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Chlorophyll synthesis related protein, coding gene and application thereof

A technology that encodes genes and chlorophyll, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve problems such as complex regulatory networks of chlorophyll synthesis and degradation, and achieve the effect of improving synthesis ability

Active Publication Date: 2016-05-11
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the synthesis of chlorophyll is regulated by internal and environmental factors, and this regulation is controlled by nuclear genes and (or) plastid genes. Therefore, there is a complex regulatory network for the synthesis and degradation of chlorophyll.

Method used

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  • Chlorophyll synthesis related protein, coding gene and application thereof
  • Chlorophyll synthesis related protein, coding gene and application thereof
  • Chlorophyll synthesis related protein, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1. Acquisition of chlorophyll synthesis-related gene ALAD

[0076] The total RNA of wild-type Arabidopsis thaliana (Col ecotype) was extracted, reverse-transcribed into cDNA, and used as a template for PCR amplification using the following primers 1 and 2.

[0077] Primer 1: 5'- CCATGG TC-ATGGCTACTACACCCCATC-3' (the underlined part is the recognition sequence of NcoI, and the sequence after - is the 1st-18th position of sequence 2);

[0078] Primer 2: 5'- AGATCT CCGCTTCTCGCCGCCACAAACAAGTAGCAG-3' (the underlined part is the recognition sequence of BglII, and the following sequence is the reverse complementary sequence of the 1262-1290th position of Sequence 2).

[0079] After the reaction, agarose gel electrophoresis was performed. The results showed that the target band with a size of about 1293bp was amplified. Further, after the PCR product was recovered, it was sent to the sample for sequencing, and the results showed that the sequence of the obtained P...

Embodiment 2

[0080] Embodiment 2, the acquisition of ALAD-amiR transgenic plants

[0081] 1. Construction of recombinant expression vector pDS1301-ALAD-amiRNA

[0082] 1. Primer design

[0083] According to the precursor mir319a sequence contained in the pRS300 vector, the following primers ALAD-F and ALAD-R were designed. In addition, according to the ALAD gene shown in Sequence 2 in the sequence listing, four oligonucleotide chains were predicted by Weigelworld software: ALAD1, ALAD2, ALAD3 and ALAD4, which were used to design ArtificialRNAiALAD.

[0084] ALAD-F: 5'-CTGCAAGGCGATTAAGTTGGGTAAC-3';

[0085] ALAD-R: 5'-GCGGATAACAATTTCACACAGGAAACAG-3'.

[0086] ALAD1: 5'-GATAACGATACTGTTTACCCCACTCTCTCTTTTGTATTCC-3';

[0087] ALAD2: 5'-GAGTGGGGTAAACAGTATCGTTATCAAAGAGAATCAATGA-3';

[0088] ALAD3: 5'-GAGTAGGGTAAACAGAATCGTTTTTCACAGGTCGTGATATG-3';

[0089] ALAD4: 5'-GAAAACGATTCTGTTTACCCTACTCTACATATATTCCT-3'.

[0090] Among them, ArtificialRNAiALAD "5'-TAACGATACTGTTTACCCCAC-3'" interacts with...

Embodiment 3

[0116] Example 3, Functional Identification of ALAD-amiR Transgenic Plants

[0117] Positive T3 generation ALAD-amiR transgenic plants (amiR-1 and amiR-2 strains) identified in Example 2, Arabidopsis plants transformed into pDS1301 empty vector, and wild-type Arabidopsis (No-0 ecotype) for the experimental material.

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Abstract

The invention discloses a chlorophyll synthesis related protein, a coding gene and application thereof. The protein provided by the invention is a) or b) as follows: a) a protein composed of an amino acid sequence shown as sequence 1 in a sequence table; b) a chlorophyll synthesis related protein obtained by subjecting the amino acid sequence of the a) defined protein to substitution and / or deletion and / or adding by one or several amino acid residues. Experiments prove that a transgenic plant can be obtained by inhibiting chlorophyll synthesis related gene ALAD through artificial microRNA (amiRNA), and the transgenic plant shows constitutive disease resistance response. Therefore, artificial inhibition of ALAD gene expression provides a brand new and effective way for genetic improvement of crops' resistance to diseases.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a protein related to chlorophyll synthesis, its coding gene and application. Background technique [0002] Photosynthesis is the process of using solar energy on the largest scale on earth, which provides organic matter, energy and oxygen for almost all life activities. About 220 billion tons of organic matter are synthesized through photosynthesis on the earth every year, which is equivalent to 10 times the annual energy consumption required by human beings. 90%-95% of plant dry matter comes from the products of photosynthesis, and photosynthesis is the material basis for crop yield formation. Photosynthesis is mainly carried out in the chloroplast of plant leaves, which requires the participation of a series of pigments and protein complexes. Chlorophyll is the main pigment that absorbs light energy. The reduction or absence of chlorophyll will make the color of leaves lighte...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N15/113A01H5/00
Inventor 林荣呈唐为江王晚晴
Owner INST OF BOTANY CHINESE ACAD OF SCI
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