Tobacco gene NtFIP1 for improving nitrogen utilization rate and cloning method and application of tobacco gene NtFIP1
A cloning method and a technology of utilization rate, applied in the application, genetic engineering, plant gene improvement and other directions, can solve the problem of not finding upstream regulatory genes, etc., and achieve the effect of improving the utilization rate of tobacco nitrogen and improving the quality of tobacco
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Embodiment 1
[0046] The clone of the tobacco gene NtFIP1 coding sequence of the present invention was obtained according to the following method:
[0047] 1. Extraction of tobacco total RNA (kit method)
[0048] Use the kit method (column type total RNA extraction kit) to extract the total RNA of the corresponding tobacco plants, the specific method is as follows:
[0049] (1) Weigh about 100mg of tobacco variety K326 leaves in a 1.5mLEppendorf tube and grind them fully in liquid nitrogen;
[0050] (2) Add 1 mL of BufferRLT (the volume of the material is less than or equal to 10% of the volume of BufferRLT), shake and mix well, and let stand at room temperature for 5 minutes;
[0051] (3) To fully remove cell wall residues, proteins, fats, polysaccharides, etc., centrifuge at 4°C and 12000rpm for 10 minutes, and transfer the supernatant to a new centrifuge tube;
[0052] (4) Phase separation:
[0053] ①Add 0.2mL chloroform, shake vigorously for 15s, and let stand at room temperature for 2min;
[0054] ...
Embodiment 2
[0083] In this example, the methods for obtaining homozygous tobacco gene NtFIP1 complementary to Arabidopsis fip1 mutants and wild-type heterocomplementary lines and overexpression lines are as follows:
[0084] 1. Construction of pENTR3C-NtFIP1 recombinant plasmid
[0085] 1. Amplify the CDS of NtFIP1 gene by PCR and connect with pENTR3C
[0086] NtFIP1 forward primer (F): 5’-CGGGGTACCATGGAAGATGACGACG-3’;
[0087] NtFIP1 reverse primer (R): 5’-CCGCTCGAGATTGCTGGTCCATCTCCT-3’;
[0088] Using the extracted cDNA as a template, PCR amplification was carried out with specific primers F and R, and KpnI and XhoI restriction sites were introduced respectively upstream and downstream of the target gene. The PCR product was recovered with Tiangen (Beijing) glue recovery kit, and the PCR product was digested with Gateway-compatible pENTR3C. The operation steps are as shown in Table 4.
[0089] Table 4 The dosage of each ingredient during digestion
[0090] ingredient Dosage Target fragment r...
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