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Drought and salt tolerant transgenic plant preparation method

A technology of transgenic plants and genes, which is applied in the field of preparation of transgenic plants, can solve the problems of lack of methods for preparing transgenic plants with little environmental impact, and achieve the effect of improving drought tolerance and salt tolerance and having little environmental impact

Inactive Publication Date: 2015-04-15
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is a lack of a method for preparing transgenic plants that are resistant to drought and salt and have little impact on the environment

Method used

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  • Drought and salt tolerant transgenic plant preparation method
  • Drought and salt tolerant transgenic plant preparation method
  • Drought and salt tolerant transgenic plant preparation method

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preparation example Construction

[0043] The preparation method of the drought-tolerant and salt-tolerant transgenic plant of the present invention comprises the following steps:

[0044] (1) obtaining the nucleotide sequence and amino acid sequence of the maize transcription factor gene ZmPIF3;

[0045] The present invention designs following primers according to the sequence of the ZmPIF3 gene:

[0046] F: 5'-ATGTCCGACAGCAGCGACTTCG-3' SEQ ID NO.1,

[0047] R: 5'-TCATGTTTCAGCCTCATTTCTTCC-3' SEQ ID NO.2.

[0048] The full-length cDNA (open reading frame) of ZmPIF3 gene was amplified from the total cDNA of maize Zhengdan 958 by RT-PCR technology. It is shown by sequencing that it is a transcription factor of the PIFs family. The full-length cDNA of ZmPIF3 is 1941bp, SEQ ID NO.5, encoding a protein consisting of 645 amino acids, SEQ ID NO:6.

[0049] The present invention designs following detection primer according to the cDNA sequence of ZmPIF3 gene:

[0050] F: 5'-CAATCCAGCCACCATTCCC-3' SEQ ID NO.3,

[0...

Embodiment 1

[0063] Obtaining Nucleotide and Amino Acid Sequences of Maize Transcription Factor ZmPIF3

[0064] Log on to the homepage of the public database NCBI, search for the amino acid sequence of the transcription factor PIF3 in Arabidopsis, and use this amino acid sequence as a query probe to obtain the nucleotide and amino acid sequences of a highly homologous PIF3 transcription factor gene ZmPIF3 in maize Such as figure 1 with figure 2 shown.

Embodiment 2

[0066] Molecular Cloning of Maize Transcription Factor ZmPIF3

[0067] Maize seedlings at the three-leaf and one-core stage, the variety Zhengdan 958, were taken, quick-frozen in liquid nitrogen, and stored in a -70°C refrigerator for extraction of total RNA. Total RNA was extracted using RNAiso Plus Kit from TaKaRa Company. Maize cDNA was synthesized according to the instructions of Revert Aid TM First Strand cDNA Synthesis Kit from Fermentas Company for first-strand synthesis.

[0068] The first strand of cDNA synthesized by the above kit was used as the amplification template, and the designed F: 5'-ATGTCCGACAGCAGCGACTTCG-3' SEQ ID NO.1 and R: 5'-TCATGTTTCAGCCTCATTTCTTCC-3' SEQ ID NO.2 were used as primers, RT-PCR was used for cDNA amplification. The amplification conditions were: 94°C preheating for 5 minutes; 94°C, 40s, 57°C, 40s, 72°C, 2min, a total of 35 cycles; 72°C, 10min. After the PCR, electrophoresis analysis was carried out, and the amplified fragment of about 19...

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Abstract

The invention discloses a drought and salt tolerant transgenic plant preparation method which comprises the following steps: (1) maize transcription factor gene ZmPIF3 nucleotide and amino acid sequences are obtained; (2) maize ZmPIF3 gene fragments are obtained by RT-PCR; (3) expression profile of maize transcription factor gene ZmPIF3 in adversity stress can be analyzed by fluorescence quantitative PCR method, and the ZmPIF3 gene fragments are constructed into plasmid vectors; (4) plasmid with ZmPIF3, which is obtained by the step (3), is transformed into agrobacterium by electroporation; (5) the agrobacterium with the transformed plasmid is transformed into a target plant; (6) target plant transgenic positive seedlings are identified; (7) target plant transgenic T2-generation positive homozygous plants are screened; and (8) stress resistance of the transgenic positive homozygous plants is analyzed. Maize PIFs family transcription factor gene ZmPIF3 improves the drought and salt tolerance capability of the transgenic plants.

Description

technical field [0001] The invention belongs to the field of crop genetics and breeding, and in particular relates to a method for preparing transgenic plants resistant to drought and salt. Background technique [0002] The arid and semi-arid areas in the world account for about half of the cultivated land. In these areas, the water supply is insufficient, the forest vegetation is sparse, the ecological environment is deteriorating, the soil erosion is serious, and natural disasters are frequent. Even in the case of abundant soil moisture, water deficit often occurs, which affects physiological processes such as photosynthesis, material transport, protein synthesis and cell elongation. Drought and high salinity will cause plants to dehydrate to varying degrees, causing a series of physiological and metabolic changes in plants. Therefore, abiotic stress such as drought and high salinity is called water stress or osmotic stress. The process of plant response to osmotic stress...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00
Inventor 高勇陈建民江薇戴毅陆怡吴美琴任晓芸
Owner YANGZHOU UNIV
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