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Rice gene BADH2 site-directed knockout system and application thereof

A technology of fixed-point knockout and rice, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., and can solve problems such as complex and long periods

Active Publication Date: 2013-04-03
TIANJIN GENOVO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the breeding of fragrant rice varieties is to use hybridization to transfer fragrant rice genes into ordinary rice. Although molecular markers can be used for assisted selection, this process is complicated and takes a long time

Method used

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  • Rice gene BADH2 site-directed knockout system and application thereof
  • Rice gene BADH2 site-directed knockout system and application thereof
  • Rice gene BADH2 site-directed knockout system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, the sequence and analysis of rice gene BADH2

[0037] The sequence of the rice gene BADH2 is shown in sequence 1 of the sequence listing. Sequence analysis shows that the gene includes 15 exons in total, which are the 1st-108th (first exon), the 208th-351st (second exon), the 1404th- 1485 (third exon), 1581-1729 (fourth exon), 2427-2519 (fifth exon), 2651-2775 (sixth exon), 2651-2775 (sixth exon) 2860-2923 (the seventh exon), 3114-3180 (the eighth exon), 3873-3949 (the ninth exon), 4305-4418 (the tenth exon) , 4819-4896 (11th exon), 4978-5129 (12th exon), 5233-5294 (13th exon), 5566-5672 (13th exon) Exon 14), positions 5771-5860 (exon 15).

[0038] In the present invention, the sequences on the second exon and the fourth exon are used as target sequences of transcription activator-like effector nucleases (TALENs).

Embodiment 2

[0039] Embodiment 2, TALENs design and construction of its recombinant expression vector

[0040] 1. Selection of TALENs target sequences

[0041] 1. Target sequence A targets the second exon of rice gene BADH2, the sequence is as follows:

[0042] 5'-GAAGAGGAACCGGGGCCgcgactgg gcgcgc gcgcCGGGCGCCGTCCGGGCC-3' (No. 273 to No. 324 of Sequence Listing Sequence 1); the lowercase letters in it are spacer sequences, and the uppercase letters on both sides are TALENs module recognition sequences (named L-a and R-a respectively); the underline is BssH Ⅱ Enzyme recognition sequence;

[0043] 2. Target sequence B targets the fourth exon of rice gene BADH2, the sequence is as follows:

[0044] 5'-GCTGGATGCTTTGAGTActttgc agatct tgcagaATCCTTGGACAAAAGGC-3' (No. 1589 to No. 1640 of Sequence Listing Sequence 1); the lowercase letters in it are spacer sequences, and the uppercase letters on both sides are TALEN module recognition sequences (named L-b and R-b respectively); the underline i...

Embodiment 3

[0056] Embodiment 3, the acquisition of recombinant Agrobacterium tumefaciens

[0057] The recombinant vector pGW3-BADH2b obtained in Example 2 was heat-shocked to transform the Agrobacterium strain AGL-1 to obtain a recombinant Agrobacterium containing the recombinant vector pGW3-BADH2b, which was named AGL-1 / pGW3-BADH2b;

[0058] The recombinant vector pGW3-BADH2a obtained in Example 2 was transformed into Agrobacterium strain AGL-1 by heat shock to obtain a recombinant Agrobacterium containing the recombinant vector pGW3-BADH2a, which was named AGL-1 / pGW3-BADH2a.

[0059] At the same time, the empty vector pGW3 was heat-shocked to transform the Agrobacterium strain AGL-1, and the recombinant Agrobacterium containing the recombinant vector pGW3-BADH2a was obtained, which was named AGL-1 / pGW3.

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Abstract

The invention discloses a rice gene BADH2 site-directed knockout system and application thereof. The site-directed knockout system is a), b) or c) as follows: a) T-BADH2b and T-BADH2a are included, b) T-BADH2b is included, and c) T-BADH2a is included, wherein T-BADH2b and T-BADH2a are both specifically spliced transcriptional activation subsample effective factor nucleases of rice gene BAHD2; and the target sequences of the both are respectively 1589-site to 1640-site nucleotide sequence and 273-site to 324-site nucleotide sequence in the sequence 1 of the sequence table. Experiments prove that the mutation frequency of the rice gene BADH2 by singly using T-BADH2b is 26.9%, the mutation frequency of the rice gene BADH2 by singly using T-BADH2a is 9.2%, and at the same time the mutation frequency of the rice gene BADH2 in the resistance callus obtained by expression of T-BADH2b and T-BADH2a in the rice Nipponbare callus is 18.1%, wherein the occurrence frequency of fragment deletion is 5.1%. The invention provides an efficient breeding method for creating the fragrant rice genetic resources and culturing the excellent fragrant rice variety.

Description

technical field [0001] The invention relates to a system for knocking out rice gene BADH2 and its application. Background technique [0002] Aroma is one of the important characteristics of high-quality rice, which has been extensively studied worldwide. The gene controlling rice aroma has been located on rice chromosome 8, within about 386kb between molecular markers RM515 and SSRJ07. Bradbury et al. (2005) found a gene with sequence differences between basmati rice and ordinary rice——BADH2, which encodes betaine aldehyde dehydrogenase (betaine aldehyde dehydrogenase), which is speculated to be related to the synthesis of rice aroma. Chen et al. (2008) proved that the BADH2 gene controls rice aroma through transgene complementation experiments. There is a natural mutation in the second or seventh exon of the BADH2 gene in fragrant rice varieties, and there is no full-length BADH2 transcript. 2AP (2-acetyl -1-pyrroline, one of the aromatic components of rice basmati rice) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/63C12N1/21C12N5/10C12N15/82C12N15/84A01H5/00
Inventor 高彩霞单奇伟陈坤玲
Owner TIANJIN GENOVO BIOTECH CO LTD
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