Tea tree PHR1 transcription factor as well as coding gene and application thereof
A technology of transcription factor and tea tree, applied in application, genetic engineering, plant genetic improvement, etc.
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Embodiment 1
[0024] Embodiment 1: the cloning of tea tree CsPHR1s gene
[0025] Use Longjing 43 cDNA as template, CsPHR1-1.F and CsPHR1-1.R as primers to amplify CsPHR1-1; use CsPHR1-2.F and CsPHR1-2.R as primers to amplify CsPHR1-2; the amplification system is as follows :
[0026]
[0027] The PCR reaction conditions were 94°C for 5min, 94°C for 30s, 58°C for 30s, 72°C for 1min, 35Cycles, and 72°C for 10min. After the PCR products were subjected to 1% agarose gel electrophoresis, SV Gel and PCR Clean-Up System kit method was used to recover, and after purification, it was connected to T-vector pClone007vector of Qingke Biological Company, introduced into Escherichia coli DH5α competent by heat shock method, and positive clones were screened on the ampicillin plate. Positive clones were tested and sent to Hangzhou Qingke Biological Co., Ltd. for sequencing. figure 1 It is the agarose gel electrophoresis image of CsPHR1s coding region sequence amplification, and the three swimming l...
Embodiment 2
[0034] Example 2: Fluorescent quantitative PCR detection of the expression of CsPHR1s in different tissues of tea tree
[0035] Trizol method was used to extract RNA from different tissues of Longjing 43, and the above RNA was reverse-transcribed according to the method of HiScript 1st Strand cDNASynthesis Kit (500 ng of Total RNA was added to 10 μl reverse transcription system) to obtain cDNA, and quantitative primers were designed by cloning the obtained CsPHR1s sequence. Prepare the reaction solution according to the following components: Hieff TMqPCRSYBR Green Master Mix 5μl, PCR Forward Primer (10μM) 0.2μl, Reverse Primer (10μM) 0.2μl, cDNA 0.5μl, ddH 2 O 4.1 μl. The amplification program was as follows: 94°C for 5min, 94°C for 10s, 58°C for 20s, 72°C for 20s, 40Cycles, and fluorescence was collected at 72°C. Depend on figure 2 It can be seen that CsPHR1-1 and CsPHR1-2 are expressed in various tissues, and the relative expression of CsPHR1-1 and CsPHR1-2 is the highest...
Embodiment 3
[0041] Example 3: Subcellular localization of CsPHR1s
[0042] Using the Ligation independent cloning (LIC) method, the PCR product of the CsPHR1 amplified fragment was recovered by gel cutting, and then the linearized dual-source expression vector was connected with DNA polymerase (the pCambia1300-GFP expression vector cut with ApaI was used in this example). ), specifically: first add an A tail to the end of the PCR product with T4 DNA polymerase, and add a T tail to the end of the linearized pCambia1300-GFP vector; then place the treated PCR product and the carrier connection system in a PCR instrument to react at 75°C 20s, 37°C and 30mim to obtain the ligation product; then transform the ligation product into DH5α Escherichia coli, plate, and culture overnight to obtain a successfully constructed recombinant plasmid. The successfully constructed recombinant plasmid and the control plasmid containing only the pCambia1300-GFP vector fragment were transformed into Agrobacteri...
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