A protein regulating leaf color at low temperature and its gene and application

A technology of leaf color and protein, applied in the field of genetic engineering, can solve the problem of unclear nuclear-cytoplasmic interaction mechanism

Active Publication Date: 2016-08-17
CHINA NAT RICE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, the key enzymes in the synthesis of Arabidopsis chlorophyll have been identified (Nagata N (2005) Identification of a Vinyl Reductase Gene for Chlorophy11 Synthesis in Arabidopsis thabliana and Implications for the Evolution of Prochlorococcus Species. The Plant Cell Online 17 :233-240 ), but only a few genes have been identified in rice, and other genes have yet to be further discovered
In addition, the regulatory mechanism of chlorophyll degradation is not yet clear, and the mechanism of nucleoplasmic interaction is still unclear, which needs to be further studied

Method used

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  • A protein regulating leaf color at low temperature and its gene and application
  • A protein regulating leaf color at low temperature and its gene and application
  • A protein regulating leaf color at low temperature and its gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Phenotypic analysis of ywl1 mutants

[0037] a) Rice material

[0038] Rice (Oryza sativa L) mutant ywl1, the original wild-type material is indica variety 93-11.

[0039] b) Determination of chlorophyll content and photosynthetic characteristics

[0040] Select plump mutant and wild-type seeds to soak and accelerate germination, plant the germinated mutant and wild-type 93-11 seeds in pots, and set the daily constant temperature in the artificial climate box to 23°C, 25°C and 28°C respectively , the light length was 12h, the dark treatment was 12h, and the light intensity was 5Lax. Observe and record mutant phenotypes. The chlorophyll content of leaves was measured at five stages (one-leaf stage, two-leaf stage, three-leaf stage, four-leaf stage and five-leaf stage) before and after greening.

[0041] Determination of chlorophyll content: Take 0.1g-0.2g of the sample to be tested and soak it in 10ml of 95% ethanol, and place it in a refrigerator at 4°C t...

Embodiment 2

[0053] Embodiment 2: Cloning of YWL1 gene

[0054] a) Genetic analysis and mapping populations

[0055] The positive and negative hybridization was used to confirm that ywl1 was a recessive mutant, and the mutant was selected for hybridization with japonica rice 02428, the F1 generation was self-crossed, and a single plant was harvested to plant the F2 population, and 4391 recessive individuals were selected from the segregated population as the positioning population . At the three-leaf stage, about 1 gram of young leaves were taken from each plant to extract total DNA for gene mapping.

[0056] b) Preliminary and fine mapping of the YWL1 gene

[0057] Genomic DNA for gene localization was extracted from rice leaves using the rapid extraction method of rice micro-DNA. The DNA extraction method was SDS method (Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version Ⅱ. Plant Mol Bio Rep 1:19-21). Take about 100mg of rice leaves, cut them into pieces and ...

Embodiment 3

[0071] Example 3: Chloroplast localization experiment of YWL1 (SEQ ID NO.3)

[0072] According to the full-length CDS sequence of YWL1 (SEQ ID NO.2), a restriction recognition site containing BamHI was designed, and recombinant primers were designed. The sequence is:

[0073] PGXhF: TTTCTCGAGATAAACCCCCTCCCACTCTCCAC (SEQ ID NO. 14)

[0074] PGSalR: TTTGTCGAGCCAAATTTGATATTGCACAATGGGA (SEQ ID NO. 15)

[0075] Using the wild-type cDNA as a template, the CDS sequence (SEQ ID NO.2) of the YWL1 gene was amplified with PrimeSTAR high-fidelity enzymes. After the amplified product was sequenced and verified to be correct, it was ligated with the PAN580-GFP vector to obtain the fusion expression vector 35S ::YWL1::GFP.

[0076] Extract and construct the fusion expression vector 35S::YWL1::GFP plasmid and the 35S::GFP control plasmid without gene fusion respectively wrapped with gold powder, and use the PDS-1000 / He type gene gun of Bio-rad company under the helium pressure of 1100psi B...

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Abstract

A protein regulating the color of leaves at low temperature and its gene and application belong to the technical field of genetic engineering. The invention discloses a nucleotide sequence of a gene regulating the leaf color protein at low temperature and an amino acid sequence of the protein; and provides an application of the gene. The invention utilizes the rice seedling stage temperature-sensitive mutant to clone the YWL1 gene through the map-site cloning method, and the gene encodes a pseudouracil synthase family protein located in the chloroplast. The function of YWL1 gene was identified by transgene complementation experiment. Therefore, the invention can regulate the development of rice chloroplasts to obtain leaf change phenotypes at the seedling stage. Lay the foundation for the further utilization of this gene.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a protein for regulating the color of leaves at low temperature, its gene and application. Background technique [0002] Leaf is the main organ for photosynthesis in plants, and more than 2 / 3 of the dry matter in rice grains is obtained through photosynthesis after flowering (Wang Xujun, Xu Qingguo, Yang Zhijian (2005) Research progress in rice leaf senescence physiology, China Agricultural Science Bulletin 21: 187-190), the efficiency of photosynthesis has a complex relationship with the integrity of chloroplast structure and function, the stability of photosynthetic complex, and the level of chlorophyll content. In recent years, the application value of leaf color has attracted much attention. Leaf color variation can be used as a marker trait, which plays an important role in rice hybrid breeding and improved rice breeding. It can be used to determine ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N5/10C12N1/21C12N1/15C12N1/19C12N15/82
Inventor 魏祥进胡培松唐绍清邵高能焦桂爱谢黎虹圣忠华宋建刘聪利
Owner CHINA NAT RICE RES INST
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