Application of Arabidopsis atgdsl Gene in Rapeseed Against Sclerotinia and Promoting Seed Germination
An Arabidopsis, gene technology, applied in the field of plant genetic engineering and biology, can solve very few problems, and achieve the effects of low cost, high speed, and improved germination speed
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Embodiment 1
[0036] Example 1: AtGDSL Gene acquisition
[0037] 1. Seedling cultivation
[0038] The wild-type Arabidopsis was used as the experimental material, and the growth conditions were as follows: temperature 20 ± 2°C; humidity 60-90%; daily photoperiod of 8 hours of light and 16 hours of darkness; light intensity of 44 μmol m –2 s–1 .
[0039] 2. RNA extraction and cDNA first-strand synthesis
[0040] RNA extraction: UNIQ-10 column Trizol kit (purchased from Shanghai Yingjun Biotechnology Co., Ltd.) was used to take a small amount of sample, freeze it with liquid nitrogen in a mortar, grind it into powder, and add 1 mL of TRNzol-A reagent ( In the 2mL EP tube of Tiangen Biochemical Technology Co., Ltd.), after fully shaking, centrifuge at 12000rpm at 4°C for 10min; then take about 800μL of the supernatant, add 300μL of chloroform (pre-cooling is easy to separate), shake vigorously for 15s, and place at room temperature for 3min centrifuge at 12000rpm at 4°C for 10min, transfer...
Embodiment 2
[0054] Example 2: Construction of overexpression AtGDSL Transgenic Rapeseed Plants
[0055] 1. AtGDSL Overexpression vector construction
[0056] The CaMV 35S strong promoter was added to the upstream EcoRI / KpnI restriction site of the vector pCAMBIA1300 (Beijing Dingguo Changsheng Biotechnology Co., Ltd.), and the CaMV Nos terminator was added to the downstream BamHI / HindIII restriction site. Transformed into recombinant vector pCAMBIA1300-35S-Nos. The specific process is as follows:
[0057] The specific process is as follows: according to the CaMV 35S sequence (Gene ID: AJ007626) published on NCBI, use PrimerPremier 5.0 software to design primers: upstream primer: 5′- GAATTC TTAATTAAGAGCTCGCATGCC-3' (SEQ ID NO.5) contains EcoRI restriction site (underlined part), downstream primer: 5'- GGTACC GTCCCCGTGTTTCTCCAA-3' (SEQ ID NO.6) contains the KpnI restriction site (underlined part), and the CaMV 35S fragment is amplified from the pEGAD vector (purchased from Treasure B...
Embodiment 3
[0082] Example 3: Overexpression AtGDSL Response of Rapeseed Rapeseed Plants to Sclerotinia Infection
[0083] Sclerotia sclerotia were cultured in PDA medium. PDA medium (1L) formula: 200g potato, 10-20g sucrose, 17-20g agar powder, 1000g double distilled water; cut the peeled potato into small pieces, add 800mL water, boil for 0.5h, filter the residue with gauze, add water Make up 1000mL, then add sugar and agar, heat to melt the agar completely, aliquot while hot, and autoclave for later use.
[0084] Take the four-leaf-one-heart stage of the above-mentioned PCR test positive AtGDSL The fourth true leaf of positive plants was inoculated in vitro. Cut the leaves and place them in a white porcelain plate. The leaves of each transgenic plant and the non-transgenic control are placed side by side in a plate, and a layer of wet gauze is laid under the leaves. Take a freshly prepared φ5mm mycelium block, with the mycelium side facing down. , inoculated at the position slightl...
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