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Protein AePDS associated with biosynthesis of abelmoschus esculentus carotenoid and stress resistance as well as encoding gene and applications of protein AePDS

A carotene, stress-resistant technology, applied in the biological field, can solve problems such as limiting and affecting food production

Active Publication Date: 2019-04-09
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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  • Protein AePDS associated with biosynthesis of abelmoschus esculentus carotenoid and stress resistance as well as encoding gene and applications of protein AePDS
  • Protein AePDS associated with biosynthesis of abelmoschus esculentus carotenoid and stress resistance as well as encoding gene and applications of protein AePDS
  • Protein AePDS associated with biosynthesis of abelmoschus esculentus carotenoid and stress resistance as well as encoding gene and applications of protein AePDS

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] The acquisition of embodiment 1 okra AePDS 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 AePDS 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 AePDS gene fragment was connected with the vector pCBGUS containing double 35S promoters by DNA ligase, and the recombinant plant expression vector pCAMBIA1301-AePDS containing the okra AePDS 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 AePDS gene

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

[0100] 1. Preparation of Agrobacterium

[0101] (1) Transform pCAMBIA1301-AePDS into Agrobacterium tumefaciens EHA105 strain (Biovector Co.,LTD) by electric shock method to obtain recombinant Agrobacterium containing pCAMBIA1301-AePDS, and spread it on 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....

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Abstract

The invention discloses key enzyme protein AePDS for biosynthesis of abelmoschus esculentus carotenoid and applications of an encoding gene of the key enzyme protein AePDS to increase of content of plant carotenoid and improvement of salt tolerance and drought resistance. The protein is as follows (a) or (b): (a), protein composed of an amino acid sequence shown in SEQ ID NO. 2; (b), protein whichis derived from the sequence SEQ ID NO. 2, associated with plant stress resistance and obtained by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid residuesequence shown in SEQ ID NO. 2 in the sequence table. Experiments prove that when the encoding gene of the protein is introduced into arabidopsis thaliana, the content of carotenoid in the transgenicarabidopsis thaliana is obviously increased, and the salt tolerance and drought resistance of transgenic arabidopsis thaliana plants are simultaneously improved. Therefore, the protein AePDS associated with biosynthesis of abelmoschus esculentus carotenoid and stress resistance and the encoding gene of the protein have great theoretical significance and practical value in regulation of biosynthesis of the plant carotenoid as well as salt tolerance and drought resistance. The protein has broad application space and market prospect in the agricultural field.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the biosynthesis of okra carotenoids, salt-tolerance and drought-resistance related protein AePDS and 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. β-carotene is the precursor of vitamin A synthesis in humans and animals. Lutein and zeaxanthi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/82A01H5/00A01H6/20
CPCC12N9/001C12N15/825C12N15/8273C12Y103/99031
Inventor 王飞兵陈新红叶玉秀周青张毅吴雨王立辉戚名扬赵慧云
Owner HUAIYIN INSTITUTE OF TECHNOLOGY
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