Rice cold-inducible promoter p-ltt7 and its application
A technology of rice and fragments, applied in the fields of application, angiosperm/flowering plants, and the introduction of foreign genetic material using vectors, which can solve problems such as genetic bottlenecks
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Embodiment 1
[0030] Example 1, Discovery of rice cold-inducible promoter p-LTT7
[0031] 1. Discovery of cold-induced genes
[0032] First, the cold-tolerant line IL112 rice and the control parent Guichao 2 rice were used as materials for microarray (the chip was the rice whole genome chip (GeneChip) from Affimetrix Company. Rice Genome Array), Cat. No.: 900599) hybridization, and on the basis of chip data analysis, combined with comparative genomics analysis, screened cold tolerance related genes, after genome comparison and cold tolerance correlation analysis, finally obtained the cold tolerance gene LTT7, under cold treatment conditions, The expression of the gene was significantly increased ( figure 1 ), is a cold-inducible gene.
[0033] 2. Discovery of the cold-inducible promoter p-LTT7
[0034] Primers were designed using primer3 primer design software, and restriction endonuclease PstI and SmaI recognition sites and protective bases were introduced at both ends of the primers. ...
Embodiment 2
[0040] Embodiment 2, the acquisition and identification of p-LTT7 transgenic rice
[0041] 1. Construction of p-LTT7 plant expression vector
[0042] 1. Synthesize the DNA shown in sequence 1 of the sequence list, use the synthesized DNA as a template, and use specific primers to perform PCR amplification on T7GUS (T7GUSF / T7GUSR). After the reaction, perform 1% agarose on the PCR amplification product Gel electrophoresis detection, recovery and purification of the 1979bp DNA fragment (p-LTT7) (the recovery kit used is the DNA product purification kit of Tiangen Biochemical Technology Co., Ltd., catalog number: DP204-02).
[0043] 2. Digest the PCR product recovered in step 1 with restriction enzymes SmaI and PstI.
[0044] 3. Digest the plant expression vector pCambia1381 with restriction enzymes SmaI and PstI, and recover the vector skeleton.
[0045] 4. Ligate the digested product of step 2 with the vector backbone of step 3 to obtain a recombinant plasmid.
[0046] 5. Th...
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