A wrky transcription factor that regulates fruit top hardening in golden pears
A transcription factor, gold technology, applied in genetic engineering, plant genetic improvement, chemical instruments and methods, etc., can solve problems such as complex pathogenesis
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Embodiment 1
[0020] Example 1, transcriptome sequencing and data analysis.
[0021] 1. Use NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB, E7490) to enrich eukaryotic mRNA; use MICROBExpress Bacterial mRNA Enrichment Kit (invitrogen,
[0022] AM1905) remove rRNA and enrich mRNA. Using mRNA as a template, use NEBNext mRNA Library PrepMaster Mix Set for Illumina (NEB, E6110) and NEBNext Multiplex Oligos for
[0023] llumina (NEB, E7500) was used to construct the library on the machine. The prepared library was electrophoresed on a 1.8% agarose gel to detect the size of the library insert (Insert Size), and then the Library Quantification Kit-Illumina GA
[0024] I Universal (Kapa, KK4824) was used for q-PCR quantification. Qualified libraries were clustered on Illumina cbot, and finally Illumina HiSeq was used in Beijing Biomark Biotechnology Co., Ltd. TM 2500 were sequenced.
[0025] 2. Perform data evaluation on the original paired-end reads sequence obtained by sequencing, r...
Embodiment 2
[0026] Example 2, the acquisition and sequencing of the cDNA sequence of WRKY gene.
[0027] 1. RNA extraction and cDNA synthesis
[0028] (1) For every 0.1-5 g of frozen ground plant tissue (approximately 8 mL), add 5 mL of RNAplantPlus Reagent, shake until thoroughly mixed.
[0029] (2) Leave at room temperature for 5 minutes (lay the centrifuge tube flat to maximize the surface area).
[0030] (3) Centrifuge at 10,000 rpm at 4°C for 3 minutes, and transfer the supernatant to a new RNAase-free centrifuge tube.
[0031] (4) Add 1 mL of 5 M NaCl to every 5 mL of supernatant and mix well.
[0032] (5) Add 3 mL of chloroform for every 5 mL of supernatant, and mix well by inverting up and down.
[0033] (6) Centrifuge at 10,000 rpm at 4°C for 15 minutes (it can be extended to make the separation of protein, DNA and RNA more thorough), and transfer the upper aqueous phase to a new centrifuge tube.
[0034] Note: To extract plant tissue rich in polyphenols or starch, repeat ste...
Embodiment 3
[0049] Example 3, Onion Subcellular Localization: Onion Endo-Epidermal Transformation
[0050] (1) Choose a fresh yellow onion, peel off the outer 3-4 layers of scales, peel off the inner scales of the onion on the ultra-clean workbench, and use a scalpel to cut the inner epidermis into 1 cm 2 For the left and right squares, use tweezers to peel off the inner epidermis, spread the peeled side down on 1 / 2 MS solid medium, and culture in the dark at 28 °C for 24 h.
[0051] (2) Preparation of infection solution: Take the previously preserved Agrobacterium bacteria solution (pCAMBIA1300-PpWRKY and empty vector pCAMBIA1300) in LB liquid medium (containing 100 mg / L Kan and 100 mg / L Rif) and place at 28 °C Shake at 200 rpm on a constant temperature shaker until the OD600 is about 0.6, transfer to a 50 mL centrifuge tube and centrifuge at 5000 rpm for 10 min, collect the bacteria, discard the supernatant and use an equal volume of 1 / 2MS liquid medium to resuspend twice, with the em...
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