The cold resistance gene ptrtzf1 of trifoliate trifoliate and its application in genetic improvement of plant cold resistance
A gene and plant technology, applied in the direction of plant gene improvement, application, plant peptides, etc., can solve the problems such as TZF that have not yet been seen, and achieve the effect of reducing agricultural production costs and achieving environmental friendliness.
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Embodiment 1
[0037]Example 1: Cloning of PTRTZF1 Gene Full length CDNA
[0038]With 枳 cDNA as a template, use high-fideli enzyme amplification, amplification system is shown in Table 1, the amplification process is shown in Table 2, the amplification primer sequence is: 5'-CactaaaaAaggccctCACCC-3 'and 5'-gagcaggccctcattattatcc-3'.
[0039]Purification of the product was purified by the Axyprep-96DNA gel recovery kit (Axygene, USA) to purify the product.The 18-T vector (japan) is connected, and the connection system is included in Table 3, 16 ° C for 30 min after 30 min, transforming E. coli sensing state trans5α.
[0040]Table 1 gene amplification system
[0041]
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[0043]Table 2 gene amplification PCR program
[0044]
[0045]After the transformation, 12-16 hours were collided on the plate on a single clone to 1.5 ml of centrifuge tube, and the LB liquid medium containing the corresponding antibiotic was added, and the shaker was turbid to the bacterial liquid to be turbid, and then positive identification was...
Embodiment 2
[0052]Example 2: Expression analysis of PTRTZF1 gene under different adverse conditions
[0053]The expression mode of the PTRTZF1 gene is analyzed by the method of real-time fluorescence quantitative PCR (QRT-PCR), and the quantitative reagent is Quantinova.TM Sybrgreen PC (QIAGEN, Germany), method reference instructions, the reaction system is shown in Table 5.
[0054]Table 5 Quantitative PCR Reaction System
[0055]
[0056]Design PRIMER 5.0 Design PTRTZF1 Real-time Quantitative Primers (forward primers: 5'-gttgcaatctcccaaccggggc-3 '; reverse primers: 5'-gactgctgctccccacga-3'), citrus Actin is an interference gene (forward primer: 5'-catccccAgccttcc 3 '; reverse primers: 5'-ccaaccttagcacttccc-3'). The reaction procedure is shown in Table 6. 2 after the reaction is completed-ΔΔCT The algorithm calculates the amount of gene expression.
[0057]Table 6 Quantitative PCR Reaction Procedure
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[0059]
[0060]The results show that the expression of PTRTZF1 is rapidly increased (about 48 times) after ...
Embodiment 3
[0061]Example 3: PTRTZF1 gene super expression vector construction
[0062]The primer was designed to amplify the PTRTZF1 gene in the middle of XBA I and SMA I on the PBI121 vector, and the primer is designed as follows, with wild type cDNA as a template.
[0063]PTRTZF1-PBI121-F: 5'-GCTctaga Atggaaggtgaactcccca-3 '
[0064]PTRTZF1-PBI121-R: 5'-TCCCCCGGG Ttatgccaccatctgctcct-3 '
[0065]Amplification fragment recovery, PMD18-T vector connection and conversion, positive cloning detection and sequencing sequencing. The PTRTZF1 is then connected from the T vector to the PBI121 vector by T4 ligase (the vector is shownfigure 2 The PBI121-PTRTZF1 is constructed and converted to GV3101 for the following examples.
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