Method for increasing carotenoid content of corn kernels by using gene editing technology
A gene editing and carotene technology, applied in the field of genetic engineering, can solve the problems of gene redundancy, time-consuming, limiting the application effect of improved materials, etc., and achieve the effect of increasing the content
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Embodiment 1
[0042] Example 1 Construction of gene editing vector
[0043] (1) starch debranching enzyme gene ZmISA2, its nucleotide sequence is as follows:
[0044] ATGGCCTCCTCCCTCCCCGCGCCGCCGGCCTCGCCCTCTTCCTCCTGGCGCGGACTCACGCCCCGCTGCCCTCCGCCTCGCTGCGGTCCCCTCCTCGCCCGCGCGGTAGCGCGTTCTTACCGTTACCGCTTCCGAACCGACGACGACGGCGTGGTGGACGTGGCCGTCGCCGGGAAAGACGGCGATGCGGGGTATGTGGTCGCTATCGAGGCTCCTACCCATGGACAGAGGGGCGGTCTTGTGCTCCGCCCCGCCGGCTCCGGCGAGGGCGTCCCTCTGGCCCCAGCCGCGCCGGGAGGTGCCCTCGTGGCTGAGTTGTCCTACGACGTGGCCCGCGCGCCGTTCCACGTCTCGTTCACGCTGGCCGACGCGATGGGAGCGGAGATACGGACACACCGCGGGACGAGCTTCCGCGTGCCTGTTGGCGTCGGACGGGGCTGCCCCTCGCCGCTCGGCCTGTCCCAGTCCAAGGATGGGGCCGCTAACTTCGCGGTTTACAGCAAGATCGCCAAGGGCATGGTGCTCTGCCTCTTCGGTGGTGGCGGCGGGGACGGACCCGCGCTGGAGATTGAGCTCGACCCGTACGTCCACCGGACCGGCGATGTCTGGCACGTCTCGATGGAGAGCGTGGAGGGGTACGCCCGCTACGGCTTCCGCAGCGGGCTGTTCGCAATGTTTGGCATTGACCGCCCGCTACTCGACCCGTACGCCAAGGTGATCGGGGACTTCGTCGCTGGCGACTCTGTTGATGAGGATGGGCTAGCTGTGCCATCCATAAGGTGTCTCGCGTCCTTGAAGAATGCACCCAACTACGATTGGGGCAGGGACAAGCACCCATGC...
Embodiment 2
[0081] Example 2 The gene editing vector was transformed into Agrobacterium LBA4404
[0082] 1) CaCl 2 Preparation of Agrobacterium tumefaciens competent cells
[0083] (1) From the YEP plate (Rif R ,Str R ), pick a fresh LBA4404 single colony and inoculate it in YEP liquid medium containing 50mg / L Str and 25mg / L Rif, 28°C, 220rpm shaking culture overnight for 24-36h;
[0084] (2) Take 2ml of the bacterial liquid in the logarithmic growth phase activated overnight, inoculate it in 50mL YEP liquid medium, and cultivate the bacterial liquid OD at 20°C 600 to about 0.4 to 0.6;
[0085] (3) Transfer the bacterial liquid to an ice-precooled 50 mL sterile centrifuge tube, ice bath for 30 min, centrifuge at 4,000×g for 10 min at 4°C, and enrich the bacterial cells;
[0086] (4) Pre-cool 0.05M CaCl with 10mL ice 2 Suspend the bacterial cells, take an ice bath for 30 min, centrifuge at 4,000 × g for 10 min at 4 °C, and enrich the bacterial cells;
[0087] (5) Pre-chill 0.05M CaC...
Embodiment 3
[0101] Example 3 Maize genetic transformation
[0102] (1) The material for embryo extraction was the corn inbred line C01. The young maize embryos were observed on the ninth day after pollination. When the embryos grew to about 1.5 mm, the ear was taken back to the laboratory for embryo extraction.
[0103] (2) Prepare the Agrobacterium infection solution, and shake the activated Agrobacterium (Agrobacterium tumefaciens containing pBUE411-1gR-ZmIsa2, or Agrobacterium tumefaciens containing pBUE411-2gR-ZmIsa3-ZmZpu1) in YEB liquid medium bacteria to a specific concentration (OD 550 = 0.5), collect the cell pellet by low-speed centrifugation, and then use inf (per liter composition: N6 salt and vitamin (sigma) 2 g, sucrose 68.5 g, glucose 36 g, L-proline 0.7 g, MES 0.5 g, 1 mg / ml 2,4-D 1.5ml)+AS (Acetosyringone, (100mM), 1ml)) liquid medium to resuspend, shake at 75r / min at 25°C for 24h, until the concentration is OD 550 =0.3-0.4.
[0104] (3) Wash the immature embryos taken...
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