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Key gene for regulating and controlling chlorophyll degradation in the senescence process of plant and application thereof

A gene, chlorotic technology, applied to the key new gene CRN1 involved in the regulation of chlorophyll degradation and its application, molecular-assisted breeding for screening/identification of chlorophyll strains, and the creation of chlorophyll strains, which can solve the problem of accumulation of chlorophyll esters , pheophorbide activity deficiency, no detection of pheophorbide accumulation and other problems, to increase the content, prolong the life of the goods, improve the green period and landscape effect.

Inactive Publication Date: 2013-04-10
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, neither altered chlorophyllase activity nor pheophorbide accumulation was detected in the mutant
However, chlorophyllin a and b did accumulate in the mutant compared to the wild type, suggesting a possible defect in phosphochelatase activity (Fang, Z., et al. J. Exp. Bot 49:503-510 , 1998)

Method used

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  • Key gene for regulating and controlling chlorophyll degradation in the senescence process of plant and application thereof
  • Key gene for regulating and controlling chlorophyll degradation in the senescence process of plant and application thereof
  • Key gene for regulating and controlling chlorophyll degradation in the senescence process of plant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: AtNYE1 co-expression gene screening

[0050] Buchanan-Wollaston et al. (Buchanan-Wollaston et al.Plant J.42, 567-585, 2005) compared the gene expression levels of Arabidopsis genome-wide under various aging conditions. We selected 827 genes in the literature results in The GO classification of genes expressed under various aging conditions showed that 67 of these genes encoded chloroplast-localized proteins, including chlorophyll degradation-related genes such as NYE1 and PaO. The expression angler program (Toufigh et al PlantJ.43, 153-163 2005) was used to analyze these 67 genes, and some genes with the expression profiles most similar to NYE1 were obtained (see attached figure 1 ).

Embodiment 2

[0051] Example 2: Analysis of T-DNA insertion mutants and identification of CRN1

[0052] From the Arabidopsis Biological Resource Center (ABRC www.arabidopsis.org / abrc / ) ordered the T-DNA insertion mutants of these genes, sowed them on the plate of 1 / 2MS+30mg / L Kan, removed the seedlings with resistance to the soil after 10 days, when the 6th rosette leaf of the plant was fully expanded , took the 3rd-4th leaf and carried out dark treatment, and found that one strain (mutant number SALK_000095) showed obvious stagnant green traits (see attached Figure 2-4 ). The results of PCR analysis of the genomic DNA showed that the T-DNA was inserted on the third exon at the position of At5g13800 of the target gene, and it was a homozygous insertion mutant (see attached Figure 5 ). Semi-quantitative PCR analysis showed that the full-length gene was not expressed in the SALK_000095 homozygous mutant (see attached Image 6 ), we named the gene CRN1 ( c o- r regulated with N YE1,...

Embodiment 3

[0092] Example 3: Phenotype analysis of insertion mutants and determination of chlorophyll content

[0093] When the sixth rosette leaf of the plant was fully unfolded, the 3rd to 4th leaves were taken for dark treatment; the leaves were placed in a petri dish with 2 layers of wet filter paper and treated for different days, then samples were taken to determine the total chlorophyll content. The results showed that the degradation rate of chlorophyll in crn1-1 was slower than that in nye1-1 (see attached Figure 7 ), showing that CRN1 has a greater application prospect in the creation of plant green strains.

[0094] Chlorophyll determination:

[0095] 0.1g of fresh leaves were ground with liquid nitrogen and extracted with 3ml of acetone. Stand under dark conditions to prevent chlorophyll from decomposing. After the chlorophyll is completely extracted and dissolved in acetone, measure the A645 and A663 values ​​with a spectrophotometer, and calculate their contents by the ...

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Abstract

The invention relates to a new key gene participating in the regulation and the control of chlorophyll degradation and application thereof, belonging to the technical field of plant gene engineering. Stay-green traits can prolong the commodity price service life of green leafy vegetables and the postharvest green period of fodder crops so as to increase the content of main nutrition constituents, i.e. chlorophylls and proteins; and the stay-green traits can also outstandingly improve the green period and the landscape effect of lawn plants. The invention provides the key gene AtCRN1 for regulating and controlling the chlorophyll degradation and metabolism of plants. An amino acid coding sequence of the key gene AtCRN1 is characterized by being SEQ ID No: 2. The invention also provides a method for establishing a stay-green plant strain, comprising the following steps of: inducing the gene AtCRN1 of a target plant to generate mutation through chemical or physical factors; and destroying or reducing the expression of the gene AtCRN1. Besides, detecting whether the gene AtCRN1 with overall length is contained in a plant genome or not can also be used as a molecular auxiliary breeding method for screening / identifying the stay-green plant strain.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a new key gene CRN1 involved in the regulation of chlorophyll degradation and its application. The invention also provides a method for creating stagnant green plant strains and a molecular assisted breeding method for screening / identifying stagnant green plant strains. Background technique [0002] After the plant leaves enter the senescence process, the yellowing phenomenon caused by the rapid degradation of chlorophyll is the common and most notable apparent feature of the senescence of almost all plant green organs. Although leaf senescence can be induced by many environmental and developmental factors, the timing and process of leaf senescence are regulated by apoptosis. In the past few decades, scholars have found that the leaf green period is delayed until maturity or senescence in many crops, that is, stay-green (stay-green) or non-yellowing...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N15/10A01H1/06C12N15/84A01H5/00
Inventor 蒯本科任国栋魏强梁宁菁
Owner FUDAN UNIV
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