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DNA molecule for expressing hairpin RNA for inhibiting wheat starch branching enzyme IIa (SBEIIa) and application thereof

A DNA molecule and molecular technology, applied in the field of DNA molecules of hairpin RNA, can solve the problems of restricting the cultivation of high-amylose wheat varieties, and achieve the effect of improving quality and increasing amylose content.

Inactive Publication Date: 2011-10-12
CHINA AGRI UNIV
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Problems solved by technology

However, there is currently no wheat variant material with high amylose screened in China, and only Japanese reports have screened a starch synthase (SGP-1) mutant (its amylose content is only about 37%) abroad, which limits the high amylose wheat. Breeding of varieties

Method used

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  • DNA molecule for expressing hairpin RNA for inhibiting wheat starch branching enzyme IIa (SBEIIa) and application thereof
  • DNA molecule for expressing hairpin RNA for inhibiting wheat starch branching enzyme IIa (SBEIIa) and application thereof
  • DNA molecule for expressing hairpin RNA for inhibiting wheat starch branching enzyme IIa (SBEIIa) and application thereof

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1, the acquisition of RNA interference carrier

[0035] 1. SbeIIa gene fragment acquisition

[0036] By comparing the mRNA homology of the wheat SBE coding gene, search the wheat SbeIIa gene mRNA sequence (GenBank NO.: Y11282, AF338431) in GenBank, and use Primer5.0 software to design primers as follows:

[0037] SBEIIa-s2: 5'-CGAGATGGTGGCTTGAAGAA-3' (SEQ ID NO: 4)

[0038] SBEIIa-a2: 5'-ATGATCAAGCCTGCTGAATCC-3' (SEQ ID NO: 5)

[0039] Trizol RNA extraction kit (Tiangen Company) was used to extract "Nongda 152" wheat (Triticum aestivum L) pollinated for 15 days. The study of the impact. Chinese Journal of Cereals and Oils, 2006, 21 (6): 27-30 "published, the public can obtain from China Agricultural University) total RNA of grain ( figure 1 , wherein 1, 2, 3, and 4 are all extracted RNA), carry out reverse transcription-PCR (RT-PCR) with the M-MLV reverse transcription kit (Promega Company) according to the two-step method of the instruction manual, and ob...

Embodiment 2

[0048] Embodiment 2, trans sbeIR wheat acquisition and functional identification

[0049] 1. Acquisition and identification of trans sbeIR wheat

[0050] Recipient: Baofeng 104 (Triticum aestivum L), a common wheat variety, is a popular wheat variety approved by the Powdery Mildew Group of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. A new variety of winter wheat——Baofeng 104. It was published in Journal of Wheat Crops, 2003, 23(4): 147, and the public can obtain it from China Agricultural University, hereinafter referred to as wild-type wheat.

[0051] Explants: callus induced by wheat immature embryos;

[0052] Strain: Escherichia coli strain DH5α;

[0053] Carrier: pBAC47P+sbeIR obtained by Example 1, pBAC35SIH3 (using the pSP72 vector of Promega Company as the starting vector, inserting the CaMV 35S promoter (GenBank NO: AF485783.1) between HindIII and Sal I), between Kpn I and EcoR The NOS terminator (GenBank NO: AF485783.1) was inserted...

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Abstract

The invention discloses a DNA molecule for expressing hairpin RNA for inhibiting wheat starch branching enzyme IIa (SBEIIa) and application thereof. The invention provides an RNA molecule, the nucleotide sequence of which is the sequence 4 in a sequence table. The invention also provides a DNA molecule for expressing the RNA, wherein the nucleotide sequence of the DNA molecule is the sequence 5 in the sequence table. Proved by experiments, positive and negative RNA interfering vectors inserted with wheat grain SBEIIa gene fragments are obtained by using an RNA interference (RNAi) technology; and when the RNA interfering vectors are transferred to wheat, the amylose content of wheat grain starch is improved and the quality of the wheat starch is improved on the premise that vegetative growth and stress resistance of the wheat are not affected.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DNA molecule expressing hairpin RNA inhibiting wheat SBEIIa and application thereof. Background technique [0002] The amylose content generally accounts for about 25% of common wheat starch and about 12-16% of wheat flour. The unique molecular structure of amylose leads to its unique physical and chemical properties, processing performance and application value: such as 1) due to its good film-forming properties, amylose is an important raw material for the production of edible food packaging materials, films, etc.; 2) due to Amylose belongs to the resistant starch, which is not easy to be degraded and digested by intestinal enzymes. After eating, the blood sugar level of the human body will not increase significantly. It is used to prepare special food for diabetics and solve the hunger of patients; for obesity , gallstone disease, intestinal dysfunction and colon cancer have go...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A01H5/00
Inventor 梁荣奇孙其信倪中福李保云尤明山彭惠茹解超杰秦丽燕王树彬姚颖垠杜金昆刘志勇
Owner CHINA AGRI UNIV
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