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Oryza sativa autophagy-related protein OsATG8b (oryza sativa autophagy-related gene 8b) and novel application of gene of oryza sativa autophagy-related protein OsATG8b

An autophagy-related protein and rice technology, applied in the fields of application, genetic engineering, virus/bacteriophage, etc., can solve problems affecting heavy metal tolerance or accumulation

Inactive Publication Date: 2016-06-01
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] At present, there is no literature reporting whether the rice autophagy-related protein OsATG8b gene can affect the tolerance or accumulation of heavy metals (especially cadmium) in rice.

Method used

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  • Oryza sativa autophagy-related protein OsATG8b (oryza sativa autophagy-related gene 8b) and novel application of gene of oryza sativa autophagy-related protein OsATG8b
  • Oryza sativa autophagy-related protein OsATG8b (oryza sativa autophagy-related gene 8b) and novel application of gene of oryza sativa autophagy-related protein OsATG8b
  • Oryza sativa autophagy-related protein OsATG8b (oryza sativa autophagy-related gene 8b) and novel application of gene of oryza sativa autophagy-related protein OsATG8b

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: The expression of OsATG8b gene is regulated by the induction of heavy metal cadmium, zinc, copper and other stress factors and hormones

[0040] The rice variety used in the present invention is Nipponbare (Oryzasativa L.japonica.cv.Nipponbare), and the seeds are preserved and provided by the South China Botanical Garden of the Chinese Academy of Sciences. The rice seeds were germinated with 1 / 2 liquid MS medium, and after soaking to accelerate germination, the rice seedlings were transferred to a 23°C culture room (16h light / 8h dark), and continued to be cultured with 1 / 2MS liquid medium for 2 weeks. Then use CdCl containing 2 (50μM), ZnSO 4 (2mM), CuCl 2 (150μM), NaCl (200mM), mannitol (300mM), H 2 o 2 (5mM) and JA (92μM) 1 / 2MS liquid medium to treat rice seedlings, collect 0.5g each of rice young leaves and radicles after stress treatment 0h, 2h, 6h, 12h, 24h and 48h, for extracting total RNA . The extraction of RNA was carried out according to the s...

Embodiment 2

[0045] Example 2: Cloning of OsATG8b gene and construction of yeast recombinant expression vector OsATG8b-pYES260

[0046] The seedling leaves of the japonica rice variety Nipponbare (preserved in the South China Botanical Garden) were taken, and the RNA of the young leaves was extracted and reverse-transcribed into cDNA. Using the cDNA as a template, primers OsATG8bYEF: 5'-TTTCAGGGCGCCATGGCCAAGAGCTCGTTCAA-3' and OsATG8bYER: 5'-CGTTACTAGTGGATCCTAGAGCAGCCCAAAGGTG- 3' (SEQIDNO.7 and SEQIDNO.8), the full-length cDNA reading frame of the OsATG8b gene was amplified by high-fidelity Taq enzyme PCR. For the PCR system used, refer to the instruction manual of PrimeSTARHSDNAPolymerasewithGCBuffer from TaKaRa Company. The amplified DNA fragment was in accordance with the instructions of HiPureGelPureDNAKits of Magen Company. The recovered fragment was used for insertion into the yeast expression vector pYES60. Saccharomyces cerevisiae expression vector pYES260 was digested with NcoI a...

Embodiment 3

[0047] Example 3: Expression of OsATG8b gene in Saccharomyces cerevisiae improves tolerance of yeast to heavy metal cadmium

[0048] Cultivate Saccharomyces cerevisiae strain Δycf1 (purchased from Euroscarf, European Yeast Research Center, http: / / web.uni-frankfurt.de / fb15 / mikro / euroscarf / , strain number Y04069), use pYES260 plasmid and recombinant OsATG8b-pYES260 to the above yeast to convert. Since the OsATG8b gene is placed under the regulation of the yeast galactose-induced promoter PGAL1 (see figure 1 shown), OsATG8b-pYES260 was transformed into Saccharomyces cerevisiae and grown on the growth selection synthetic medium (Selective Growth Synthetic Medium, SDmedium) supplemented with galactose to induce heterologous overexpression of OsATG8b in yeast.

[0049] The method used for yeast transformation is the lithium acetate conversion method, and the specific steps are as follows:

[0050] 1) Inoculate a single colony of the yeast strain Δycf1 to be transformed into 5 mL o...

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Abstract

The invention discloses an oryza sativa autophagy-related protein OsATG8b (oryza sativa autophagy-related gene 8b) and application of a coding gene of the oryza sativa autophagy-related protein OsATG8b to improvement of heavy metal tolerability or accumulation of plants. Expression of the gene in engineered saccharomyces cerevisiae can improve cadmium tolerability of yeasts and reduce the content of cadmium in the yeasts, so that the protein OsATG8b shows a heavy metal cadmium detoxication capability. If the gene is expressed in engineering bacteria in a transgenic manner, on the one hand, heavy metal cadmium tolerability of the engineering bacteria can be improved, on the other hand, heavy metal cadmium accumulation in transgenic saccharomyces cerevisiae can be reduced, and accordingly the engineering bacteria can be applied to microorganism genetic modification engineering aiming at heavy metal cadmium pollution. The gene has a potential to be applied to cadmium-resistant genetic engineering breeding of plants, and by regulation of expression of the gene in the plants, heavy metal cadmium tolerability of transgenic plants can be changed, heavy metal cadmium accumulation in the transgenic plants can be changed as well, so that the problem of quality decline caused by cadmium enrichment of agricultural products acquired from heavy metal cadmium polluted soil is solved. The gene has a potential to be applied to low-cadmium or cadmium-resistant genetic engineering breeding of crops.

Description

technical field [0001] The invention belongs to the field of biological genetic engineering, in particular to rice autophagy-related protein OsATG8b ( O ryza s ativa A u t ophagy-related G ene 8b ) and its coding gene and its application in regulating the tolerance and accumulation of Saccharomyces cerevisiae to cadmium. Background technique [0002] Autophagy is a conserved metabolic pathway in eukaryotes, which is widely found in eukaryotes and mainly mediates the decomposition of intracellular substances and the recycling of nutrients. In plants, studies have shown that the autophagy pathway is involved in multiple physiological processes such as plant growth, development and stress response. Autophagy-related protein ATG8 is one of the key factors involved in the formation of autophagosomes. ATG8 is a ubiquitin-like protein whose sequence is typically characterized by two α-helices at the amino-terminus and a ubiquitin-like core at the carboxy-terminus. ATG8 is c...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/82A01H5/00
CPCC12N15/81C12N15/8271C12N15/8273C12N15/8279C12N2800/102
Inventor 张美莫辉李静
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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