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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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existence of large areas of salinized land has seriously affected food p

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

Examples

Experimental program
Comparison scheme
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Example Embodiment

[0073] Example 1 Okra AeZDS protein and its encoding gene acquisition

[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 the chalcone synthase gene AeCHS of okra. Journal of Plant Genetic Resources, 2014, 15(3): 561- 567] method, take off the leaf material of okra variety Taiwan Wufu plant, quick-freeze in liquid nitrogen, and store at -80℃.

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

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

Example Embodiment

[0096] Example 2 Construction of AeZDS gene overexpression vector

[0097] The DNA fragments containing the nucleotides shown in SEQ ID NO. 1 in the sequence list identified by the sequencing in Example 1 were double digested with BamH I and Sac I. The DNA fragments were recovered by 1% agarose gel and passed through T 4 DNA ligase ligated the recovered AeZDS gene fragment with the pYPx245 plasmid containing the double 35S promoter, and the recombinant plasmid AH128 containing the grape AeZDS gene was obtained by restriction enzyme identification and sequence analysis. The expression vector also contains the gusA reporter gene and the kanamycin resistance marker gene with introns, such as figure 1 Shown.

Example Embodiment

[0098] Example 3 Transformation of AeZDS gene into Arabidopsis

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

[0100] 1. Preparation of Agrobacterium

[0101] (1) Transform pCAMBIA1301-AeZDS into Agrobacterium tumefaciens strain EHA105 (Biovector Co., LTD) strain by electroporation to obtain recombinant Agrobacterium containing pCAMBIA1301-AeZDS, and spread it on a plate containing kanamycin resistance. Turn.

[0102] (2) Pick a single Agrobacterium and inoculate it in 5mL LB liquid medium (rifampicin 50μg / mL, chloramphenicol 100μg / mL), culture at 28°C, 250rpm for 20h.

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

[0104] (4) The cells were collected by centrifugation at 8000rpm, 4°C, 10...

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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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