Key gene for regulating and controlling chlorophyll degradation in the senescence process of plant and application thereof
A chlorophyll degradation and key gene technology, applied in the direction of plant genetic improvement, application, plant products, etc., can solve the problems of accumulation of chlorophyll ester, no detection of pheophorbide accumulation, and deficiency of pheophorbide activity. , to increase the content, improve the green period and landscape effect, and prolong the life of the goods
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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 Pa. The expression angler program (Toufigh et al Plant J.43, 153-1632005) 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 flat 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 Figure 6 ), we named the gene CRN1 ( c o~ r regulated with ...
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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