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Waxy protein subunits of wheat variety Chinese spring, and coding genes and application thereof

A technology for encoding genes and varieties, which is applied to the expression of wheat protein and its application field

Inactive Publication Date: 2014-09-10
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, there have been no reports on the in vitro expression of the gene encoding Waxy in Chinese spring, the purification of the Waxy expressed protein, and the effect of the protein on dough quality.

Method used

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  • Waxy protein subunits of wheat variety Chinese spring, and coding genes and application thereof
  • Waxy protein subunits of wheat variety Chinese spring, and coding genes and application thereof
  • Waxy protein subunits of wheat variety Chinese spring, and coding genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Total RNA extraction and cDNA synthesis of wheat variety Chinese spring seeds

[0042] Total RNA was extracted from Chinese spring wheat grains according to the instructions of EasyPureTM Plant RNA Kit, and cDNA was synthesized according to EasyScriptTM First-Strand cDNA Synthesis SuperMix Kit (Quanshijin Biotechnology Co., Ltd., Beijing).

[0043] For the specific operation method, please refer to the operating instructions of the instrument.

Embodiment 2

[0044] Example 2: Design of primers and amplification and purification of target gene

[0045] According to the known wheat Waxy sequence, design cloning primers, the upstream primer is 5'-ATGGCGGCTCTGGTCACGT-3' (SEQ ID NO7) , the downstream primer is 5'-TCAGGGAGCGGCGACGTT-3' (SEQ ID NO8) , the above primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0046] PCR system: cDNA 100ng, 0.1μmol·L-1 F / R 1μL each, IA Taq DNA polymerase 0.25μL, 0.05mmol·L-1 dNTP mix 12.5μL, add ddH 2 O to the reaction volume of 25 μL.

[0047] The PCR reaction program was: 95°C for 5 min; 94°C for 40 s, 68°C for 8 min, 35 cycles; 72°C for 8 min; storage at 10°C.

[0048] A DNA purification and recovery kit (Tiangen Biochemical (Beijing) Co., Ltd., spin column type DP214) was used to recover the amplified product, which was detected by electrophoresis for later use.

Embodiment 3

[0049] Embodiment 3: target fragment ligation and transformation

[0050] First, the recovered target fragments were ligated with pEASY-E1 vector, and then the ligated products were transformed into E. coli DH5α competent cells, and positive clones were screened on LB plates coated with ampicillin (AMP). The specific ligation reaction system adopted is shown in Table 1 below:

[0051] Table 1 ligation reaction system

[0052]

[0053] Ligation at 25°C for 30 min or at 16°C overnight.

[0054] In the above method, E.coli DH5α competent cells can be purchased or conventional CaCl can be used 2 method to prepare;

[0055] The transformation method of the ligation product is to mix 5 μL of the ligated product and 50 μL of DH5α competent cells into a 1.5 ml centrifuge tube, let stand on ice for 30 min, then heat-shock transformation in a water bath at 42°C for 45 s, and then ice-bath for 2 min; add 500 μL of liquid LB medium , Shake the bacteria at 190 rpm at 37°C for 40-50m...

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Abstract

The invention discloses full length coding sequences of coding genes of three Waxy protein subunits, and the corresponding coding sequences and amino acid sequences of coded protein subunits are shown as SEQ ID NO1-SEQ ID NO6. Correspondingly, the invention discloses cloning and in-vitro expression method of the above coding sequences. Finally, the invention also discloses application of the protein subunits to improve flour quality.

Description

technical field [0001] The invention relates to the expression and application of wheat protein, and belongs to the field of biotechnology. Background technique [0002] Common wheat grain endosperm contains 3 Waxy protein subunits with molecular weights of 62.8kD, 56.7kD and 58.7kD, namely Wx-A1, Wx-B1 and Wx-D1, composed of genes Wx-7A, Wx located in 7AS, 4AL and 7DS. -4A and Wx-7D codes (Ainsworth and Clark, 1993). Common wheat varieties, such as Chinese Spring, have not been able to isolate complete and accurate coding sequences so far. [0003] Since the content of amylose in wheat grains is highly positively correlated with the content of Waxy protein (Yao Danian, 1999), the content of amylose is an important factor affecting the quality of noodles. For many years, the field has concentrated on the study of the content of amylose protein by Waxy protein, etc. There is no research on the effect of the Waxy protein on the quality of the quality. [0004] Waxy protein ...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/10C12N15/70A21D2/26
CPCC07K14/415
Inventor 陈其皎高翔孟敏董剑赵万春
Owner NORTHWEST A & F UNIV
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