ZmGPDH5 (Zea Mays Glycerol-3-Phosphate Dehydrogenase 5) and application of encoding gene thereof in regulating stress tolerance of plant
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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: 2019-04-05
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 ZmGPDH5 (Zea Mays Glycerol-3-Phosphate Dehydrogenase 5) and application of an encoding gene thereof in regulating the stress tolerance of a plant. A member ZmGPDH5 of a ZmGPDHgene family is used as a study object, and is transferred into wild type Arabidopsis to obtain T3-generation homozygous transformants; two transformants of OE-1 and OE-2 in the T3-generation homozygous transformants are selected for identifying the salt-tolerant function; taking the wild type Arabidopsis as a control, the germination rate, the root length and the fresh weight change of ZmGPDH5 transgenic Arabidopsis under salt stress can be studied; a result shows that under the treatment condition of the salt stress, the seed germination rate of the ZmGPDH5 transgenic Arabidopsis is remarkably higher than that of the wild type Arabidopsis, and the root length, the fresh weight and the growth vigor of the ZmGPDH5 transgenic Arabidopsis are remarkably superior to those of the control; the result shows that the ZmGPDH5 is capable of remarkably increasing the salt tolerance of a transgenic plant, and the ZmGPDH5 as a salt-tolerant gene can be applied to breeding of stress-tolerant varieties of zea mays.
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...
Claims
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