Tea tree MYC2 transcription factor and application thereof
A technology of transcription factor, tea tree, applied in the direction of application, genetic engineering, plant genetic improvement, etc.
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Embodiment 1
[0021] Embodiment 1: the cloning of tea tree CsMYC2s gene
[0022] With the Longjing 43 cDNA shown in SEQ ID No.1 as a template, CsMYC2.1 is amplified with CsMYC2.1.F and CsMYC2.1.R as primers; with the Longjing 43 cDNA shown in SEQ ID No.2 as a template, Using CsMYC2.2.F and CsMYC2.2-R as primers to amplify CsMYC2.2; using Longjing 43 cDNA as shown in SEQ ID No.3 as template, using CsMYC2.3-F and CsMYC2.3-R as primers to amplify Increase CsMYC2.3; use Longjing 43 cDNA as shown in SEQ ID No.4 as a template, and use CsMYC2.4-F and CsMYC2.4-R as primers to amplify CsMYC2.4; the amplification system is as follows:
[0023]
[0024] 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 PCRClean-Up System kit method was used to recover, and after purification, it was connected to T-vector pClone007 vector of Qingke Biological C...
Embodiment 2
[0038]Example 2: Fluorescent quantitative PCR detection of the expression of CsMYC2s in different tissues of tea tree
[0039] Trizol method was used to extract RNA from Longjing 43 roots, stems, old leaves, mature leaves, young leaves and terminal bud tissues, and the above RNA was reverse transcribed according to the method of HiScript 1st Strand cDNA Synthesis Kit (10μl reverse transcription system was added with 500ng Total RNA ) to obtain cDNA, and design quantitative primers based on the cloned CsMYC2s sequence. Prepare the reaction solution according to the following components: HieffTMqPCR SYBR Green Master Mix 5μl, PCR Forward Primer (10μM) 0.2μl, Reverse Primer (10μM) 0.2μl, cDNA 0.5μl, dH2O 4.1μl. 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. The analysis results showed that the expression level of CsMYC2.1 was higher in roots and terminal buds, followed by new le...
Embodiment 3
[0049] Example 3: Subcellular localization of CsMYC2s
[0050] Using the Ligation independent cloning (LIC) method, the PCR products of the CsMYCs amplified fragments were recovered by gel cutting, and the linearized dual-source expression vectors were respectively connected with DNA polymerase (pCV-GFP-N1 digested with ApaI was used in this example). expression vector), 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 pCV-GFP-N1 vector; then place the processed PCR product and the carrier connection system in a PCR instrument Reaction at 75°C for 20s, 37°C for 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 pCV-eGFP-N1 vector fragment were transformed into Agroba...
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