| Betalain is a kind of natural pigment with tyrosine as its precursor,which has physiological properties such as anti-oxidation and anti-cancer properties.Betalain in nature are mainly found in plants of the order Dianthus and Amanita fungi.Betanins includes two categories,betacyanins and betaxanthin according to different colors.Amaranth is rich in betalain.Because of it’s advantages of fast growth,large biomass and high pigment content,it is expected to become the main source plant of natural betalain.Interestingly,betalain and anthocyanin,which have similar functions,have been found to be unable to co-exist in a plant.Compared with anthocyanins,people still do not fully understand the synthesis mechanism of betalain,and the research on the key genes for the synthesis of betalain in amaranth is even less known.Therefore,studying the synthesis mechanism of betalain in amaranth is helpful to improve the synthesis and metabolism pathway of betalain,and provides a theoretical basis for the utilization of amaranth resources.This study mainly includes the following aspects:1.Select the pure red variety(Jing Xian No.1,JX1),the red and green double color variety(Jing Xian No.3,JX3),and the pure green variety(Jing Xian No.4,JX4),transcriptome sequencing of its leaves and stems.After analyzing the transcriptome data,the key genes related to betalain synthesis and metabolism are identified: encoding cytochrome P450 gene: Atr CYP76AD1,encoding dioxygenase gene: Atr DODA1 and encoding glycotransferase gene: Atr DOPA5 GT.Through bioinformatics analysis,their protein signal peptides,hydrophilicity prediction,subcellular localization of gene expression proteins were predicted and experimentally verified,etc.2.The expression of key genes for betalain synthesis in different tissue parts of amaranth varieties JX1,JX3,and JX4 were detected.Real-time quantitative PCR results show that the two genes Atr DODA1 and Atr CYP76AD1 have the highest expression levels in JX1,followed by JX3,and JX4 have the lowest expression levels.It is positively correlated with the content of Betalain in JX1,JX3 and JX4,and it is speculated that they are the main key bases for regulating the synthesis of Betalain.It was found that the Atr DOPA5 GT gene has higher expression levels in JX1,JX3,and JX4.3.The functions of Atr DODA1,Atr CYP76AD1 and Atr DOPA5 GT were verified by using tobacco transient expression and stable expression system.Transient expression experiment of genes related to betaine synthesis in tobacco leaves.The results table showed that the single genes of Atr DODA1,Atr CYP76AD1,Atr DOPA5 GT could not produce betalain after being injected into tobacco leaves,but the three genes could produce betalain after being expressed at the same time.Construction of the three-gene co-expression vector PTC1300-Atr DODA1-Atr DOPA5GT-Atr CYP76AD1 for transgenic work in dicotyledonous model plants tobacco(Nicotiana tabacum L.)and tomato(Solanum lycopersicum).So far,transgenic tobacco plants and tomato callus that stably express betalain have been obtained,and mass spectrometry has shown that the increased red pigment in the transgenic plants is betalain.The results verified that Atr DODA1,Atr CYP76AD1 and Atr DOPA5 GT are key genes for betaine synthesis in plants.4.Enzyme activity detection of key enzymes in betalain synthesis.Construction of yeast expression vectors PESC-HIS-Atr DODA1,PESC-LEU-Atr CYP76AD1 and PESC-HIS-Atr DODA1-Atr CYP76AD1,and transfer them into WAT11 yeast respectively.The beet genes Bv DODA1 and Bv CYP76AD1 were also constructed with PESC-HIS-Bv DODA1,PESC-LEU-Bv CYP76AD1 and PESC-HIS-Bv DODA1-Bv CYP76AD1 vectors as positive controls.The experimental results show that the color change of yeast fermentation broth transferred with Atr DODA1 and Atr CYP76AD1 genes is consistent with the change of samples expressing sugar beet genes.It shows that Atr DODA1 and Atr CYP76AD1 have corresponding enzymatic activities,further verifying that they are involved in the pathway of betalain biosynthesis. |
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