| Taurine,also known as bovine choline and Bilin,is one of the most abundant free amino acids in vertebrates.It is mainly distributed in the brain,heart,liver,kidney,muscle and blood of animals.It is mainly synthesized with taurine as the substrate in higher animals under the catalysis of flavin monooxygenase(FMO).Taurine plays an important role in the development of visual nerve,the function of liver and gallbladder,the regulation of osmotic pressure,antiinflammation and anti-oxidation of many higher animals,but there is little research on taurine in plants.Our previous study found that the content of taurine and hypotaurine in maize ocd1 mutant was significantly higher than that of wild type.Therefore,it is speculated that taurine can be synthesized in corn,and hypotaurine may also be used as the substrate.In this study,the key enzyme flavin monooxygenase,which catalyzes the conversion of hypotaurine to taurine in maize was systematically analyzed and its function was identified in order to explore the biosynthesis of taurine in plants.According to the genome annotation,17 flavin monooxygenase family genes were screened and named as Zm FMO1-17.(1)The phylogenetic tree of 17 flavin monooxygenase family genes was constructed using Mega 6.0.The results showed that FMO family was mainly divided into two clusters during the evolution of monocotyledons and Zm FMOs had close evolutionary relationship with Os FMOs and At FMOs;(2)The results of RT-PCR showed that half of Zm FMOs were expressed in all parts of corn and Zm FMO10 was the most specific in maize tissues;(3)Compared with the wild type,seven genes of Zm FMOs family were up-regulated at transcriptional level.The average expression level of Zm FMO6 transcript in ocd1 was about3.6 times higher than that of the wild type;(4)Zm FMO6 were selected as the main research objects.There is a typical PLN02171 domain was found in its protein sequences which was unique to flavin monooxygenase family;(5)The prokaryotic expression vectors of Zm FMO6 were constructed and transformed into E.coli successfully.In vitro enzyme activity assay using NADH as a cofactor showed that Zm FMO6 encoded an enzyme protein with strong activity and could catalyze the conversion of hypotaurine to taurine;(6)The expression vector of Zm FMO6-35S:GFP1300 was constructed.After agrobacterium tumefaciens was transformed into tobacco,the transient expression of Zm FMO6 was observed under laser confocal microscope;(7)Pro UBI-Zm FMO6-GFP overexpression vector was successfully constructed by homologous recombination;(8)The mutant zmfmo6 was identified by EMS mutation and finally obtained 5 pure mutant plants.At present,the content of taurine in maize was detected in the process of propagation. |
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