Application of Zea mays CRR1 protein and coding gene thereof in regulating and controlling low-temperature stress tolerance of Zea mays
A technology that encodes genes and corn, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of reduced photosynthetic activity, affecting the transport of assimilates, etc., and achieve the effect of strong low temperature tolerance
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Embodiment 1
[0045] Construction and detection of embodiment 1CRR1 gene overexpression vector
[0046] Total RNA was extracted from B73 corn (Zea mays L.), and cDNA was obtained by reverse transcription. The cDNA was used as a template, and F and R were used as primers to amplify the CRR1 gene. on the carrier. CRR1 gene overexpression vector construction method is as follows:
[0047] (1) The total RNA of B73 corn was extracted with the RNA extraction kit of Magen Company, and the specific steps were referred to the instructions of the kit.
[0048] (2) Use the reverse transcription kit from Thermo Company to reverse transcribe the RNA into cDNA, and refer to the kit instructions for specific steps.
[0049] (3) Using cDNA as a template and F and R as primers, amplify the cDNA of the CRR1 gene (as shown in SEQ ID NO.1, its encoded amino acid sequence is as shown in SEQ ID NO.2), and run electrophoresis on the amplified product The gel was cut and recovered. For the recovery method, refe...
Embodiment 2
[0055] Construction and detection of embodiment 2CRR1 gene overexpression plant
[0056] The pBCXUN vector containing the CRR1 gene described in Example 1 was transformed into the Agrobacterium EHA105 strain (Ma et al., 2009, Enhanced tolerance to chilling stress in OsMYB3R-2 transgenic rice ismediated by alteration in cell cycle and ectopic expression of stressgenes.Plant Physiol .150,244–256), and then infect maize callus to obtain transgenic seedlings. The specific method is: inoculate the Agrobacterium containing the target carrier in 100 mL LB liquid culture solution (Kan 50 μg / mL, Rif 50 μg / mL, Gen 50 μg / mL), shake and culture at 28°C overnight until the OD6 value is 1.0 -2.0, with 50g, centrifuge at room temperature for 15min, collect the cells; use 2mL transformation solution (1 / 2MS, 5% sucrose, 40μL Silwet L-77) to suspend the cells; soak the corn callus in the transformation solution of Agrobacterium ,seal. Put it back on the light culture rack and grow normally un...
Embodiment 3
[0064] Example 3 Detection of Low Temperature Resistance of Plants Overexpressing CRR1 Gene
[0065] First sow the seeds of OE-2, OE-3, OE-5, OE-6 obtained in CK group (wild type corn) and embodiment 2 in black soil, import soil and vermiculite (mass ratio 1:1:1 ) in small pots with a length of 10 cm, a width of 10 cm, and a height of 10 cm, put 5 grains in each pot, and then cover with 2 cm of soil and place them in a tray, water until the soil is completely wet, and place in a cultivation room at 23°C for 16 hours Light, 8 hours dark. After 14 days of growth, low temperature treatment at 4°C was performed for 2-3 days until the second leaf shriveled and wilted, and then it was taken out and placed in a 23°C culture room to recover for two days, then took pictures and collected materials for ion leakage rate statistics.
[0066] The plant growth of the CK group and the maize overexpression line after low temperature treatment were recovered as follows: figure 2 shown. The...
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