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Application of gene CDA1 in regulation and control of chloroplast development

A chloroplast and gene technology, applied in the field of plant genetic engineering and cell biology, can solve the problem of unclear regulatory molecular mechanism

Inactive Publication Date: 2020-05-12
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chloroplast is an important organelle in plants. Although its development and regulatory mechanism have been widely concerned by researchers, due to its complexity, there are still many regulatory molecular mechanisms that are still unclear.

Method used

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  • Application of gene CDA1 in regulation and control of chloroplast development
  • Application of gene CDA1 in regulation and control of chloroplast development
  • Application of gene CDA1 in regulation and control of chloroplast development

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Construction of CDA1 gene interference strains, CDA1 gene mutants and tag vectors

[0050] 1. Construction of CDA1 gene interference strain:

[0051] 1.1 Construction of interference carrier:

[0052] The backbone interference vector pCAMBIA2300RNAi-CDA1 was obtained by transforming pCAMBIA2300. Primers (CDA1IF and CDA1IR) were designed according to the CDS sequence of the CDA1 gene to amplify a 256bp specific fragment (SEQ ID NO.5) on the CDA1 gene. After the fragment was recovered by agarose gel electrophoresis, primers CDA1-PstL and CDA1-KpnR were designed to amplify the specific fragment, and then the PCR fragment and the pCAMBIA2300RNAi-CDA1 vector were digested with KpnI and PstI. pCAMBIA2300RNAi-CDA1 of the chain exogenous fragment. Take 1 μL of the ligation product and transform it into Escherichia coli DH5α by the freeze-thaw method, and coat the transformed product with kanamycin-resistant (50 μg / mL) LB medium. Cultivate overnight at 37°C, and p...

Embodiment 2

[0089] Embodiment 2: Electron microscope observation of chloroplast

[0090] Fresh leaves from the same part of the intervention line (2i-27) and wild type (WT) were selected at the seedling and bolting stages. The sample was fixed with 2.5% glutaraldehyde (25% glutaraldehyde was diluted to 2.5% with pH 7.2, 0.2M phosphate buffer), vacuumed until the leaves all sank to the bottom of the tube, and kept overnight at 4°C. After washing three times with 0.2M phosphate buffer (pH 7.2), fix with 1% osmic acid fixative (pH 7.0) for 2h. After the fixation was completed, the cells were washed again with phosphate buffer for 3 times, each time for 10 min. Enter the dehydration link afterwards, alcohol gradient dehydration at all levels, each 30min, alcohol gradient is successively 30%, 50%, 70%, 80%, 90%, 100% ethanol (twice). When dehydration is complete, discard the alcohol. The material is infiltrated sequentially with the mixture of alcohol and SPI-812 resin according to the grad...

Embodiment 3

[0092] Example 3: Subcellular Localization Experiment

[0093] The principle of the subcellular localization experiment is to transfer the high-purity, transfection-grade target gene-GFP fusion expression plasmid into the isolated Arabidopsis thaliana mesophyll cell protoplasts, and use PEG-Ca 2+ In the mediated method, after the fusion protein is expressed in protoplasts, the localization of the target gene can be determined by observing the localization of the green fluorescence in the cell with a Leica confocal microscope. Extraction of subcellular localization vector plasmids QIAGEN Plasmid Midi Kit (http: / / www.qiagen.com / ) was used to prepare transfection-grade plasmids. Isolation of protoplasts was performed according to the method reported by Yoo et al. (2007). with PEG-Ca 2+ Mediated method After the expression vector was transformed into Arabidopsis protoplasts, cultured overnight at room temperature in the dark. Since the target gene in this study is predicted to ...

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Abstract

The invention discloses an application of a gene CDA1 in regulation and control of chloroplast development, and belongs to the technical field of plant genetic engineering and cell biology. Accordingto the invention, an interference vector of an Arabidopsis thaliana CDA1 gene is constructed and then wild Arabidopsis thaliana is transfected to obtain an interfered strain. A research finds that cotyledons of the interfered strain show different degrees of green loss, and chloroplasts have no thylakoid membrane structure and are severe in vacuolation. According to the invention, the mutant cda1is obtained by inserting T-DNA into the Arabidopsis thaliana CDA1 gene, and the accumulation amount of photosynthetic proteins of the mutant cda1 is greatly reduced compared with the accumulation amount of photosynthetic proteins of the wild Arabidopsis thaliana. Experiments prove that the CDA1 gene participates in regulation and control of chloroplast development, influences accumulation of plantphotosynthetic protein, can be used for researching development and photosynthesis of plant chloroplasts, and provides theoretical guidance for research of development of chloroplasts of the plant and regulation and control of photosynthetic performance of the plant.

Description

technical field [0001] The invention relates to the technical fields of plant genetic engineering and cell biology, in particular to the application of a gene CDA1 in regulating the development of chloroplasts. Background technique [0002] Photosynthesis provides a source of material and energy for the growth of plants. Under the irradiation of visible light, photosynthetic pigments are used to convert carbon dioxide (or hydrogen sulfide) and water into organic matter and release oxygen (or hydrogen). Photosynthesis Function is the sum of a series of complex metabolic reactions, which is the basis for the survival of the biological world and an important medium for the earth's carbon-oxygen cycle. [0003] Chloroplast is an important place for photosynthesis, and it is also the carrier of photosynthetic pigments, which are widely distributed in the green tissue cells of leaves. The formation of chloroplasts is a complex regulatory process of nucleoplasmic genes, and any mu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00A01H6/20
CPCC07K14/415C12N15/8269
Inventor 李慧秦晓春祝利霞李秀秀
Owner UNIV OF JINAN
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