Application of Sesame siwrky67 Gene in Regulation of Melatonin Synthesis
A technology of melatonin and sesame, applied in the field of plant genetic engineering, can solve the problems of few reports on melatonin synthesis regulation genes, and achieve the effect of increasing the content of melatonin and improving the nutritional quality of sesame
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Embodiment 1
[0023] Embodiment 1 Sesame SiWRKY67 gene acquisition and overexpression vector construction
[0024] 1. Acquisition of Sesame SiWRKY67 gene
[0025] (1) From the 7910 domestic and foreign resources preserved in the National Sesame Medium-term Bank, according to the geographical origin and genetic diversity detection results, a step-by-step sampling strategy was adopted, and 500 sesame materials were selected for resequencing analysis;
[0026] (2) Using the Illumina Hiseq 2500 sequencing platform, the 2×76 paired-end sequencing method was used to resequence the whole genome of 500 sesame materials with low coverage, and obtained a genome sequence with 2.6 times coverage;
[0027] (3) Genome-wide association analysis was performed on the melatonin content of sesame seeds in the two environments using the EMMAX software package, and a SNP located at 6839561bp on linkage group 6 was detected in both environments and the seed melatonin content significant association (P-20 )(Such...
Embodiment 2
[0041] Example 2 Functional Verification of Sesame SiWRKY67 Gene in Sesame Hairy Roots
[0042] The vector pCAMBIA1301S-SiWRKY67 prepared in Example 1 was transferred into Agrobacterium rhizogenes K599, and then the sesame cotyledon explants were infected to induce the occurrence of transgenic hairy roots, and the expression of SiWRKY67 gene in the transgenic hairy roots and the degeneration of the gene were detected. Melanin content. The specific operation is as follows:
[0043] 1. Induction and cultivation of transgenic SiWRKY67 sesame hairy roots
[0044] (1) The recombinant vector is transformed into Agrobacterium K599:
[0045] 1) Add 2 μg of plasmid DNA to each 100 μL of K599 Agrobacterium competent cells, beat the bottom of the tube by hand to mix, and then place it on ice for 5 minutes, liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, and ice bath for 5 minutes.
[0046] 2) Add 700 μL of LB liquid medium without antibiotics, and culture with shaking at...
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