Coenzyme Q synthetic pathway benzene ring 6-site hydroxylase and application thereof
A technology of hydroxylase and benzene ring, applied in the field of genetic engineering, can solve the problem of incomplete analysis of synthetic pathways
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Embodiment 1
[0031] Example 1: Cloning of Arabidopsis thaliana CoqF gene and complementation of Escherichia coli ΔubiF mutant strain
[0032] The genes of the plant coenzyme Q synthesis pathway have the characteristics of co-expression, and some candidate hydroxylase genes were screened through co-expression analysis with the known genes of the Arabidopsis coenzyme Q pathway. The activity of these candidate genes was detected by the method of complementing Escherichia coli 6-hydroxylase gene UbiF mutant strain. It was found that after transferring one of the candidate genes (At1g24340, NP_173844.2, GeneID: 839050, its nucleotide sequence such as SEQ ID NO.1, and its amino acid sequence such as SEQ ID NO.2), it can complement the growth of the UbiF mutant strain, Hence the name CoqF. CoqF is a gene of unknown function in Arabidopsis. The specific experimental process is as follows.
[0033] To obtain Escherichia coli ΔubiF mutant strains, the UbiF gene of Escherichia coli MG1655 strain w...
Embodiment 2
[0040] Example 2: Arabidopsis thaliana CoqF gene complementation of Saccharomyces cerevisiae Δcoq7 mutant strain
[0041] 将拟南芥CoqF基因(扩增引物ACGAGATGTAAGAGCACAATGGCGATTCTAGGGCTT,ATCGAATTCCTGCAGCCCTCATTGTTTCCCTAGTAT)N端融合酵母Coq3的线粒体信号肽(扩增引物TTCGATATGGGATCCCCCATGTTGTTAAGATCTAGA,TGTGCTCTTACATCTCGT)并克隆至酵母表达载体pRS426,以酵母Coq8的启动子(TTGCGGCCGCGATCCGGGTGTTCGG,CGCGGATCCCATATCGAACGATATCTT) and terminators (GACGTCGACAATACTTCCCCGCTATTTG, CGCTCGAGGCTATTGGCAGAAGGATT) regulate expression.
[0042] The yeast Coq8 gene (CCGCGGCCGCATGGTTACAAATATGGTGAAAT, GGCTCGAGTTAAACTTTATAGGCAAAAATC) was cloned into the expression vector pRS423.
[0043] The above two vectors were co-transformed into Saccharomyces cerevisiae Δcoq7 mutant strain (Y12381, purchased from EUROSCARF), and the obtained positive transformants were respectively inoculated on SD medium with 2% glucose or 3% glycerol and 2.5% ethanol as carbon sources ( Figure 5 In A), the results show that it can grow on the medium with glycerol and ethanol a...
Embodiment 3
[0046] Example 3: CoQ content decreased in RNAi transgenic plants of Arabidopsis CoqF
[0047] Construction of RNAi vector: PCR amplification of about 300 bp fragment of AtCoqF coding region (GGGGACAAGTTTGTACAAAAAGCAGGCTTCAACAGTTGGTTCGATCCT, GGGGACCACTTTGTACAAGAAAGCTGGGTTTCTTGAACCTGGTTCAGC), cloned into vector pDonr207 by BP reaction, and then reverse cloned the two fragments into vector pK7GWIWG2R(II) by LR reaction. The wild-type Arabidopsis Col-0 was transformed by flower dipping method. Obtain 3 significantly down-regulated lines and detect CoQ 9 The content was significantly lower than that of the wild type, indicating that AtCoqF was involved in the synthesis of CoQ in plants.
[0048]As shown in Figure 6, A shows the relative expression of the AtCoqF gene detected by qRT-PCR in the three transgenic lines. B shows the coenzyme Q9 content in the aerial part of the plant detected for 4 weeks, and the data are the mean ± standard deviation of four biological replicates. ...
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