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A Study on Introducing the Blast Resistance Gene Pi-d2 into the Main Parents of Hybrid Rice Shuhui527 Mediated by Agrobacterium Tumefaciens

Author: HuYan
Tutor: LiShiGui
School: Sichuan Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: Agrobacterium-mediated Pi-d2 gene Rice blast Indica
CLC: S511
Type: Master's thesis
Year: 2008
Downloads: 150
Quote: 2
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Abstract

Rice is one of the most important food crops, nearly half of the population is the staple food of rice worldwide. Rice blast by ascomycetes (Magnaporthe grisea, asexual generation Pyricularia grisea) major fungal diseases caused by the widespread occurrence of the rice producing areas of the world, due to a high degree of differentiation of pathogenicity, makes difficult effective control of rice blast. The long-term practice proved that nurture and use of resistant varieties rice blast effective prevention and treatment of the most useful way. With the development of genetic engineering, the use of transgenic technology exogenous gene improved blast resistance has become a new way to use the technology to existing cultivars exogenous resistance genes not subject to a restraint to expand use of the source of the gene. Indica cultivated rice varieties, compared with japonica, a large number of indica rice varieties with poor tissue culture indice and low conversion efficiency, this situation has severely limited the application of transgenic technology in indica rice breeding. Blast resistance gene Pi-d2 in rice roots, stems, leaves are the constitutive expression of the encoded protein contains a 17 amino-terminal amino acids of the extracellular domain of the signal peptide and signal identification zone, the carboxy-terminal serine / threonine threonine protein kinase activity area, the middle of a hydrophobic transmembrane structure may be a new class of resistance genes. In this study, for the first time using Agrobacterium-mediated Pi-d2 gene into indica varieties Shuhui 527 and japonica varieties flower 11, molecular biology, GUS activity detection and disease identification, suitable for efficient plant regeneration system of Shuhui527 preliminary materials to improve the rice blast resistance, laid the foundation for the rice and other gramineous plant disease resistance molecular breeding. The main findings are as follows: 1. Different receptors material, the different explants callus induction rate, Shuhui 527, Shuhui 498, spend 11 and four varieties of Taipei 309 immature embryo callus induction rate 88.7%, 87.5%, 91.4% and 96.0%, respectively, mature embryo callus induction rate was 78.5%, 75.3%, 85.1% and 86.3%, respectively. For four rice materials, the overall performance of the immature embryo callus higher than mature embryos, higher than indica japonica callus. 2. Efficient plant regeneration system suitable for indica varieties Shuhui527 genetic transformation. Experimental results show that 2.0 mg / L 2,4-D in basic medium NMB add favor Shuhui527 callus induction and subculture; MS 2.0 mg / L 6-BA 2.0 mg / L KT 0.25 mg / L NAA, differentiation medium conducive to the differentiation of the callus; Following on behalf of two callus highest rate, partially dried before processing differentiation of callus favor differentiation. 3 the rate and the rate of emergence of resistant callus impact of transgenic efficiency, especially in the latter. Found in the experiment, Shuhui 498 after hygromycin resistant callus can get a lot of, but very difficult to differentiate into seedlings. Transgenic rice plants Pi-d2 gene. Immature embryos of different rice varieties and mature embryos as explants by Agrobacterium tumefaciens in three different target gene expression vector (pCB6.3kb, pCB5.3kb, pZH01-2.72kb) import rice, the final total 292 regenerated plants, its PCR detection of the target gene, 176 showed positive Shuhui 527 87 Shuhui 498 a flower 11 37 51 Taipei 309. Pi-d2 gene RT-PCR analysis, the results showed that the Pi-d2 gene expression at the transcriptional level. 5. Hygromycin-resistant transgenic plants T ₁-generation analysis showed that, Shuhui 527 and Taipei 309, respectively, 64.6% and 68.2% of the strains performance 3:1 segregation Mendelian single gene inheritance, that form of integration of exogenous genes most is the unit point. 6. Transfer of blast resistance in transgenic plants to improve. 25 independent transgenic lines T ₁-generation plants were inoculated with results showed that the transgenic plants resistance to rice blast physiological races ZB15 improved lesion size and reduce the number of resistance was Mendel and non-Mendelian segregation. The objective gene three expression vectors in the receptor performance ZB15 Resistance no significant difference. Agronomic traits of transgenic plants without significant genetic variation. Important agronomic traits of transgenic rice contemporary and T ₁-generation examine the results show that the transgene insertion and tissue culture process may result in some variation of agronomic traits of the receptor, but the vast majority of non-genetic, in the progeny can be restored.

A Study on Introducing the Blast Resistance Gene Pi-d2 into the Main Parents of Hybrid Rice Shuhui527 Mediated by Agrobacterium Tumefaciens

Abstract

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