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Method for improving characters of gossypol in cotton, and use thereof

A technology of gossypol and cotton, which is applied in the fields of plant bioengineering and plant improvement genetic engineering, and can solve problems such as high yield and uncoordinated use of by-products, restrictions on cotton production and development, and cottonseed waste

Inactive Publication Date: 2013-07-10
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the improvement of people's living standards and the incomplete utilization of a large number of cottonseeds, the production problems such as high yield and uncoordinated use of by-products have restricted the further development of cotton production in my country.

Method used

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  • Method for improving characters of gossypol in cotton, and use thereof
  • Method for improving characters of gossypol in cotton, and use thereof
  • Method for improving characters of gossypol in cotton, and use thereof

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Experimental program
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preparation example Construction

[0071] The present invention has no special limitation on the preparation method of the polynucleotide, including but not limited to: chemical synthesis method, in vitro transcription method and so on. It should be understood that those skilled in the art can prepare the polynucleotide interfering molecule in various ways.

[0072] construct

[0073] In order to specifically down-regulate the content of gossypol in cotton seeds, the inventors conducted extensive research and found suitable regulatory elements.

[0074] Therefore, the present invention provides a construct comprising the following structures in sequence (5'→3'): cotton seed-specific promoter; Seq 正向 -X-Seq 反向 ; among them, Seq 正向 To form the nucleotide sequence of a molecule that specifically interferes with the expression of the juniperene-8-hydroxylase gene CYP706B1 after being introduced into cotton, Seq 反向 for Seq 正向 A substantially complementary nucleotide sequence; X is located at Seq 正向 and Seq 反向...

Embodiment 1

[0094] Embodiment 1, ProGlobulin and LP132-1 fragment acquisition

[0095] 1. DNA Extraction from Cotton

[0096] The following method was adopted: take 0.5 g of Asian cotton (G. arboreum) leaves, grind them into powder with liquid nitrogen, transfer them to an 8 mL centrifuge tube, add 5 mL of grinding buffer (1M glucose, 0.1M citric acid, 5% Triton X- 100 (pH 5.0)), mix well. Centrifuge at 1000g at 22°C for 10 min to collect the precipitate, resuspend in grinding buffer, and centrifuge again, repeating three times until the precipitate appears off-white. Wash the precipitate with 5 mL of washing buffer (0.5M glucose, 0.05M citric acid (pH 5.0)), centrifuge at 22°C and 1000g for 10 min, remove the supernatant, and repeat 2-4 times until the precipitate becomes milky white. Add 5 mL of lysis buffer (1% SDS, 1.4M NaCl, 0.1M EDTA (pH 8.3)), and lyse in a water bath at 60° C. for 15 min. Centrifuge at 5000g at 22°C for 10min, collect the supernatant, add 2 times the volume of ...

Embodiment 2

[0112] Embodiment 2, vector construction and Agrobacterium transformation

[0113] 1. Vector Construction

[0114] The dsRNA carrier structure required to be constructed is as shown in Figure 2, including a seed-specific promoter ProGlobulin, a forward (i.e. Sense, S) gene segment, an internal element of the Arabidopsis RTM gene (i.e. Intron, about 120bp , see Figure 1B (SEQ ID NO: 2)) and an inverted gene segment (ie Antisense, AS) and NOS terminator. It is constructed by inserting the sequence containing Sense-Intron-Antisense into the pCambia2301 vector (purchased from Cambia Company) between the BamHI and SacI sites.

[0115] First, the internal element (about 120bp) of the Arabidopsis RTM gene (AT2G43730) was used with specific primers RTM+(5'-TCTAGAACGTTGTAAGTCTATTTTTG-3'(SEQ ID NO: 8)) and RTM-(5'-GCGGCCGCTCTGCTGGGTCCAAATCACA- 3' (SEQ ID NO: 9)) was amplified by high-fidelity enzyme pfu, the PCR product was double-digested with XbaI and NotI restriction endonucleases,...

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Abstract

The invention relates to a method for improving characters of gossypol in cotton, and a use thereof. In the invention, expression of a cadinene-8-hydroxylase gene CYP 706 B 1 in cotton seeds is down-regulated specifically thus synthesis of gossypol in cotton seeds is inhibited effectively without an influence on synthesis of gossypol in other tissues. Through the method, characters of gossypol ina cotton plant are improved and an integrated utilization value of cotton is increased greatly.

Description

technical field [0001] The invention belongs to the fields of plant bioengineering and plant improvement genetic engineering; specifically, the invention relates to a method and material for specifically reducing gossypol content in cotton seeds (cotton seeds) without affecting gossypol in other tissues of cotton plants, and Transgenic cotton plants obtained by this method. Background technique [0002] Gossypol is a sesquiterpene aldehyde derivative that accumulates in a large amount in the glands and root epidermal cells of the aboveground parts of cotton plants, and also in cottonseeds. As a phytoprotectin of cotton, it plays an important role in the response to disease and insect resistance. Gossypol is a kind of polyphenolic compound with universal toxicity. It is a toxic substance that is harmful to cells, blood vessels and nerves, and seriously affects the edible and feed value of cottonseed oil and cottonseed cake. [0003] Cotton is a multipurpose crop that integr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82A01H4/00A01H5/00
Inventor 陈晓亚杨长青骆萍武秀明王凌健林芝萍毛颖波
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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