Oligopeptide for improving germination rate of plant seeds under salt stress and application
A technology for plant seeds and salt stress, which is applied to oligopeptides for improving the germination rate of plant seeds under salt stress and the application field, can solve the problems of low frequency of beneficial mutations, residual effects, low efficiency, etc. Effect
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Embodiment 1
[0037] Example 1 Cloning and Vector Construction of Peanut AhCEP3 Gene
[0038] The present invention downloads the gene AhCEP3 through the peanut genome database website PeanutBase (https: / / www.peanutbase.org), utilizes Pfam (Protein family: http: / / pfam.sanger.ac.uk / ) and SMART (Simple Modular Architecture Research Tool :http: / / smart.embl-heidelberg.de / ) analysis shows that this gene encodes a typical CEP small peptide (CDS 252bp as shown in SEQ ID NO: 1, amino acid 83aa as shown in SEQ ID NO: 2), containing N-terminal signal peptide and the conserved CEP domain at the C-terminus.
[0039] SEQ ID NO:1
[0040]
[0041] SEQ ID NO:2
[0042]
[0043] Design primers according to the CDS sequence of the peanut AhCEP3 gene found, and clone. The cloning method is as follows:
[0044] 1. Peanut RNA Extraction
[0045] The total RNA of peanuts was extracted using Tiangen Plant Total RNA Extraction Kit (DP419).
[0046] (1) Grind Tifrunner peanut seedlings grown in hydropon...
Embodiment 2
[0072] Example 2 Induction of Peanut Hairy Roots
[0073] 1. Peanut Explant Acquisition
[0074] The peanut seeds of Luhua No. 11 were planted in pots in an artificial climate chamber (25°C, 16h light / 8h dark). Peanut seedlings grow to the stage of 3-6 compound leaves, and 2-4 compound leaves are selected from the bottom up as experimental materials. Rinse the peanut leaves with tap water for 10 minutes, wash them gently with sterile water for 2 minutes, soak them in 4% sodium hypochlorite solution for 2 minutes, and take the slightly white incision of the petiole as the standard. Then wash it with sterile water for 3-4 times, soak it in sterile water for 10 minutes, and dry it for later use.
[0075] 2. AhCEP3-R1601 infected peanut leaves
[0076] AhCEP3-R1601 and R1601 Agrobacterium were respectively inoculated in 100 mL of YEP liquid medium containing kanamycin (100 μg / mL) and hygromycin (200 μg / mL), cultured at 28°C with shaking at 180 rpm for 18 h, and continuously act...
Embodiment 3
[0077] Example 3 Screening of Transgenic Peanut Hairy Root Positive Seedlings
[0078] Transgenic and non-transgenic (hairy roots transfected with empty R1601, as a control) hairy roots of more than 2 cm were selected, transferred to MS solid medium for subculture for 2 weeks, and some hairy roots were intercepted to extract RNA. Transcribed to obtain cDNA.
[0079] The fluorescence quantitative PCR instrument is Icycler real-time PCR system (Bio-Rad, USA), and the qRT-PCR reaction conditions are as follows:
[0080] (1) 95.0°C for 60s; (2) 95.0°C for 10s; (3) 58.0±5.0°C for 10s; (4) 72.0°C for 15s; (5) PlateRead; (6) Incubate at 65°C for 20s; (7) Melting curve From 65°C to 95°C, read every 0.5°C, hold 1s; (8) End; (2) (3) (4) 50-60cycles.
[0081] Using peanut actin11 as an internal reference, adjust the concentration of each template so that the difference between the internal reference Ct values is less than 2, using 2 -ΔΔCt Roughly calculate the relative expression le...
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