Intraspecific gene improvement method for common tobacco mosaic virus resistance

A mosaic virus and tobacco technology, applied in the biological field, can solve the problems of difficulty in cultivating germplasm materials, long period of hybrid breeding, etc.

Inactive Publication Date: 2012-09-26
GUIZHOU TOBACCO SCI INST
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Problems solved by technology

The breeding of TMV-resistant tobacco germplasm is mainly through traditional cross breeding, but it is difficult to effectively break the linkage between resistance genes and unfavorable trait genes. In addition, the long cycle of cross breeding makes it difficult to cultivate high-quality and usable germplasm materials. Therefore, tobacco Intraspecific gene transformation for TMV resistance combined with marker-free gene selection to improve tobacco will have broad application potential

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  • Intraspecific gene improvement method for common tobacco mosaic virus resistance
  • Intraspecific gene improvement method for common tobacco mosaic virus resistance

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Embodiment Construction

[0039] A method for improving the resistance of tobacco common mosaic virus in-species, comprising the following steps:

[0040] (1) Clone Tobacco Ubi.u4 Constitutive expression of the gene promoter Pubi.u4:

[0041] Genomic DNA was extracted from the leaves of common tobacco variety K326. According to the characteristics of the Pubi.u4 promoter sequence provided in Genbank Accession (Genbank Accession): X77456, the amplification primers were designed and synthesized, and the upstream primer Ubi-1 was introduced Hind III enzyme cleavage site, introduction of downstream primer Ubi-2 Xba I restriction site. Tobacco cloned by PCR method Ubi.u4 Gene 796 bp of Pubi.u4 promoter.

[0042] The amplification primers for the Pubi.u4 promoter are:

[0043] Ubi-1: 5'-CCC AAG CTT GGA GGC TAA CTA CGT TAG AGC-3';

[0044] Ubi-2: 5'-TGC TCT AGA TCT GTA TAT ACA GAA AAG GTT-3';

[0045] (2) Synthetic terminator Tubi.u4 and LB-MCS-RB sequence:

[0046] According to the Tubi.u4 termina...

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Abstract

The invention discloses an intraspecific gene improvement method for common tobacco mosaic virus resistance, belonging to the technical field of biology. The method comprises the following steps of: (1) building a Pubi.u4 promoter from the common tobacco to drive the TMV-N (tobacco mosaic virus) gene expression and the tobacco intraspecific gene expression vector pSH-P-N-T of which expression is stopped by Tubi.u4; (2) carrying out co-transformation on the tobacco explant by the intraspecific gene expression vector pSH-P-N-T and the expression vector pSh737 which only contains the marker gene through sonication-assisted agrobacterium-mediated transformation, and screening to obtain T0 generation transformed tobacco plant; and (3) carrying out T1 generation genetic segregation and screening test to obtain the intraspecific gene improvement plant of the marker gene-free common tobacco mosaic virus resistance (TMW). According to the method, the maker-free transformation of the common tobacco can be realized, and the influence of the non-tobacco source gene sequence can be eliminated, so that the transgenosis safety can be furthest realized.

Description

Technical field [0001] The present invention is a biotechnology field. Specifically, it is a method that involves a way to improve the general tobacco species using gene expression regulatory sequences and functional genes in ordinary tobacco species. [0002] Background technique [0003] GM technology breaks the boundaries of the species, so that genes between different species can perform unprecedented new combinations. However, in genetically modified crop breeding, the functional genes, promoters, and terminate sub -expression of other species are often introduced.Β-glucosaconase (GUS) gene, green fluorescent protein (GFP) gene, antibiotics or herbicide resistance genes and other selection marks.While showing the excellent characteristics of these genetically modified crops, they also bring environmental safety and food safety such as environmental safety and food safety. In particular, genetically modified plants are used to produce functional foods, medicines and industria...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00C12Q1/68C12Q1/34
Inventor 赵杰宏王轶韩洁赵丹
Owner GUIZHOU TOBACCO SCI INST
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