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Novel paddy rice D-lactate dehydrogenase, coding gene and application thereof

A lactate dehydrogenase and coding gene technology, applied to a new type of rice D-lactate dehydrogenase and its coding gene and application fields, can solve problems such as aggravation of pests and diseases, pollution, crop yield reduction and the like

Inactive Publication Date: 2014-05-28
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, global climate conditions have deteriorated, natural disasters have occurred frequently, saline-alkali land has increased, waterlogging and drought have occurred from time to time, and pests and diseases have intensified in some areas; and with the process of industrialization, the land is seriously polluted by heavy metals such as copper and cadmium. These conditions will have a serious impact on the growth of crops, eventually resulting in reduced crop yields, affecting farmers' income and national food security

Method used

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  • Novel paddy rice D-lactate dehydrogenase, coding gene and application thereof
  • Novel paddy rice D-lactate dehydrogenase, coding gene and application thereof
  • Novel paddy rice D-lactate dehydrogenase, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the cloning of rice OsD-LDH gene

[0036] The young leaves and roots of indica rice 93-11 were taken, ground with liquid nitrogen, and the total RNA was extracted according to the Trizol method (Invitrogen), and the mixed DNA was digested with DNase I (Fermentas); 18 As primers, cDNA was obtained by reverse transcription with Superscript II (Invitrogen). All the above experiments were carried out according to the instructions of the kit.

[0037] Based on Arabidopsis D-lactate dehydrogenase (AtD-LDH) sequence (Engqvist M, Drincovich MF, Flügge U-I, Maurino VG (2009) Two d-2-hydroxy-acid dehydrogenases in Arabidopsis thaliana with catalytic capacities to participate in the last reactions of the methylglyoxal and β-oxidation pathways.Journal of Biological Chemistry284:25026-25037) BLAST the rice genome sequence to obtain the corresponding homologous sequence, design primers using cDNA as template, and use high-fidelity amplification enzyme pfu for RT-PCR am...

Embodiment 2

[0042] Example 2, OsD-LDH induced expression purification and catalytic activity identification

[0043] (1) Construction of prokaryotic expression vector

[0044] Using the correctly sequenced OsD-LDH clone as a template, high-fidelity amplification enzyme pfu was used to amplify OsD-LDH by PCR. The amplification reaction system and procedure were the same as in Example 1, and the primers used were shown in the table below.

[0045]

[0046] The fragment was recovered, digested with Sal I and Not I, and connected into the prokaryotic expression vector pGEX-6P-1, transformed into Escherichia coli DH5α, and the positive clone was sent for sequencing, and the sequenced correct clone was transformed into the Escherichia coli expression strain BL21(DE3 ).

[0047] (2) Induced expression and purification of OsD-LDH protein

[0048] 1) Pick a single colony containing the recombinant plasmid and put it into 15mL LB (ampicillin (Amp) 50μg / mL), culture overnight at 37°C and 220rpm...

Embodiment 3

[0076] Example 3, Analysis of the expression pattern of OsD-LDH in different tissues at different developmental stages of rice

[0077] Samples of indica rice 93-11 at different development stages were taken, ground with liquid nitrogen, and total RNA was obtained by removing DNA according to the method in Example 1. After chloroform extraction to remove DNase I, Oligod(T) 18 As primers, cDNA was obtained by reverse transcription with M-MLV (Promega). All the above experiments were carried out according to the instructions of the kit.

[0078] Using the obtained cDNA as a template, design primers for real-time fluorescent quantitative PCR (qRT-PCR), the instrument used in the experiment is Bio-Rad CFX96 TM Real-time system (Real-time system), the reagent is iQ TM SYBR Green Supermix, quantitative PCR results were analyzed by Bio-Rad CFX Manager software, and all quantitative expression results were compared with at least 3 internal reference genes.

[0079] The real-time f...

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Abstract

The invention discloses a novel paddy rice D-lactate dehydrogenase (OsD-LDH), a coding gene and application thereof, and belongs to the field of plant bioengineering. The novel paddy rice D-lactate dehydrogenase is D-lactate dehydrogenase which is cytochrome C depended, the amino acid sequence of the novel paddy rice D-lactate dehydrogenase is shown in SEQ ID NO.1, the nucleotide sequence of the D-lactate dehydrogenase coding gene is shown in SEQ ID NO.2. The OsD-LDH gene provided by the invention participates in the pyruvic aldehyde pyruvaldehyde metabolism generated under a stress condition in a plant body, and can be used for improving the adversity stress resistance of the plant and cultivating high stress resistance plants. The OsD-LDH gene provided by the invention can be further used as a selection marker and applied in transgenic selection through adopting pyruvic aldehyde pyruvaldehyde or D-lactate acid as selection pressure. The D-lactate dehydrogenase provided by the invention can be used for cultivating high stress-resistance plants and can be used for transgenic selection, and has great importance for increasing agricultural production and improving transgenic methods.

Description

technical field [0001] The invention relates to the field of plant bioengineering, in particular to a novel rice D-lactate dehydrogenase and its coding gene and application. Background technique [0002] Rice is one of the most important food crops in the world. In agricultural production, rice often encounters adversity conditions such as various biotic or abiotic stresses during the growth period. Plants under these stress conditions produce methylglyoxal, a mutagen and genotoxic agent that inhibits cell growth at high concentrations (Ray S, Dutta S, Halder J, Ray M (1994) Inhibition of electron flow through complex I of the mitochondrial respiratory chain of Ehrlich ascites carcinoma cells by methylglyoxal. Biochem J.303:69–72), in plants, the glyoxalase system is used to detoxify methylglyoxal, and finally produce D-lactic acid (Yadav SK , Singla-Pareek SL, Sopory SK (2008) An overview on the role of methylglyoxal and glyoxalases in plants. Drug metabolism and drug int...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N15/11C12N15/82A01H5/00C12Q1/68
CPCC12N9/0006C12N15/8271C12N15/8273C12Y101/01028
Inventor 安保光李阳生邓小龙兰杰
Owner WUHAN UNIV
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