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Cloning and application of major gene GS5 capable of controlling width and weight of rice grain

A technology of major genes and grains, which is applied in the fields of application, genetic engineering, and plant genetic improvement, and can solve problems such as interference of QTL phenotypic traits

Inactive Publication Date: 2010-11-10
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 GS5 capable of controlling width and weight of rice grain
  • Cloning and application of major gene GS5 capable of controlling width and weight of rice grain
  • Cloning and application of major gene GS5 capable of controlling width and weight of rice grain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Construction of GS5 near isogenic line

[0030] 1 Backcross and Selection

[0031] as attached figure 2 As shown, from the double haploid population derived from the combination of the wide-grain rice variety Zhenshan 97B and the narrow-grain variety H94, a family containing the GS5 gene and 55% of the genetic background is consistent with Zhenshan 97B was selected, and Zhenshan 97B was crossed with Zhenshan 97B. 97B is the reincarnated parent, backcrossed continuously for 3 generations, in BC 1 f 1 and BC 2 f 1 In the next generation, only positive selection was carried out on GS5, that is, individual plants whose target segment was Zhenshan 97B / H94 heterozygous genotype were selected for the next round of backcrossing. The target segment was identified in two SSR (Simple Sequence Repeat) markers RM593 and RM574 with reference to previous QTL mapping results (see: ( http: / / www.gramene.org / ) within the bounded range. Save BC 3 f 1 In the next gener...

Embodiment 2

[0035] Example 2: Fine mapping and map-based cloning of GS5

[0036] 1 grain wide phenotypic measurements

[0037] After the grains are naturally dried, they are placed at room temperature for at least 3 months to ensure the drying of the grains and the relative consistency of the water content among the strains. Randomly select 10 full grains from each individual plant, arrange them side by side in the same direction, without overlapping, and without gaps, and arrange them in a row. Use a vernier caliper to read the width, and find the average value to get the grain width. . Grain thousand-kernel weight is estimated from the weight of 200 filled kernels randomly selected.

[0038] 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 traits such as grain width of each individual plant in Zhenshan 97B, H94 and random populations were investigated, and the genotypes of each plant were deter...

Embodiment 3

[0047] Embodiment 3: Transgenic complementation test of GS5

[0048] 1 GS5 transgenic technology route:

[0049] Real-Time PCR expression profiling analysis found that the gene was expressed in various tissues during the whole growth period, and the expression level of the wide-grain variety (Zhenshan 97B) was higher than that of the small-grain variety (H94) in the glume endosperm ( Figure 5 ), the predicted expression level plays an important role, so according to the predicted full-length cDNA sequence of the candidate gene, a pair of RT-PCR specific primers (Table 4) with restriction endonuclease BamHI and PstI linkers were designed to amplify the cDNA of H94 The fragments were connected to the TA clone Promega T vector, and the correct clone containing the candidate gene without mutation was selected, and the positive clone was digested with restriction endonucleases BamHI and PstI, subcloned, and then connected to the binary overexpression vector pCAMBIA1301S) (pCAMBIA...

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Abstract

The invention belongs to the technical field of plant gene engineering, disclosing a separated and cloned major gene GS5 capable of controlling the width and the weight of a rice grain and the DNA sequence of the allelic gene of the major gene GS5. The DNA sequence is shown as SEQ ID NO.1 (Zhenshan 97B) and SEQ ID NO.3 (H94) and contains 10 exons. The amino acid sequence of the major gene and the amino acid sequence of the allelic gene are shown as SEQ ID NO.2 and SEQ ID NO.4. By using two large-grain rice varieties and two small-grain rice varieties for comparative sequencing, in an approximately 6.1kb range, 22 common base differences exist between the large-grain variety and the small-grain variety, wherein 18 mutations are in a promoter area, 4 mutations are in a code area and 5 amino acids are caused to be changed. By using a transgenic technology, GS5 transgenic rice plants are obtained and express that the width and the weight of the rice grain are obviously improved when being compared with the control width and the control weight of the rice grain. The character changes are quite coincident with the two genotype expressions of a Zhenshan 97 near-isogenic line and a GS5 near-isogenic line. The invention additionally discloses a method of near-isogenic line breeding, gene cloning and gene transfer and application thereof.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering. It specifically involves the cloning and application of a major QTL (GS5) located on the short arm of the fifth chromosome of rice that controls grain width and grain weight. Background technique [0002] Rice grain size is an important agronomic trait: (1) Grain size is controlled by the three sub-characters of grain length, grain width, and grain thickness, which are the main determinants of thousand-grain weight, and grain weight is the three main components of rice yield one of the factors. Therefore, grain size is an important yield trait; (2) Grain weight is extremely significantly positively correlated with grain length, grain width, and grain thickness (Xing Yongzhong et al., 2001, Acta Botanica Sinica 43: 840-845), so Grain size is also an important rice appearance quality trait. The quality of rice is more and more concerned by the world. People's requirements for r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/113C07K14/415A01H5/00
Inventor 何予卿李一博邢永忠范楚川张启发
Owner HUAZHONG AGRI UNIV
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