Application of miR1850, miR1850 precursor and encoding genes thereof in regulation and control of plant stress tolerance
A technology of plant stress tolerance and transgenic plants, which can be applied in the fields of application, plant products, genetic engineering, etc., and can solve the problems that miR1850 research has not been reported yet
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Embodiment 1
[0061] Example 1. Discovery of osa-miR1850 and cloning of its precursor sequence Pre-miR1850
[0062] 1. Discovery of osa-miR1850
[0063] Using the rice sequencing variety Nipponbare as the experimental material, the seedlings were treated with cold stress at the three-leaf stage, and the miRNA chip expression profile of rice seedlings under cold stress was constructed, and a differentially expressed miRNA was obtained through analysis and screening, and its name was osa-miR1850. The sequence of the mature body is sequence 1.
[0064] 2. Cloning of osa-miR1850 precursor sequence
[0065] 1. Design of primers
[0066] Search keyword miR1850 on miRBase (http: / / www.mirbase.org / ) website, get osa-miR1850, its accession number is MI0008227, and obtain its precursor sequence Pre-miR1850 (as shown in sequence 1), precursor The size of the sequence Pre-miR1850 is 133bp. Replace the base U in the precursor sequence Pre-miR1850 with T, and use this as a template to do a Blast compa...
Embodiment 2
[0072] Example 2, Analysis of the expression pattern of Pre-miR1850 under cold stress
[0073] 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 with Pre-miR1850-RT-F and Pre-miR1850-RT-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:
[0074] Pre-miR1850-RT-F: 5'-GTGATGGAGATGCGATGGAAA-3';
[0075] Pre-miR1850-RT-R:5'-TTGGAGGAAGATTGATGTGAACTAA-3';
[0076] osa-ELF1-α-F:5'-GCACGCTCTTCTTGCTTTCAC-3';
[0077] osa-ELF1-α-R:5'-TCTTGTC...
Embodiment 3
[0079] Example 3, the acquisition and cold tolerance analysis of Pre-miR1850 transgenic rice
[0080] 1. Construction of Pre-miR1850 plant overexpression vector
[0081] 1. Using the genomic DNA of rice Kongyu 131 as a template, PCR amplification was carried out with Pre-miR1850-u-F and Pre-miR1850-u-R primers to obtain the Pre-miR1850 fragment. The primer sequences are as follows (the underline represents the linker sequence required for vector construction, where U is the USER restriction site):
[0082] Pre-miR1850-u-F:5'- GGCTTAAU GCTGTAGTGCTGCATCTGTGGATA-3';
[0083] Pre-miR1850-u-R:5'- GGTTTAAU TGCATTCAATGTTCACGTTTCTTCA-3'.
[0084] 2. Carry out double enzyme digestion on pCAMBIA330035Su vector with restriction endonuclease PacI and Nt.BbvCI to obtain vector digestion products. Incubate the obtained vector digestion product, USER enzyme (NEB, M5505S) and the Pre-miR1850 fragment obtained in step 1 for 20 min at 37°C, and use USER enzyme to cut the uracil of the Pr...
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