Related gene of drought resistant medicago sativa as well as encoding protein and application of gene and protein
An alfalfa, gene coding technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem of less research on alfalfa drought resistance breeding and so on
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Embodiment 1
[0041] Real-time quantitative PCR analysis of bZIP gene
[0042] In order to further study the expression of the bZIP gene (the gene shown in SEQ ID NO: 1) obtained from the suppression subtractive hybridization library, the real-time quantitative PCR method was used for analysis. The specific steps are as follows:
[0043] Using 50-day-old alfalfa plants (Medicago sativa L.cv. Baoding alfalfa, Beijing Zhonghu Dongfang Grass Technology Co., Ltd.) as experimental materials, 30% (w / v) PEG 8000 was used to treat 0, 15, and 30 respectively. Minutes and 1, 6, 12, and 24 hours, extract total RNA. 5μg of total RNA was used to prepare the first-strand cDNA. The specific steps are as follows: first add 0.5μl of 50μM oligo(dT) (Invitrogen), 1μl of 10mM dNTP Mix and 5μl of DEPC-treated water, heat shock at 65°C for 5 minutes, Cool on ice for 1 minute; secondly, add 4μl 5×First Strand Buffer, 2μl 0.1M dithiothreitol (DTT), 1μl 40U / μl RNaseOUT (Invitrogen) and 200U SuperScript II Reverse Trans...
Embodiment 2
[0050] Subcellular localization of bZIP gene
[0051] Construction of plant expression vector: 1) RNA extraction (extracted by Trizol method), 2) reverse transcription (M-MLV, promega company), 3) PCR amplification: using reverse transcribed cDNA as a template, using Taq enzyme for PCR , Connect the designed restriction sites (NcoI and SpeI) into the PCR product, 4) The PCR product and the vector are subjected to restriction treatment, 5) The restriction product is connected, the vector structure is as follows figure 2 Shown.
[0052] Transform Escherichia coli for detection, use gene gun to transfer the empty expression vector that does not contain the target bZIP gene into onion epidermal cells, and observe its subcellular location (such as image 3 (Shown); use the gene gun to introduce the vector containing the target gene into onion epidermal cells and observe its subcellular location (such as Figure 4 Shown).
[0053] image 3 In the comparison between A and B, it can be seen...
Embodiment 3
[0055] Agrobacterium-mediated bZIP gene transformation of alfalfa plants
[0056] 1. Materials and reagents
[0057] 1. Plant materials
[0058] The tested alfalfa variety was alfalfa (Medicago sativa L.cv. Baoding alfalfa, Beijing Zhonghu Dongfang Grass Technology Co., Ltd.).
[0059] Cotyledons of sterile seedlings germinated for 7 days are the recipient materials for genetic transformation.
[0060] 2. Agrobacterium strains and plasmid vectors
[0061] The Agrobacterium strain used was Agrobacterium tumefaciens: GV3103 (Beijing Tianenze Gene Technology Co., Ltd.), Agrobacterium culture medium:
[0062]
[0063] Plasmid vector: pCAMBIA1302 (purchased from Shanghai Sipiji Biotechnology Co., Ltd.). Insert the bZIP gene into pCAMBIA1302, the specific steps are as follows:
[0064] 1) RNA extraction (extracted by Trizol method), 2) reverse transcription (M-MLV, promega company), 3) PCR amplification: using reverse transcribed cDNA as a template, amplify the bZIP gene sequence (primer: 5) '...
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