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Application of aba 8'-hydroxylase gene OsABA8ox2 in plant photomorphogenesis and root development

A technology for photomorphogenesis and hydroxylase, which is applied in the field of plant genetic engineering and genetic improvement to achieve the effect of improving shade tolerance, improving shade tolerance and good photomorphogenesis

Active Publication Date: 2020-03-10
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In summary, there is no OsABA8ox Related reports of family genes involved in plant photomorphogenesis and root development

Method used

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  • Application of aba 8'-hydroxylase gene OsABA8ox2 in plant photomorphogenesis and root development
  • Application of aba 8'-hydroxylase gene OsABA8ox2 in plant photomorphogenesis and root development
  • Application of aba 8'-hydroxylase gene OsABA8ox2 in plant photomorphogenesis and root development

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] OsABA8ox2 Construction of knockout vectors and gene-edited plants ( OsABA8ox2 knockout rice line)

[0038] one, OsABA8ox2 Knockout vector pHUN4c12- OsABA8ox2 build

[0039] A 19 bp specific target sequence was selected by E-CRISP (http: / / www.e-crisp.org / E-CRISP / designcrispr.html) to generate sgRNA (single-guide RNA). The sequence (SEQ ID No: 6) is located at OsABA8ox2 92-110 bp of the full-length 1533 bp CDS sequence (SEQ ID No: 4) (5'-GGAGGAGAGATGTTGGACA-3'). synthetic primer pair OsABA8ox2-CRIS (Table 1), add a phosphate group to the 5' end of the sequence using the following system:

[0040] OsABA8ox2-CRISF (10 mM) 1 μl

[0041] OsABA8ox2-CRISR (10 mM) 1 μl,

[0042] 10x T4 DNA Ligase Buffer (with 10 mM ATP) 2μl,

[0043] T4 polynucleotide kinase (3’ phosphatase plus; NEB) 1μl,

[0044] h 2 O 15 μl.

[0045] 37°C for 40 min, 65°C for 20 min (enzyme heat inactivation), 94°C for 5 min, 50°C for 2 min.

[0046] Plasmid pHUN4c12 (In Vitro Mutagenesis, ...

Embodiment 2

[0060] OsABA8ox2 Obtaining and identification of overexpression rice lines

[0061] for build OsABA8ox2 overexpression vector, complete OsABA8ox2 Coding sequence (SEQ ID No: 4) with primer pair OsABA8ox2-OE (Table 1) Perform RT-PCR (reverse transcription polymerase chainreaction) amplification. The amplified fragment verified by sequencing was integrated into the transformed and BamHI(NEB) and Sac Ⅰ (NEB) The pCAMBIA1303 vector after double digestion, making it in Ubi-1 The 3' end of the promoter is driven by the promoter. Homologous recombination was performed using GBclonart Seamless Cloning Kit (GBI, Suzhou). As in Example 1, the overexpression transgenic line of the japonica rice variety Kitaake was obtained by using Agrobacterium-mediated transformation. Using genomic DNA as a template, primer pairs OsABA8ox2-C (Table 1) Amplified by PCR exogenous OsABA8ox2 CDS fragments to identify positive transgenic lines ( figure 2 A). figure 2 Quantitative RT-PCR ...

Embodiment 3

[0063] OsABA8ox2 Phenotyping of photomorphogenesis, leaf length and plant height determination and analysis of shade tolerance in knockout rice lines

[0064] Plants were grown under weak (2100 lux) light conditions. Compared with wild type (WT), OsABA8ox2 The third leaf and leaf sheath of the overexpression (OE) line were slender and weak ( image 3 A), especially the leaf sheath length was significantly higher than that of the wild type ( image 3 B), the third leaves of the knockout lines KO1 and KO2 were significantly shorter ( image 3 A, B). The leaf color of OE was lighter than that of the wild type, showing light green, while the leaves of KO were dark green. OsABA8ox2 Involved in leaf development. The 25-day-old WT seedlings were at the 3-leaf stage, while the 4th leaf of the OE seedlings had stretched out ( image 3 A), the growth rate of overexpressed seedlings is faster than that of wild type. because OsABA8ox2 The leaves of OE seedlings are slender, the ...

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Abstract

The present invention relates to abscisic acid (abscisic acid, ABA) 8'-hydroxylase gene OsABA8ox2 The application in plant photomorphogenesis and root development belongs to plant genetic engineering and genetic improvement. Rice ABA 8'‑hydroxylase gene OsABA8ox2 The amino acid sequence of the protein product is shown in SEQ ID No:1. Knockout by gene editing technology in the present invention OsABA8ox2 Finally, it can positively regulate the photomorphogenesis under weak light or shade conditions, alleviate the shade avoidance response, and improve the shade tolerance of plants; it can optimize the root structure under drought stress to improve the drought tolerance of plants. The improvement of these traits has great application value for crop production and urban three-dimensional greening.

Description

technical field [0001] The present invention relates to plant genetic engineering and genetic improvement, in particular to ABA 8'-hydroxylase gene OsABA8ox2 Applications in plant photomorphogenesis and root development. Background technique [0002] Plants, as photoautotrophs, are very sensitive and dexterous to the light environment. Photomorphogenesis and dark morphogenesis are two distinct sets of developmental programs adopted by plants in response to environmental light (Trends in plant science, Xu et al. 2015). Sichuan and Guizhou in southwestern China are under low light conditions almost all year round (Chinese Journal of Oil Crops, Fan Yuanfang et al., 2016). The continuous or frequent rainy weather in Huanghuaihai and other regions has a very negative impact on the production of rice, corn and other crops. The close planting or intercropping adopted in modern agricultural production will intensify the competition of crop groups for light. With the acceleration...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/82A01H5/00A01H5/12A01H5/06A01H6/46
CPCC12N9/0073C12N15/8261C12N15/8273C12Y114/13093
Inventor 张艳
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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