress.We believe that the increase of resistance to salt stress in transgenic tobacco plays a role in the increase of melatonin content in transgenic tobacco.4.Construction of prokaryotic expression vector for melatonin synthesisThe recombinant prokaryotic expression vector p Cold-TF-MnTDC-TF-MnT5H2-TFMnSNAT5-TF-MnASMT12 was successfully constructed through homologous recombination,and it was transformed into E.coli expression strain and induced expression.It was found that the protein encoded by the target gene can be found on the cell.Soluble expression in the serum,the recombinant strain consumes a large amount of substrate L-tryptophan,but cannot promote a significant increase in melatonin content.After the recombinant protein was extracted and purified,the enzyme activity experiment in vitro showed that TDC enzyme played a normal role,but T5 H,SNAT and ASMT enzyme activity experiments were not carried out smoothly.We think that this is also the reason why the recombinant vector strain did not have a positive effect on melatonin production in vivo.To sum up,the preliminary functional exploration of MnTDC and MnT5H2 genes under salt stress showed that overexpression of these two genes alone could promote the synthesis of melatonin and improve the tolerance of plants to salt stress.We think that these two results are inseparable.It is due to the increase of melatonin content in transgenic plants that the salt tolerance of plants is improved.And we have successfully constructed p Cold-TF-MnTDC-TF-MnT5H2-TFMnSNAT5-TF-MnASMT12 recombinant prokaryotic expression vector,but the expression of this recombinant vector needs to be further explored.