A kind of bahd acyltransferase gene and its application
A technology of use and gene fragment, applied in the application field of BAHD acyltransferase gene, to achieve the effect of increasing value
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Embodiment 1
[0033] Example 1 Isolation and prokaryotic expression of HVUL2H29914 gene
[0034] This example is mainly about the method of HVUL2H29914 gene acquisition, vector construction and prokaryotic expression.
[0035] (1) Construction of gene fragments and vectors
[0036] Weigh 1 gram of fresh barley leaves, extract barley RNA, synthesize cDNA with M-MLV Reverse Transcriptase from Thermo Fisher Company, and design primers as follows:
[0037] F: CCGAGATCTATGAAGATCACCGTGCACTCTTC
[0038] R: CCGGAATTCCTAAAAGAGTGGCACGTAGGCGTCC
[0039] Perform PCR amplification to obtain fragments of the target band size (results such as figure 1 shown). The PCR product was purified with a gel extraction kit (Gel Extraction Kit D2500-02, OMEGA).
[0040] The nucleotide sequence (SEQ ID NO.1) of the amplified target fragment HVUL2H29914 gene is:
[0041]ATGAAGATCACCGTGCACTCTTCCAAGGCCGTCAAGCCCGAGTACGGCGCCTGCGGCCTCGCTCCGGGGTGTACCGCCGACGTCGTCCCGCTCACCGTGCTCGACAAGGCCAACTTCGACACGTACATCTCGGTGATCTACGCC...
Embodiment 2
[0057] The construction of embodiment 2 transgenic tobacco
[0058] ①Transform the transient expression vector containing the target gene (transient expression vector pEAQ, from John InnesCentre) into Agrobacterium (EHA105);
[0059] ②Pick positive Agrobacterium clones in 500ul LB containing corresponding antibiotics (kn), and culture them for 20-24 hours;
[0060] ③Transfer 200ul into 5ml LB containing the corresponding antibiotic (kn), shake at 28°C at 220rpm to about OD=2.0.
[0061] ④ Centrifuge at 10000rpm at room temperature for 2 minutes to collect the bacteria, resuspend the bacteria with the transformation buffer prepared in advance, and shake on the shaker for 3 hours; the composition and concentration of the buffer working solution are as follows: 10mM MES (pH5.7), 10mM MgCl2, 100μM acetosyringone (acetosyringone, AS).
[0062] ⑤Take the prepared 1ml syringe, remove the needle, select a syringe with a smooth outlet to inhale the bacterial liquid, take the Nicotian...
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