Protein PHD-Finger 17 related to plant stress resistance as well as coding gene and application of protein PHD-Finger 17
A technology that encodes genes and plant stress tolerance, applied in the biological field, can solve problems such as poor insemination, infertility, and poor germination of seeds
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Embodiment 1
[0082] Cloning of embodiment 1, PHD-Finger17
[0083] 1. Cloning of CDS region of PHD-Finger17 gene
[0084] 1. Design of primers
[0085] PHD-Finger17 cloning primers were designed using Primer Premier 5.0. The primer sequences are as follows:
[0086] PHD-Finger17-F: 5'-ATGGGGAAGGGAGGGGAAGG-3';
[0087] PHD-Finger17-R: 5'-CTAGACACCTTCAGTTCCTTG-3'.
[0088] 2. Cloning of PHD-Finger17
[0089] Mature, plump rice seeds (Nipponbare) were selected and treated at 42°C for 3-5 days to break dormancy. Sterilized with 10% NaClO for 30 minutes, washed 5 times with sterile distilled water, soaked the seeds in the dark for 1 day, then transferred the germinated rice seeds to 30°C for 1 day in the dark, and then transferred the germinated rice seedlings into Yoshida solution at 28°C for 12h under light (illumination intensity 250μmol m -2 the s -1 ), cultured at 25°C and 12h in the dark until the three-leaf stage. Take the leaves of the three-leaf stage seedlings, use Trizol (In...
Embodiment 2
[0091] Example 2, Analysis of the expression pattern of PHD-Finger17 under cold stress
[0092] The rice seedlings at the three-leaf stage in Example 1 were cold-treated at 4°C, and the above-ground tissues were taken after 0h, 0.5h, 1h, 3h, 6h, 9h, 12h and 24h of treatment, and the total RNA was extracted by the Trizol method. Transcription Kit SuperScript TM cDNA was obtained by reverse transcription with III Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA). The obtained cDNA was diluted 5 times, and the diluted cDNA was used as a template to carry out RT-PCR using PHD-Finger17-qRT-F and PHD-Finger17-qRT-R primers. At the same time, rice elongation factor 1-α gene (osa-ELF1-α) was used as an internal reference gene. The primer sequences used for detection are as follows:
[0093] PHD-Finger17-qRT-F: 5'-TGTGCAGGGTGTGCTGGATT-3';
[0094] PHD-Finger17-qRT-R: 5'-CCTTTGACGACTTTGTGATTGTTG-3';
[0095] osa-ELF1-α-F: 5'-GCACGCTCTTCTTGCTTTCAC-3';
[0096] osa-ELF1-α-R:...
Embodiment 3
[0098] Embodiment 3, the acquisition and cold tolerance analysis of PHD-Finger17 overexpression rice
[0099] 1. Construction of PHD-Finger17 plant overexpression vector
[0100] 1. The cDNA of rice Nipponbare was used as a template, and the PHD-Finger17-u-F and PHD-Finger17-u-R primers were used for PCR amplification to obtain the CDS sequence of PHD-Finger17. The primer sequences are as follows (the underline represents the linker sequence required for vector construction, where U is the USER restriction site):
[0101] PHD-Finger17-u-F: 5'- GGCTTAAU ATGGGGAAGGGAGGGGAAGG-3';
[0102] PHD-Finger17-u-R: 5'- GGTTTAAU CTAGACACCTTCAGTTCCTTGGAA-3'.
[0103] 2. Carry out double enzyme digestion on pCAMBIA330035Su vector with restriction endonuclease PacI and Nt.BbvCI to obtain vector digestion product. Incubate the obtained vector digestion product, USER enzyme (NEB, M5505S) and the PHD-Finger17 fragment obtained in step 1 for 20 min at 37°C, and use USER enzyme to cut the ...
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