Mutated della protein and its application
A mutated protein and protein technology, applied in the fields of application, recombinant DNA technology, DNA / RNA fragments, etc., can solve the problems of reducing corn plant height, reducing the ability to recognize and bind, repressing the transcription and expression of growth-related genes, etc., to achieve plant height reduction Effect
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Embodiment 1
[0108] Example 1. Target design and vector construction
[0109] Firstly, the D8 (Dwarf8) gene, the gene encoding DELLA protein in maize, was analyzed through the Maize Gene and Genome Database (maizeGDB) and NCBI, the sequence of the D8 gene was obtained, the relevant gene sequence information was marked, and the downloaded D8 gene sequence was annotated. The motifs of each domain and the range of exons and introns are shown.
[0110] The purpose of this study was to change the amino acid sequence of the DELLA domain of DELLA protein, so we chose to edit the N-terminal amino acid 1-105 (including DELLA motif and VHYNP motif) region of DELLA protein. The 1-105th amino acid sequence of DELLA protein N-terminal coded by D8 gene of maize B73 inbred line provided by NCBI is as follows: MKREYQDAGGSGGDMGSSKDKMMAAAAGAGEQEEEDVDELLAALGYKVRSSDMADVAQKLEQLEMAMGMGGVGGAGATADDGFVSHLATDTVHYNPSDLSSWVES, see SEQ ID No.1 in the sequence table. Different maize inbred lines were sequenced, and th...
Embodiment 2
[0120] Embodiment 2, genetic transformation
[0121] In this example, the maize inbred lines Zheng 58, Qi 319 and XCW175 were selected, and the vectors in Example 1 were used for gene editing respectively.
[0122] 2.1 Transformation of Agrobacterium
[0123] The vector in Example 1 was transferred into Agrobacterium strain EHA105 by the heat shock method, and single clones were picked, subjected to liquid culture and PCR identification, and stored in a -80°C refrigerator for later use.
[0124] 2.2 Activation of strains
[0125] The bacteria were removed from the refrigerator and streaked on YEP solid medium.
[0126] 2.3 Prepare Agrobacterium infection solution
[0127] Scrape fresh bacterial cells from the newly activated bacterial plate and resuspend in the infection solution.
[0128] 2.4 Take young corn embryos
[0129] Take the corn ear about 10 days after pollination, remove the bracts and filaments, pick the young embryos, and put them into the infection medium (...
Embodiment 3
[0140] Example 3. Screening of positive seedlings
[0141] For regenerated seedlings, a small amount of leaves were taken to extract genomic DNA by CTAB method. Identify whether the constructed maize D8 gene editing vector is transferred into maize embryos, and design primers for the elements that are not in the maize genome but contained in the vector, and use primer pairs Cas-jc-F1 (AAGAAGCGGAAGGTCGGTAT) and Cas-jc-R1 (CTCAGGTGGTAGATGGTGGG), Cas-jc-F2 (CAGAAAGAGCGAGGAAACCA) and Cas-jc-R2 (CCTCAAACAGTGTCAGGGTCA), ZmU6P-F (AAACAGCAGTCCGTAGGTG) and ZmU6T-R (AGAATTGGCGAGGACTGA) and other three pairs of primers were detected separately, any pair of primers can be amplified to The target product, the regenerated seedling is the transgenic positive seedling.
[0142] The corresponding fragments of the D8 gene were amplified for transgenic positive shoots with primer pairs ZmD8-jc-F1 (CCCTCCCCTACCCTTTCCT) and ZmD8-jc-R1 (TGTGACGGTGGACGATGTGG). The amplified product was sequenced b...
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