Fast-growing hydrophyte nitrate transporter GeNRT1.1 and coding gene and application thereof
A nitrate and encoding technology, applied in the direction of plant genetic improvement, application, microorganisms, etc., can solve the problem that strains cannot be transported or grown
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Embodiment 1
[0061] Embodiment 1, the acquisition of GeNRT1.1
[0062] 1. Discovery of GeNRT1.1
[0063] 1. Total RNA extraction and cDNA synthesis of dwarf pearls
[0064] Using the RNAprepPurePurePlant kit from TIANGEN Company, the total RNA of dwarf pearls was extracted according to the kit instructions, and cDNA was obtained by reverse transcription.
[0065] 2. PCR amplification
[0066] Using the cDNA obtained by reverse transcription in step 1 as a template, PCR amplification was performed using degenerate primers H5 and H3 to obtain a PCR product. The primer sequences are as follows: H5: GGNGCNGAYCARTTYGAYG; H3: AVYTCYTCNACYTGNGTNAC. The PCR amplification program was: 95°C pre-5min; 94°C denaturation 30s; 52°C annealing 30s; 72°C extension 1min, 35cycle; 72°C, 10min.
[0067] 3. Electrophoresis and sequencing
[0068] After the amplification was finished, 1% agarose gel electrophoresis was detected, and the PCR product was connected to the T carrier, and the sequencing results...
Embodiment 2
[0126] Embodiment 2, verification of GeNRT1.1 gene function
[0127] In this example, the function of the GeNRT1.1 gene was verified by using the Δynt-Leu double mutant Hansenula polymorpha.
[0128] 1. Construction of GeNRT1.1-transferred yeast
[0129] 1. Construction of the shuttle plasmid
[0130] Insert the DNA molecule shown in Sequence 4 in the sequence listing between the speI and salI restriction sites of the pYNR-EX vector, and keep other sequences of the pYNR-EX vector unchanged to obtain the recombinant vector pYNR-GeNRT1.1.
[0131] 2. Linearization
[0132] pYNR-GeNRT1.1 was digested with BstpI and linearized to obtain linearized pYNR-GeNRT1.1, which was subjected to 1% agarose gel electrophoresis ( Figure 4 ), recover and purify the target product, use a spectrophotometer for quantification, and set aside.
[0133] 3. Preparation of double mutant Hansenula polymorpha (△ynt-Leu) competent cells
[0134] ①Pick a single colony of △ynt-Leu double mutant Hansen...
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