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Application of protein AeZDS and encoding gene thereof in promoting accumulation and stress resistance of plant carotenoids

A technology of carotene and transgenic plants, applied in the direction of botany equipment and methods, applications, plant peptides, etc., can solve problems affecting food production, restrictions, etc.

Active Publication Date: 2019-04-26
HUAIYIN INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existence of large areas of salinized land has seriously affected food production and has become the main factor restricting agricultural production

Method used

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  • Application of protein AeZDS and encoding gene thereof in promoting accumulation and stress resistance of plant carotenoids
  • Application of protein AeZDS and encoding gene thereof in promoting accumulation and stress resistance of plant carotenoids
  • Application of protein AeZDS and encoding gene thereof in promoting accumulation and stress resistance of plant carotenoids

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] The acquisition of embodiment 1 okra AeZDS protein and its coding gene

[0074] 1. Experimental materials

[0075] Refer to Wang Xu et al. (2014) [Wang Xu, Han Chunle, Zhou Yanan, Wang Chunguo, Song Wenqin, Chen Chengbin. Cloning and expression analysis of chalcone synthase gene AeCHS in okra. Journal of Plant Genetic Resources, 2014, 15(3): 561- 567] method, the leaves of the okra variety Taiwan Wufu plant were removed, quick-frozen in liquid nitrogen, and stored at -80°C.

[0076] 2. Extraction and purification of total RNA from leaves

[0077] Take about 2.0 g of Taiwan Wufu leaves, grind them into powder in liquid nitrogen, put them into a 10 mL centrifuge tube, and extract the total RNA from the leaves with the Applygen Plant RNA Extraction Kit (Applygen Technologies Inc, Beijing). The kit includes: Plant RNA Reagent, Plant tissue lysis, RNA isolation, and removal of plant polysaccharides and polyphenols; Extraction Reagent, organic extraction to remove protein, ...

Embodiment 2

[0096] Embodiment 2 Construction of AeZDS gene overexpression vector

[0097] The correct DNA fragment containing the nucleotide shown in the sequence table SEQ ID NO.1 identified by sequencing in Example 1 is double-digested with BamH I and Sac I, and the DNA fragment is reclaimed with 1% agarose gel, passed through T 4 The recovered AeZDS gene fragment was connected with the pYPx245 plasmid containing double 35S promoters by DNA ligase, and the recombinant plasmid AH128 containing grape AeZDS gene was obtained through enzyme digestion identification and sequence analysis. The expression vector also contains a gusA reporter gene and a kanamycin resistance marker gene with an intron, such as figure 1 shown.

Embodiment 3

[0098] Example 3 Transformation of Arabidopsis thaliana with AeZDS gene

[0099] The plant expression vector pCAMBIA1301-AeZDS of the okra AeZDS gene constructed in Example 2 was transformed into Arabidopsis thaliana by dipping flowers, and the specific method is as follows:

[0100] 1. Preparation of Agrobacterium

[0101] (1) Transform pCAMBIA1301-AeZDS into Agrobacterium tumefaciens EHA105 strain (Biovector Co., LTD) by electric shock method to obtain recombinant Agrobacterium containing pCAMBIA1301-AeZDS, and apply it to a plate containing kanamycin resistance for screening Turn.

[0102] (2) A single Agrobacterium was picked and inoculated in 5 mL of LB liquid medium (rifampicin 50 μg / mL, chloramphenicol 100 μg / mL), and cultured at 28° C. and 250 rpm for 20 h.

[0103] (3) Transfer 1 mL of bacterial liquid into 20-30 mL of LB liquid medium (rifampicin 50 μg / mL, chloramphenicol 100 μg / mL), culture at 28°C, 250 rpm for about 12 hours, and measure OD 600 ≈ 1.5.

[0104] (...

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Abstract

The invention discloses an application of protein AeZDS and encoding gene thereof in promoting accumulation and stress resistance of plant carotenoids. The invention provides a protein (a) or (b) as follows: (a) the protein composed of an amino acid sequence shown as sequence SEQ ID NO.2 in a sequence table, and (b) the protein with one or more substituted and / or deleted and / or added amino acid residue in the amino acid sequence shown as sequence SEQ ID NO.2 in the sequence table. When the encoding gene of protein is introduced into arabidopsis, the content of carotenoid in transgenic arabidopsis is obviously increased and the stress resistance of transgenic arabidopsis plant is promoted. The protein AeZDS and the encoding gene thereof related to biosynthesis and stress resistance of carotenoids have important theoretical significance and practical value in regulating biosynthesis and stress resistance of plant carotenoids.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the protein AeZDS related to biosynthesis and stress resistance of okra carotenoids, its coding gene and application. Background technique [0002] Okra is rich in protein, free amino acids, carotenoids, various vitamins, mineral elements such as phosphorus, iron, potassium, calcium, and viscous substances composed of pectin and polysaccharides. It has multiple health functions and is popular among consumers. Those welcome. In animals, carotenoid substances also play a particularly important role, but animals cannot synthesize carotenoids themselves and can only be ingested from their daily diet. Studies have shown that carotenoids are closely related to human health, and are indispensable nutrients in human diet structure. aspects play a very important role. [0003] The synthesis of carotenoids in higher plants starts from the isoprenyl pyrophosphate (IPP) pathway. T...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/20
CPCC12N15/8243C12N15/8273C07K14/415
Inventor 王飞兵陈新红叶玉秀周青张林青戚名扬赵慧云李威张毅王立辉吴雨
Owner HUAIYIN INSTITUTE OF TECHNOLOGY
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