Application of ga3ox1 protein in regulation of alfalfa plant type
A technology of alfalfa and plant type, applied in the biological field, can solve the problems of limiting the effectiveness and convenience of agricultural activities, plant height, etc.
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Embodiment 1
[0122] Example 1. Obtaining and Phenotypic Detection of Medicago truncatula MtGA3ox1 Gene Knockout Mutant
[0123] 1. Construction of vector p6401-MtGA3ox1
[0124] 1.a. Gene editing target design
[0125] The coding sequence (CDS) of Medicago truncatula R108 is the MtGA3ox1 coding gene shown in sequence 4 in the sequence listing, which encodes the protein MtGA3ox1 whose amino acid sequence is shown in sequence 2 in the sequence listing.
[0126] Through the online target prediction website ( http: / / crispor.tefor.net / ) to generate a target list, select target 1 as 5'-ACACCATCTGGGGAACGAA-3', and target 2 as 5'-AAGCCATTCGTTCCCCAGA-3', such as figure 1 shown.
[0127] 1.b, sgRNA module construction
[0128] As shown in Table 1, the primers MtGA3ox1-BsF, MtGA3ox1-F0, MtGA3ox1-R0, MtGA3ox1-BsF and MtGA3ox1-BsR were synthesized by BGI.
[0129] The p5CBC-Target amplification reaction was carried out by bridging PCR, and the sgRNA module p5CBC-MtGA3ox1 with the target sequence...
Embodiment 2
[0161] Example 2. Obtaining and phenotypic detection of alfalfa MsGA3ox1 gene knockout mutants
[0162] 1. Construction of alfalfa MsGA3ox1 gene CRISPR / Cas9 system vector
[0163] 1.a. Gene editing target design
[0164] The coding sequence (CDS) contained in Medicago sativa is the MsGA3ox1 coding gene shown in sequence 3 in the sequence listing, and the protein MsGA3ox1 whose amino acid sequence is shown in sequence 1 in the sequence listing is encoded.
[0165] Through the online target prediction website ( http: / / crispor.tefor.net / ) to generate a target list, select target 1 as 5′-ACACCATCCGGGGAACGAA-3, and target 2 as 5′-AAGCCATTCGTTCCCCGGA-3′.
[0166] 1.b, sgRNA module construction
[0167] Primers MsGA3ox1-BsF, MsGA3ox1-F0, MsGA3ox1-R0 and MsGA3ox1-BsR were synthesized by BGI Corporation.
[0168] The p5CBC-Target amplification reaction was carried out by bridging PCR, and the sgRNA module p5CBC-MsGA3ox1 with the target sequence was amplified. The reaction system ...
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