Corn 3-glyceraldehyde phosphate dehydrogenase ZmGPDH1 and application of coding gene of corn 3- glyceraldehyde phosphate dehydrogenase ZmGPDH1 to regulation and control over plant stress tolerance
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A technology of plant stress tolerance and transgenic plants, applied in application, plant products, genetic engineering, etc., can solve the problems of few research reports
Active Publication Date: 2019-03-01
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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Except for the model plant Arabidopsis thaliana and some algae, the GPDH family has rarely been reported in other higher plants, especially in maize.
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Embodiment 1
[0074] Expression pattern of ZmGPDH1 gene in maize root under embodiment 1, salt and osmotic stress
[0075] 1. Processing of plant samples
[0076] 200mM NaCl and 400mM mannitol were used to treat the three-leaf and one-heart period corn (Maize He 344 inbred line) seedlings, and the roots were sampled at different time points (0, 1, 3, 6, 12 and 24h), and liquid nitrogen quick-frozen , stored at -80°C for RNA extraction.
[0077] 2. RNA extraction and reverse transcription
[0078] Total RNA was extracted using TRIzol reagent (Invitrogen). cDNA was obtained by reverse transcription using ReverTra Ace qPCR RT MasterMix with gDNA Remover (TOYOBO).
[0079] 3. Real-time quantitative PCR
[0080] Real-time quantitative PCR was performed using the Bio-Rad Chromo4real-time PCR system. Maize ZmGAPDH (XM_020551757) and ZmACTIN (XM_008656735.2) were used as internal references to standardize data. Primer sequences are shown in Table 1. Relative expression level using 2 -△△CT m...
Embodiment 2
[0086] Example 2, the acquisition of ZmGPDH1 transgenic Arabidopsis and its stress tolerance analysis
[0087] 1. Obtaining and identification of ZmGPDH1 transgenic Arabidopsis
[0088]1. Taking maize root cDNA as a template, using ZmGPDH1-GFP-F and ZmGPDH1-GFP-R primers for PCR amplification to obtain PCR amplification products. The primer sequences are as follows (the underlined sequence is the enzyme recognition site):
[0091] 2. Carry out double digestion and ligation of the pBI121-GFP vector and the PCR amplification product obtained in step 1 with restriction endonucleases XbaI and SalI to obtain the recombinant plasmid 35S:ZmGPDH1-GFP. and validated by sequencing.
[0092] Sequencing results show that the recombinant vector 35S:ZmGPDH1-GFP replaces the DNA fragment between the XbaI and SalI restriction sites of the pBI121-GFP vector wi...
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Abstract
The invention discloses corn 3-glyceraldehyde phosphate dehydrogenase ZmGPDH1 and application of a coding gene of corn 3- glyceraldehyde phosphate dehydrogenase ZmGPDH1 to regulation and control overplant stress tolerance. With a corn GPDH gene family member ZmGPDH1 being a research object, the corn GPDH gene family member ZmGPDH1 is transferred into wild type Arabidopsis to obtain T3 generationhomozygous transformants. The transformant OE-1 and the transformant OE-2 are selected from the T3 generation homozygous transformants for salt tolerance and drought enduring functional identification. With wild type Arabidopsis being a comparison, ZmGPDH1 transgenic Arabidopsis is researched for the germination rate, root length and fresh weight changes under salt and drought stress. Results showthat under the salt stress treatment condition, the seed germination rate of ZmGPDH1 transgenic Arabidopsis is remakrbaly higher than that of wild type Arabidopsis, and the root length, fresh weightand growing vigor of ZmGPDH1 transgenic Arabidopsis are remarkably superior to these of the comparison. It is shown that ZmGPDH1 can remarkably improve the capacity of salt tolerance and drought enduring of transgenic plants, and can be applied to cultivation of corn stress-resistant varieties as a stress-resistant gene.
Description
technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the application of corn 3-phosphate glycerol dehydrogenase ZmGPDH1 and its coding gene in regulating plant stress tolerance. Background technique [0002] Improving the stress tolerance of crops has become a hot and difficult point in the current technical research in the field of agriculture and animal husbandry, and it is also a major problem that needs to be solved urgently. In recent years, with the development of functional genomics and molecular biology, excavating key genes of stress tolerance and using genetic engineering technology to breed new crop varieties with good stress tolerance have become one of the means to effectively improve crop stress tolerance. [0003] Glycerol-3-phosphate (G-3-P) is an important intermediate product in the process of lipid metabolism and participates in various physiological and biochemical processes in plants. Glycerol 3-phospha...
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