Method for promoting plant seed augmentation and cotton fibre growth by using RDL1 gene
A gene and seed technology, applied in the fields of plant bioengineering and plant improvement genetic engineering, can solve problems such as less functional research
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Embodiment 1
[0095] Example 1. Isolation of RDL1 cDNA
[0096] Cotton RNA was extracted by cold phenol method:
[0097] 2g of material (fibers on the ovule surface of upland cotton 9 days after flowering) was ground into powder in liquid nitrogen, transferred to a 50ml centrifuge tube, and 8ml of extraction buffer (1M Tris HCl, 50mM EDTA, 1% SDS, pH9.0) and an equal volume of water-saturated phenol:chloroform:isoamyl alcohol (25:24:1), vortexed and mixed, placed on ice for 1h, and mixed every 10 minutes. Centrifuge at 13000g for 20 minutes at 4°C. Repeat phenol: chloroform: isoamyl alcohol extraction 2 to 4 times, and finally extract once with chloroform: isoamyl alcohol (24:1). Take the supernatant, add 1 / 2 volume of high salt solution (0.8M sodium citrate, 1.2M NaCl) and 1 / 2 volume of isopropanol, mix well, and place at -70°C for 1h. Centrifuge at 13,000g for 20 minutes at 4°C, remove the supernatant, dissolve the pellet in 1ml of DEPC-treated water, and centrifuge at 13,000g for 10 m...
Embodiment 2
[0103] Construction and Agrobacterium transformation of embodiment 2.RDL1 and GFP fusion vector
[0104] The GFP gene used was derived from pEGFP-1 (Clontech, Palo Alto, USA). Appropriate enzyme cleavage sites were introduced by PCR. The stop codon of GFP was removed, and an unfolded sequence of 6 amino acids (GPGGGG) was added.
[0105] Using GS1: 5'-CTAGTCTAGAATGGTGAGCAAGGGCGAGGAG-3' (SEQ ID NO: 9) and GA1: 5'-GTCCCCCGGGCGTCCTCCTCCTCCTGGTCCCTGGTACAGCTCGTCCATGCC-3' (SEQ ID NO: 10) as primers, pEGFP-1 vector as template, and using Pyrobest DNA polymerase (purchased from TAKARA company) amplified the GFP coding region, recovered from rubber tapping, double-digested with SmaI and NcoI, and connected between the EcoRV and NcoI sites of pET32c to construct the GFP-32c intermediate vector.
[0106] The target fragment was amplified with Pyrobest DNA polymerase, and the corresponding restriction sites BamHI and SacI were introduced at the same time (see Example 1). After checking ...
Embodiment 3
[0109] Example 3. Plant Transformation and Screening of Transgenic Progeny
[0110] a. Transgenic cotton
[0111] After culturing the Agrobacterium containing the vector plasmid on YEB bacterial medium supplemented with kanamycin 50 mg / L, rifampicin 100 mg / L and streptomycin 300 mg / L for 2 to 3 days, pick a single colony and inoculate it in the culture medium containing the same antibiotic. Suspension culture was carried out overnight at 28°C on a shaker at 200 rpm / min in YEB liquid medium. Centrifuge the bacterial solution at 4000rpm / min for 10 minutes, resuspend the pellet with 1 / 2MS liquid medium containing 30g / L glucose and 100μmol / L acetosyringone, and adjust the OD 600 The value is about 0.4 to 0.6, which is used as an infection solution for later use.
[0112] Cotton R15 (a tetraploid wild-type upland cotton, as the transgenic female parent) seeds were placed in 1 / 2MS0 medium [1 / 2MS salt (purchased from DUCHEFA M0221) + 5g / L glucose + 7g / L agar powder, pH 6.0], ger...
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