Applications of rice OsPCF7 gene in culturing high-tillering rice varieties
A technology for high tillering and rice, applied in the directions of application, genetic engineering, plant genetic improvement, etc., can solve problems such as no high tillering rice varieties, and achieve the effect of promoting stable rice yield and increasing the number of tillers.
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Embodiment 1
[0030] Embodiment 1, the cloning of rice OsPCF7 gene reading frame fragment
[0031] According to the OsPCF7 gene candidate sequence (NM_001050819) found in GenBank, use the sequence processing online toolkit (SMS) biological software network http: / / www.bio-soft.net / sms to find its largest open reading frame sequence, using PrimerPremier 5.0 The software designed primers OsPCF7-f: 5'-catgccatgggcatgcgcaacgccaagg-3' (SEQ ID No.1, the underlined part is the NcoI restriction site) and OsPCF7-r: 5'-gggtaacctcacctgatcacctcacttcc-3' (SEQ ID No.2, the underlined part is the BstEII restriction site). Take different Zhonghua No. 11 rice seedlings, extract total RNA, and then synthesize the first strand of cDNA according to the instructions of the TaKaRa RNA PCR Kit (AMV) Ver.3.0 kit, and then use the first strand of cDNA as a template , using primers OsPCF7-f and OsPCF7-r to amplify the reading frame sequence of OsPCF7 gene. The PCR amplification system consists of: 2×Taq Mix 25μl, O...
Embodiment 2
[0033]Embodiment 2, the construction of rice OsPCF7 gene plant expression vector
[0034] The purified PCR amplified product was digested with NcoI and BstEII to obtain the open reading frame fragment of the OsPCF7 gene, which was then ligated with the backbone of the vector pCAMBIA1301 that had undergone the same double digestion to obtain the plant overexpression binary vector pCAMBIA1301-OsPCF7. The constructed overexpression binary vector pCAMBIA1301-OsPCF7 was amplified with primers OsPCF7-f and OsPCF7-r for PCR identification, the results were as follows figure 2 shown. The results showed that a DNA fragment with a length of 833bp was obtained, which was consistent with the size of the open reading frame fragment of OsPCF7 gene. At the same time, the constructed overexpression binary vector pCAMBIA1301-OsPCF7 was identified by double enzyme digestion with NcoI and BstEII, and the results were as follows image 3 shown. The lengths of the digested products were 9771bp...
Embodiment 3
[0035] Embodiment 3, the preparation of rice PCF7 gene Agrobacterium engineering strain
[0036] The overexpression binary vector pCAMBIA1301-OsPCF7 was transformed into Agrobacterium tumefaciens AGLO competent cells by liquid nitrogen cold shock method, and spread on YEB solid medium (containing 1.5% (w / w) agar, 50 mg / L kana (Kan), 500 mg / L streptomycin (Sm) and 50 mg / L rifampicin (Rif)), pH 7.0), cultured at 28±2°C in the dark for 2 days, picked single Bacterial colonies were cultured with YEB liquid medium (containing 50mg / L of Kan, 500mg / L of Sm and 50mg / L of Rif, pH7.0) for 2 days, and the bacterial liquid was taken for PCR identification. The positive recombinant was the rice OsPCF7 gene Engineering strains of Agrobacterium, frozen at -80°C, for later use.
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