Gene and method for altering maize flowering period
A technology of flowering and mutating genes, applied in the field of molecular genetics, can solve problems such as incomplete clarity
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Embodiment 1
[0058] Example 1 Identification of changes in maize flowering period after knockout of candidate genes by gene editing
[0059] The present invention uses the CRISPR-Cas9 gene editing technology to perform fixed-point editing on 15 genes among the 34 genes related to maize flowering period shown by the association analysis results.
[0060] Embodiments include the construction of gene editing vectors, genetic transformation of maize, and functional verification of editing effects. details as follows:
[0061] 1. Construction of gene editing vector
[0062] The gene editing vector of the present invention is G08943-CPB-ZmUbi-hspCas9, and its vector diagram is as follows figure 1 shown. The basic vector of this vector is CPB-ZmUbi-hspCas9. In the present invention, the dual-target U6-sgRNA is obtained by OverlapPCR and then cloned into the basic vector by homologous recombination. The specific construction process is as follows:
Embodiment 2
[0075] Example 2 In-depth analysis of corn flowering traits and identification of mutant genes
[0076] Conduct more in-depth trait identification of gene-edited materials with altered flowering traits and analyze specific editing sites. After the gene editing of GRMZM2G148453, the maize flowering period of the T0 material was earlier than that of the recipient control KN5585. The anthesis of T1 material from this transformant was further characterized and analyzed for specific editing sites.
[0077] There are two target sites designed during gene editing of GRMZM2G148453, as shown in SEQ ID NO.2 and SEQ ID NO.3 respectively. The gRNA sequences expressed by the vectors containing these two targets are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively.
[0078] Extract seedling DNA to detect gene editing. Primers were designed, and the primer sequences are shown in SEQ ID NO.7 and SEQ ID NO.8. Amplify the target editing segment, the amplification system is: DNA: 3 μL, bid...
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