Bt protein as well as coding gene and application thereof

A technology that encodes genes and bt proteins, applied in the field of genetic engineering, can solve the problems that the insecticidal activity of Bt proteins needs to be further improved, and achieve the effects of ensuring high and stable yields, improving resistance, and enhancing expression activity

Active Publication Date: 2014-09-24
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the insecticidal activity of existing Bt proteins still needs to be further improved

Method used

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  • Bt protein as well as coding gene and application thereof
  • Bt protein as well as coding gene and application thereof
  • Bt protein as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The acquisition of embodiment 1mBt gene

[0029] The modification and transformation of the mBt gene is achieved by utilizing techniques well known to those skilled in the art, i.e. PCR primer site-directed introduction of mutation base technology PCR site-directed mutagenesis technology, and its specific steps are:

[0030] (1) A polypeptide stabilizing the protein is inserted between the 3rd and 4th amino acid residues of the wBt hybrid protein, and the amino acid sequence of the polypeptide is NPNINECI;

[0031] (2) Carry out amino acid substitution modification on the 97th, 480th and 521st residues that play a key role in insecticidal activity and specificity, wherein, the D at the 97th position is replaced with Q, and the R at the 480th position is replaced with K, the N at the 521st position is replaced by H;

[0032] (3) according to the codon bias of the rice gene, without changing the amino acid sequence, the coding sequence obtained in step (2) is codon-optim...

Embodiment 2

[0035] Embodiment 2 prepares transgenic rice

[0036] 1 Construction of recombinant vector

[0037] (1) Preparation of mBt gene insert fragment: through primer design and PCR amplification, restriction sites Nco I and Kpn I were introduced at the 5-end and 3-end of the mBt gene coding sequence, respectively, and purified using the AxyPrep PCR cleaning kit from Axygen Company PCR products, the operation steps are as follows:

[0038] 1). Add 3 volumes of Buffer PCR-A to the PCR reaction solution (if Buffer PCR-A is less than 100 μl, add to 100 μl); after mixing, transfer to the preparation tube, and place the preparation tube in a 2ml centrifuge tube (provided in the kit), centrifuge at 12,000 rpm for 1 min, and discard the filtrate.

[0039] 2). Put the preparation tube back into a 2ml centrifuge tube, add 700μl Buffer W2, centrifuge at 12000rpm for 1min, and discard the filtrate; Note: Make sure that absolute ethanol has been added to the original solution of Buffer W2 acco...

Embodiment 3

[0084] Identification of insect resistance of embodiment 3 transgenic mBt rice

[0085] The mBt gene-transferred Nipponbare rice was used as the object to identify the resistance of the mBt gene to the rice leaf roller and Chilo suppressalis, and the wBt-transgenic and non-transgenic Nipponbare plants were used as positive and negative controls, respectively.

[0086] The resistance identification of rice leaf roller was carried out by the detached leaf method: that is, in the middle stage of rice tillering, 6 leaves (one leaf) with a length of 2 to 4 cm were randomly cut from 3 tillers on each of the rice plants to be tested. And press on the small filter paper sheet soaked with 0.1g / L benzomidazole fresh-keeping liquid on blade two ends. Then, the leaves were placed in small flat-bottomed glass tubes (9.5cm×1.5cm), and 5 ant borers were inserted into each tube, and the mouth of the tube was tightly plugged with a degreased cotton plug, and 3 strains / transformants were repeat...

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Abstract

The invention discloses a Bt protein as well as a coding gene and application thereof. An amino acid sequence of the Bt protein is as shown in SEQ ID No.1, and a base sequence of the coding gene of the Bt protein is as shown in SEQ ID No.2. The application refers to application of the Bt protein in improving insect resistance of plants. Expression activity of an mBt gene of mBT transgenic rice obtained by transferring rice through the coding gene is 20%-50% stronger than that of a wBT gene; resistance on lepidopteron ice leaf folder and striped rice borer is also greatly improved as follows: the rice leaf folder can form a certain withered and yellow hazard symptoms on leaves of the wBt gene ruce, but the leaves of the mBt transgenic gene rice are not formed or only formed with withered and yellow hazard symptoms or only extremely small withered and yellow symptoms are formed near a shear mouth; on the wBt transgenic rice, a white head rate caused by striped rice borer is 1.1 + / -0.1%, and the white head rate on the mBt transgenic rice is 0.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a Bt protein, its coding gene and application. Background technique [0002] Lepidoptera pests such as rice stem borer are one of the important restrictive factors for high and stable rice yield. So far, no rice seed resources resistant to lepidopteran pests such as rice borer have been found. In the past, the control of rice borers was mainly based on spraying chemical pesticides, but a large amount of pesticide residues not only caused serious environmental pollution, but also reduced the hygienic quality of rice and affected people's health. As a major rice producing country, Lepidoptera pests cause huge losses to rice production in my country every year. [0003] Bt protein is a crystal protein produced by Bacillus thuringiensis during its cell production process, which has a specific poisonous effect on Lepidoptera insects. In the 1980s, the first ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/325C12N15/32C12N15/82A01H5/00
CPCC07K14/325C12N15/8286
Inventor 凃巨民陈浩张集文
Owner ZHEJIANG UNIV
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