Application of maize 3-phosphate glycerol dehydrogenase zmgpdh5 and its coding gene in regulating plant stress tolerance
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A technology of plant roots and nucleic acid molecules, which can be used in applications, plant products, genetic engineering, etc., and can solve the problems of few research reports
Active Publication Date: 2021-11-23
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] Example 1, the expression pattern of ZmGPDH5 gene in maize root and leaf under salt stress
[0075] 1. Processing of plant samples
[0076] Maize seedlings (maize He 344 inbred line) at the three-leaf and one-heart stage were treated with 200mM NaCl, and the leaves and roots were sampled at different time points (0, 1, 3, 6, 12 and 24h) of the treatment, quick-frozen in liquid nitrogen, and -80 ℃ 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 ...
Embodiment 2
[0086] Example 2, the acquisition of ZmGPDH5 transgenic Arabidopsis and its stress tolerance analysis
[0087] 1. Obtaining and identification of ZmGPDH5 transgenic Arabidopsis
[0088]1. Taking maize root cDNA as a template, using ZmGPDH5-GFP-F and ZmGPDH5-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:ZmGPDH5-GFP. and validated by sequencing.
[0092] Sequencing results show that: the recombinant vector 35S:ZmGPDH5-GFP replaces the DNA fragment between the XbaI and SalI restriction sites of the pBI121-GFP vector with...
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Abstract
The invention discloses the application of corn 3-phosphate glycerol dehydrogenase ZmGPDH5 and its coding gene in regulating plant stress tolerance. The invention takes ZmGPDH5, a member of the maize GPDH gene family, as the research object, and transforms it into wild-type Arabidopsis thaliana to obtain homozygous transformants of T3 generation. Among them, two transformants, OE‑1 and OE‑2, were selected for functional identification of salt tolerance. Using wild-type Arabidopsis as a control, the germination rate, root length and fresh weight of ZmGPDH5 transgenic Arabidopsis under salt stress were studied. The results showed that under the salt stress treatment, the seed germination rate of ZmGPDH5 transgenic Arabidopsis was significantly higher than that of wild type Arabidopsis, and the root length, fresh weight and growth vigor of ZmGPDH5 transgenic Arabidopsis were significantly better than those of the control. It shows that ZmGPDH5 can significantly improve the salt tolerance of transgenic plants, and ZmGPDH5 as a salt tolerance gene can be used to breed stress-tolerant maize varieties.
Description
technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the application of corn 3-phosphate glycerol dehydrogenase ZmGPDH5 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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