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Transgenic method capable of controlling ALA synthesis in plants and promoting growth and stress resistance

A technology for promoting growth and stress resistance, applied in the field of genetic engineering, can solve the problems of no practical value, photobleaching of transgenic plants, etc., and achieve the effects of increased biological yield and economic yield, normal growth and development, and strong stress resistance.

Inactive Publication Date: 2011-08-31
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, all transgenic plants, whether tobacco or rice, can only grow under low-light conditions
Once transferred to strong light (1 / 6 of natural light intensity), transgenic plants will appear photobleaching phenomenon, therefore, such transgenic plants have no practical value in agricultural production

Method used

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  • Transgenic method capable of controlling ALA synthesis in plants and promoting growth and stress resistance
  • Transgenic method capable of controlling ALA synthesis in plants and promoting growth and stress resistance
  • Transgenic method capable of controlling ALA synthesis in plants and promoting growth and stress resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, the acquisition of transphotosensitive promoter AtHemA1P and YHem1 plants

[0033] 1. Construction of the recombinant expression vector used for the transgene

[0034] 1. Acquisition of Arabidopsis HemA1 photosensitive promoter (AtHemA1P)

[0035] According to the nucleotide sequence of the Arabidopsis thaliana HemA1 gene promoter registered in Genbank (Genebank No: AF295364), the total DNA of the extracted Arabidopsis thaliana was used as a template, and the primer (AtHemA1PZ1: 5 '-CCCAAGCTTACCGAAATGTAGGAATCCCACTTC-3' and primers (AtHemA1PF1: 5'-AGGATCCCAAAATCTCAATCTCCTCTCTGTC-3') added with BamHI restriction sites were used for PCR amplification. The amplification system was 50μl, containing 10mmol / L Tri-HCl pH 8.3, 50mmol·L -1 KCl, 2mmol L -1 Mg 2 Cl, 250 μmol L -1 Various dNTPs, 25pmol of each primer, 200-500ng of DNA, and 1U of Taq polymerase, the amplification conditions were 94°C / 10min pre-denaturation, 94°C / 40s denaturation, 72°C annealing and ...

example 1

[0077] Example 1, the functional identification of the light-sensitive promoter AtHemA1P controlling the YHem1 gene in tobacco

[0078] 1. Changes of YHem1 gene expression in transgenic tobacco with light time

[0079] In order to detect the light-sensitive promoter AtHemA1P controlling the expression of YHem1 gene under light, the T cells transfected with YF8631 3 In the next generation tobacco plants, the expression of YHem1 gene was detected by semi-quantitative and real-time fluorescent quantitative PCR with the change of light time.

[0080] The result is as figure 2 As shown, whether semi-quantitative PCR detection or real-time fluorescence quantitative PCR detection was used, the expression of YHem1 gene increased rapidly when the transgenic tobacco was changed from dark conditions to light conditions, reached the maximum value after 5 hours of light, and then gradually decreased. After 2.5 hours in the dark, the transcription level of YHem1 gene was very low, indica...

example 2

[0106] Example 2, functional identification of YHem1 Arabidopsis controlled by the photosensitive promoter AtHemA1P

[0107] 1. Study on improving salt tolerance of transgenic Arabidopsis

[0108] The independent T of the positive transgenic Arabidopsis that example 1 obtains 2 generation strain (P 0 , P 3 , P 12 ) seeds are sterilized (carried out according to the method of step 2 in method two in example 1), and the transgenic Arabidopsis T thaliana after disinfection 2 Generation (P 0 , P 3 , P 12 ) seeds were spread on MS medium containing 0, 50mM, 100mM, 150mM NaCl, cultured in the dark at 4°C for two days, then transferred to 22±2°C, cultured in 16hr light, and grown under salt stress for two weeks, and observed, cultured under the same conditions Wild type Arabidopsis (WT) (WT Arabidopsis, 'Columbia' ecotype) was used as a control.

[0109] It was found that the growth of the transformed YK3840-YHem1 Arabidopsis lines and wild-type Arabidopsis (WT) was inhibited...

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Abstract

The invention discloses a transgenic method capable of controlling ALA biosynthesis in plants and promoting growth and stress resistance of plants belonging to genetic engineering field. The method is to do the trangenosis with an Arabidopsis thaliana HemAi gene photosensitive type promoter and a saccharomyces cerevisiae hansen 5-aminolevulinic acid (ALA) synthase gene (YHemi) in the plant through a plant expression vector. The invention can promote the synthesis of chlorophyll and heme and avoid the accumulation of ALA and other porphyrin intermedium in the dark through increasing ALA synthetic amount in plants, thereby avoiding plant photobleaching phenomenon and improving tolerances of plants to low temperature, high temperature, highlight, dim light and salt stress. Therefore, the method not only does not influence normal growth of the transgenic plants, but also can raise biological yield and economical yield of plants. The method can used for herbaceous plant like Arabidopis thaliana, tobacco, tomato, rape, paddy rice, watermelon, strawberry, etc, and can also used for woody plant like apple, pear, etc.

Description

technical field [0001] The invention relates to a transgenic method for controlling ALA synthesis in plants, promoting growth and improving stress resistance, and belongs to the field of genetic engineering. It is a kind of application of microbial genes in agricultural production, and it is a method of cultivating new germplasm of high-yield and multi-resistant plants by using bio-genetic engineering technology. Background technique [0002] 5-Aminolevulinic acid (5-Aminolevunilic acid, ALA) is a 5-carbon compound similar to an amino acid that is ubiquitous in all organisms. 12 etc.) are key precursors for biosynthesis. In plants, it is converted from glutamic acid through a three-step enzymatic reaction; in animals and yeast, it is catalyzed by succinyl CoA and glycine through ALA synthase, and can be produced in one step. [0003] Through a large number of exogenous treatment experiments, we and other research groups have shown that ALA can significantly improve the lev...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/113C12N15/52
Inventor 汪良驹张治平姚泉洪
Owner NANJING AGRICULTURAL UNIVERSITY
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