Maize waterlogging-tolerant gene ZmWST1
A flood-resistant and corn technology, applied in the field of genetic engineering, can solve problems such as corn production loss, and achieve the effect of improving the flood-resistant ability
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Embodiment 1
[0020] Example 1 Acquisition of maize waterlogging resistance gene ZmWST1
[0021] 1. Discovery of ZmWST1, a candidate gene for waterlogging resistance in maize
[0022] 1. 400 maize inbred lines introduced from the International Maize and Wheat Improvement Center (CIMMYT) were planted in the greenhouse, and waterlogged at the stage of 3 leaves and 1 heart. After 8 days, the relative plant height, relative root length and relative secondary root were counted. 6 traits including number, relative root dry weight, relative stem dry weight and survival rate. The phenotype data of 378 inbred lines were selected, and 39797 SNPs were used to carry out association analysis on the above six traits. The mixed linear model MLM (PCA+K) was used for calculation, and the significance threshold of association analysis was 0.05 / 39797=1.25E-6. It was found that under waterlogging stress, 13 loci were associated with relative root dry weight, and the 5 loci with the smallest P values were l...
Embodiment 2
[0044] Example 2 Construction and Genetic Transformation of ZmWST1 Gene Transformation Vector
[0045] 1. Construction of transformation vector
[0046] The stain-resistant gene ZmWST1 cloned in Example 1 was connected to the pCAMBIAubi1300 (provided by our laboratory) plasmid by homologous recombination to construct a plant expression vector, which was named pCAMBIAubi1300-ZmWST1, and the specific operations were as follows:
[0047] 1. Using the recovered product in Example 1, use the pENT-D kit to clone the recovered band into the pENT-D vector. Reaction system: PCR product 4.5μl (40-50ng), diluted salt 1μl, pENT-D 0.5μl. The reaction product was mixed, centrifuged, and placed at 25°C for 1h.
[0048] 2. Transform DH10B Escherichia coli competent cells by electric shock method. The transformed Escherichia coli was cultured at 37°C for 1 hour, then spread evenly on a 100mg / Lkm LB plate, and cultured at 37°C for 20h.
[0049] 3. Extract single clones, detect, sequence, an...
Embodiment 3
[0054] Implementation of 3 Waterlogging Resistance Test of ZmWST1 Transgenic Plants
[0055] 18 ZmWST1 corn plants transferred in Example 2 were selected to carry out waterlogging stress experiment. The specific steps are as follows: the transgenic ZmWST1 corn was planted in nutrient pots, and 6 plants were planted in each pot, and repeated 3 times. The inbred line A188 was used as a control, and they were grown normally to 3 leaves and 1 heart in the greenhouse. Then, waterlogging treatment was carried out for 8 days, and the growth status of the transgenic plants was observed.
[0056] The result is as Figure 6 As shown, it was found that the ZmWST1-transformed maize grew well, while the growth of the control A188 was severely inhibited. After 3 repeated experiments, the maize plants transfected with ZmWST1 gene showed strong waterlogging tolerance. Therefore, our experimental results prove that the ZmWST1 gene can significantly improve the waterlogging tolerance of maiz...
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