Protein for adjusting rice chlorophyll synthesis and encoding gene and application thereof
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A technology encoding gene and chlorophyll is applied to the protein regulating chlorophyll synthesis in rice and its encoding gene and application field, and can solve problems such as plant death and blocked chlorophyll biosynthesis pathway.
Pending Publication Date: 2018-11-20
CHINA NAT RICE RES INST
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Problems solved by technology
T-DNA insertion OsChlH lead to blockage of chlorophyll biosynthetic pathway and subsequent plant death (Zhang at al., 2006)
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Embodiment 1
[0032] Example 1: YL-1 gene cloning
[0033] a) Rice material
[0034] Rice ( Oryza sativa L) Mutant ys-1 ( yellow leaves 1 ), the original wild-type material is the super early rice variety Zhong Jiazao 17 (YK17).
[0035] b) Genetic analysis and mapping of populations
[0036] Determined by forward and reverse hybridization ys-1 is a recessive mutant. Select mutants to cross with D50, F 1 F 2 Group, 1500 recessive individuals (seedlings with macular stripes) were selected from the isolated group as the positioning group. At the four-leaf stage, about 0.2 grams of young leaves were taken from each plant to extract total DNA for gene mapping.
[0037] c) YS-1 Preliminary and fine mapping of genes
[0038] Using the rapid extraction method of rice trace DNA to extract genes for gene mapping from rice leaves
[0039] Group DNA, the DNA extraction method is SDS method (Dellaporta SL, Wood J, Hicks JB (1983) Aplant DNA minipreparation: version II. Plant Mol Bio...
Embodiment 2
[0055] Embodiment 2: transgenic experiment
[0056] 1. Vector Construction
[0057] Design a pair to completely cover the entire YS-1 Gene ORF primers, and respectively design enzyme cutting sites KpnI and SpeI sites on the primers, PCR amplifies the wild-type genomic DNA, electrophoresis detection gel cutting recovery, the recovered products are digested with KpnI and SpeI, and connected to the same digestion On the pUbi-1390 vector, sequencing confirmed that no base mutation occurred. The constructed vector structure diagram is pUbi1390-YS-1. The constructed vector was transferred into the Agrobacterium tumefaciens strain by electric shock.
[0058] The primer sequence for amplifying the ORF sequence is:
[0059] OG2-KpnI:5'-TTACTTCTGCACTAGGTACCATGGCGACCATCACCACGC-3' (SEQ ID NO.4)
[0060] OG2-SpeI: 5'-TAGCGTTAACACTAGTCGCGGCCTGCAAAACTTCC-3' (SEQ ID NO.5)
[0061] 2. Genetic transformation:
[0062] (1) Selection of transformed receptors
[0063] Will ys-1 The callus...
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Abstract
The invention discloses a protein for adjusting rice chlorophyll synthesis and an encoding gene and application thereof. The protein has an amino acid sequence as shown in SEQ ID NO: 3. The encoding gene of the protein has a nucleotide sequence as shown in SEQ ID NO: 1. The mutation of the encoding gene of the protein related to the rice chlorophyll precursor synthesis leads to yellow stripe leaves of the rice plants, and severe mutation leads to death of whiten seedlings. The gene in a normal planting is knocked out through a Crisp/ cas9 gene encoding technology, and the normal green plant phenotype can be mutated into whiten seedlings and dies after trefoil stage. The encoding gene is an important indicating gene in the works such as the work of genetic improvement of the plant, and theencoding gene can be treated as a target gene to be applied to the seed production of hybrid rice; particularly the encoding gene can rapidly conveniently detect the purity of filial generation F1 inthe seedling stage during the seed production of two-line system hybrid rice; and meanwhile, the carried target gene sterile line etiolated seedling can be quickly removed in the seedling stage duringplanting the hybrid rice.
Description
technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a protein for regulating rice chlorophyll synthesis, its coding gene and its application. Background technique [0002] Rice is an important food crop and a model crop for studying higher plants. Rice yield is directly related to its leaf shape and leaf function. The change of rice leaf color is due to the mutation of related genes controlling chloroplast development and chlorophyll synthesis pathway, which affects the yield of rice due to the influence of photosynthesis efficiency (Wang Pingrong et al., 2006). [0003] At present, there are mainly two types of rice leaf color mutants: artificial mutation and natural variation, and many genes related to leaf color have been cloned. The metabolic pathways related to chlorophyll synthesis and chloroplast development have been basically clarified in the model plant Arabidopsis thaliana, and any mutatio...
Claims
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