Application of chlorosis seedling lethality 1 (CSL1) gene to regulation of development of rice chloroplasts

A chloroplast and gene technology, applied in the application field of CSL1 gene in regulating rice chloroplast development, can solve problems such as unclear MAPK cascade

Active Publication Date: 2020-09-25
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is unclear whether the MAPK cascade is involved in rice chloroplast development

Method used

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  • Application of chlorosis seedling lethality 1 (CSL1) gene to regulation of development of rice chloroplasts
  • Application of chlorosis seedling lethality 1 (CSL1) gene to regulation of development of rice chloroplasts
  • Application of chlorosis seedling lethality 1 (CSL1) gene to regulation of development of rice chloroplasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Isolation and Identification of T-DNA Insertion Mutants

[0045] (1) An etiolated seedling lethal rice mutant was isolated from the transformed population of japonica rice Zhonghua 11 transformed with plasmid pDsBar1300 (provided by the Institute of Plant Physiology and Ecology, Shanghai Academy of Biological Sciences, Chinese Academy of Sciences), and it was named csl1 mutant. Then the csl1 mutant genomic DNA was extracted, the genomic DNA was digested with HindIII restriction endonuclease, and the fragment was then cyclized with DNA ligase. Then, the circularized DNA was used as a template, and the left border primers H1 and H2 of the T-DNA fragment, and the right border primers C1 and C2 were used as nested PCR primers to amplify the T-DNA flanking sequence of the csl1 mutant. Among them, the sequences involved are as follows:

[0046] C1:5'-TGGCGTAATAGCGAAGAGGCC-3' (SEQ ID NO.3);

[0047] C2: 5'-AATGGCGAATGCTAGAGC-3' (SEQ ID NO.4);

[0048] H1: 5'-AATA...

Embodiment 2

[0059] Embodiment 2: Determination of chlorophyll content

[0060] The japonica rice Zhonghua 11 seeds of csl1 mutant plants were sown in the field (mutations include homozygous mutation (homozygous mutation lethal) and heterozygous mutation, and the seeds here are seeds of heterozygous mutation) to observe the phenotype of the materials, and wild-type japonica rice Zhonghua 11 (WT) was used as the control. Some plants showed yellowing in the field, and these plants died after the three-leaf stage in follow-up observation.

[0061] Use ultraviolet spectrophotometry to detect the chlorophyll a and b contents in the mutants respectively. The specific measurement methods are as follows: cut 0.1g of fresh rice leaves or leaf sheaths (three-leaf stage) into small pieces and place them in a 10ml centrifuge tube, add 2ml of extraction buffer (ethanol :propanol:H 2 0, volume ratio=4.5:4.5:1), and stood at 4° C. in the dark for 12 hours until the green leaves precipitated and the lea...

Embodiment 3

[0067] Embodiment 3: Chloroplast Ultrastructure Electron Microscopic Observation

[0068] The csl1 mutant rice leaves at the three-leaf stage (mutant plants isolated in Example 1) were fixed in glutaraldehyde and embedded in resin to observe the ultramicroscopic structure of chloroplasts with a transmission electron microscope. The specific experimental method is as follows:

[0069] The second leaf of rice at the three-leaf stage was taken, cut into small pieces with a width of 0.5 mm and a length of 2 mm, and fixed with 2.5% (v / v) glutaraldehyde at 4° C. for 24 hours. After washing 3 times with 0.1M PBS buffer, wash with 1% (w / v) OsO 4 (osmium tetroxide) for secondary fixation, and washed 3 times with 0.1M PBS buffer after 2 hours. Then use 30%, 50%, 70%, 80, 90%, 100%, 100% acetone solution by volume percentage to carry out gradient dehydration at room temperature, each gradient is 15 minutes. Utilize 6 gradients to gradually carry out resin infiltration to the material,...

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Abstract

The invention discloses application of a chlorosis seedling lethality 1 (CSL1) gene to regulation of development of rice chloroplasts. According to the invention, a lethal rice mutant at an etiolatedseedling stage is isolated from a rice T-DNA mutant library; the lethal rice mutant is inserted into a seventh intron of the CSL1 gene by means of T-DNA insertion; a 109bp sequence is deleted at a T-DNA insertion position; the chlorophyll content of the CSL1 mutant decreases; single-layer or multi-layer autophagosomes appear in the mesophyll cells; starch grains in the chloroplasts increase; a thylakoid structure is disordered; and the expression quantity of chloroplast-related genes is changed. meanwhile, the invention further constructs a knockout plant (the CSL1 gene has a deletion of one base A at the site 46 of exon 5, or a deletion of one base A at the site 47 and substitution of the base A with the base T at the site 48) by means of gene knockout, and the knockout plant has the samephenotype as the CSL1 mutant, thus showing that the CSL1 gene is involved in regulation of development of the rice chloroplasts.

Description

technical field [0001] The invention belongs to the field of rice genetic engineering, in particular to the application of CSL1 gene in regulating the development of rice chloroplast. Background technique [0002] The chloroplast is the place for photosynthesis of rice, which uses light energy to assimilate carbon dioxide in the air into organic matter to maintain growth and development, and finally form yield. There are more than 2000 proteins localized in chloroplasts, except 300-400 of which are encoded by chloroplasts themselves, and the others are all encoded by the nucleus. Therefore, the growth and development of chloroplasts involves a complex gene regulatory network. It is an important topic of genetic breeding to improve photosynthesis by regulating the development of chloroplasts, thereby increasing crop yield. [0003] As an important food crop and monocot model plant in the world, rice has been extensively studied on the regulation of chloroplast development. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N9/12A01H5/00A01H6/46
CPCC12N9/12C12N15/8269
Inventor 张泽民梁嘉燕张秋馨
Owner SOUTH CHINA AGRI UNIV
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