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Cloning and application of major gene Chalk5 for chalkiness rate of paddy rice

A major gene, chalky rate technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems such as QTL phenotypic trait interference

Inactive Publication Date: 2015-04-22
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the primary population, QTLs segregate simultaneously with many other non-QTL loci, and these non-QTL loci, like environmental factors, will have a great interference effect on the phenotypic traits of QTL

Method used

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  • Cloning and application of major gene Chalk5 for chalkiness rate of paddy rice
  • Cloning and application of major gene Chalk5 for chalkiness rate of paddy rice
  • Cloning and application of major gene Chalk5 for chalkiness rate of paddy rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Construction of Chalk5 near-isogenic lines

[0033] 1 Backcross and selection

[0034] as attached figure 1 As shown, a family containing the Chalk5 gene and 55% of the genetic background was selected from the double haploid population derived from the combination of the rice variety Zhenshan 97B with a high chalkiness rate and the variety H94 with a low chalkiness rate. Hybridization, with Zhenshan 97B as the recurrent parent, backcrossed continuously for 3 generations, in BC 1 f 1 and BC 2 f 1 In each generation, only forward selection was performed on Chalk5, that is, individual plants whose target segment was Zhenshan 97B / H94 heterozygous genotype were selected for the next round of backcrossing. The target segment refers to the previous QTL mapping results (Fan Chuchuan, 2006, Ph.D. dissertation, Huazhong Agricultural University Library) identified two SSR (Simple Sequence Repeat) markers RM593 and RM574 (see: ( http: / / www.gramene.org / ) within ...

Embodiment 2

[0038] Example 2: Fine mapping and map-based cloning of Chalk5

[0039] 1 Phenotypic measurement of chalkiness

[0040] The grains of each transgenic and near-isogenic line were dried naturally and placed at room temperature for at least 3 months to ensure that the grains were dry and the water content of the lines was relatively consistent. The chalkiness rate is expressed by the percentage of chalky rice grains among 100 randomly selected brown rice grains.

[0041] from BC 3 f 1 single plant derived BC 3 f 2 288 individuals were randomly selected from the population to form a random population. The grain chalkiness rate of each individual plant in Zhenshan 97B, H94 and random populations was investigated, and the genotypes of each plant were determined by molecular markers RM593 and RM574 at both ends (see Table 4 for the sequence). There were significant differences in the white rate between the two parents. In random populations, the grain chalkiness rate showed a ...

Embodiment 3

[0049] Embodiment 3: Transgene complementation test of Chalk5

[0050] 1 Chalk5 transgenic technology route:

[0051] Real-Time PCR expression profiling analysis found that the Chalk5 gene was specifically expressed in the endosperm tissue during the whole growth period, and the expression level of the high chalky variety (Zhenshan 97B) was higher than that of the low chalky variety (H94) in the endosperm tissue 5 days after fertilization high( Figure 5 ). Therefore, according to the predicted full-length ORF2 sequence of the candidate gene, a pair of PCR-specific primers Chalk5 ORF2 (Table 4) with restriction endonuclease KpnI and PstI adapters were designed to amplify the ORF2 fragment of Zhenshan 97B and ligated to the TA clone Promega On the T vector, select the correct clone containing the candidate gene without mutation, use the restriction endonuclease KpnI and PstI to digest the positive clone, carry out subcloning, and then connect it to the binary overexpression v...

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Abstract

The invention belongs to the technical field of plant genetic engineering. The invention discloses the DNA sequences of a separated and cloned major gene Chalk5 for regulating and controlling chalkiness characters of paddy rice and an allele (Chalk5-H94) of the major gene Chalk5, wherein the nucleotide sequence of the major gene is represented by SEQ ID No. 1, and the nucleotide sequence of the allele of the major gene is represented by SEQ ID No.3 in a sequence table. A high-chalkiness variety and two low-chalkiness varieties of paddy rice are utilized for comparison and sequencing, and it is discovered that the high-chalkiness variety and the low-chalkiness varieties have 39 common base differences in a whole-genome range, wherein 10 mutations are located in a promoter region, and 5 mutations are located in a coding region, resulting in changes of two amino acids. According to the invention, transgenic technology is employed to obtain paddy rice plants with a trans-Chalk5 gene, and the chalkiness rate of the transgenic plants is substantially increased compared with that of control groups. Character changes of the transgenic plants are identical to those of two genotypes of a nearly isogenic line of Chalk5 of Zhenshan 97.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering. It specifically relates to the cloning and application of a major QTL (Chalk5) located on the short arm of rice chromosome 5 controlling the chalkiness rate. Background technique [0002] Rice chalkiness is an important quality trait: (1) Rice chalkiness is an important appearance quality trait (Tan et al., 2000, Theor.Appl.Genet.105:248-257). The quality of rice is more and more concerned by the world. People's requirements for rice quality are not only comfortable taste, but also beautiful appearance. Therefore, the appearance quality of rice is directly related to the commodity of rice. In our country The high-quality rice standard also makes specific regulations on the chalkiness of rice, and it is believed that the chalkiness of first-grade high-quality rice should not be higher than 1%. (2) Rice chalkiness will greatly affect the processing quality, cooking and eating qu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N9/14C12N15/84A01H5/00
Inventor 何予卿李一博邢永忠范楚川张启发
Owner HUAZHONG AGRI UNIV
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