A plant leaf highly expressing at2g34420/at2g34430 bidirectional promoter and its application
A bidirectional promoter, plant leaf technology, applied in angiosperms/flowering plants, applications, plant peptides, etc., can solve the problems of high price, increased workload, and low efficiency, and achieve high-efficiency expression and improve work efficiency.
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Embodiment 1
[0034] Example 1: Analyze and obtain bidirectional promoters highly expressed in leaves through transcriptome data of Arabidopsis leaves.
[0035] The total RNA of 4-week-old Arabidopsis Col-0 leaves in 9 experimental groups was extracted, and a cDNA library was constructed by adding adapters, and 9 sets of transcriptome sequencing data were obtained by RNA-seq. From 9 sets of data, select genes with FPKM (Fragments Per Kilobase of transcript per Million mapped reads, in every 1 million reads, the number of fragments per 1K exon bases) value greater than 100, and found that AT2G34420(6125.53,8560.40,6312.31,2534.41,1820.29,2783.31,1887.70,2455.50,2147.33)及AT2G34430(3258.33,6392.27,3604.50,1582.90,772.91,3010.30,1474.32,1835.43,1562.17)为邻近基因。 The stably expressed internal reference genes AT4G05320 (UBQ10, Ubiquitin 10) and AT1G13320 (PP2AA3, PROTEIN PHOSPHATASE2ASUBUNIT A3) were used as benchmarks to evaluate the expression levels of target genes (Wang et al., 2014). In the 9 ...
Embodiment 2
[0037] Example 2: RT-PCR and qRT-PCR validation
[0038] Using a plant RNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Plant Total RNA Extraction Kit, DP432), the total RNA of Arabidopsis Col-0 leaves was extracted, and a reverse transcription kit was used to synthesize cDNA (Tiangen Biochemical Technology Co., Ltd., FastKing one-step removal of genomic cDNA first-strand synthesis master mix reagent, KR118-02), RT-PCR (ReverseTranscription-Polymerase Chain Reaction, reverse transcription-polymerase chain reaction) and real-time qRT-PCR (Quantitative Reverse Transcription-PCR) , real-time quantitative reverse transcription polymerase chain reaction) detection.
[0039] The RT-PCR amplification reaction system is 20 μl: cDNA template: uniformly adjust the loading amount to 300 ng; RT-F (10 μM): 0.2 μl; RT-R (10 μM): 0.2 μl; 2×Super PfxMasterMix: 10 μl (Kang for the century, CW2965); ddH 2 O: Make up to a total volume of 20 μl.
[0040] The reaction program is: d...
Embodiment 3
[0065] Example 3: Synthesis of fluorescent protein gene empty vector
[0066] In order to further verify the activity of the bidirectional promoter in plants, an empty vector containing eGFP and mCherry genes was constructed first. like Figure 4 As shown, the eGFP gene and the mCherry gene are distributed on the complementary strands in a head-to-head manner, and are separated by multiple cloning sites (MCSs). The 'end is the 35S transcription terminator sequence.
[0067] The specific synthesis steps are:
[0068] First, an eGFP-MCS-mCherry fragment containing multiple cloning sites was artificially synthesized. The sequence information is as follows:
[0069]
[0070]
[0071]The capital letters and underlines indicate the recognition sites of EcoR I (5' end) and Hind III (3' end), respectively, and the shaded sequence indicates the multiple cloning site region (from 5' to 3' end, the corresponding restriction endonucleases) The enzymes are: Srf I-Apa I-AfeI-Spe I...
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