Methods for synthesizing ribonucleotide sequence and acquiring transgenic plant

A technology for nucleotide sequences and transgenic plants, applied in the field of synthesizing nucleotide sequences and obtaining transgenic plants

Inactive Publication Date: 2008-05-07
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But a variety of insects have now been found to develop resistance to crystalline insecticidal proteins

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The detection of insecticidal protein and the mensuration of insecticidal activity in embodiment 1, Bt host:

[0024]Z2-1 is cloned from the isolated strain HJC-1 (Bacillus thuringiensis) of Bacillus thuringiensis. HJC-1 was cultured in Luria Broth (LB) medium at 280C, shaking at 250 rpm. Samples were taken at 12, 24, 36, 48, 60, 72, 84, and 96 hours after the start of cultivation, and bioassays were performed on the insecticidal activity against corn borer and beet armyworm, respectively. Meanwhile, these samples were detected by Western analysis method using Z2-1 antibody. The results showed that isolated HJC-1 had no obvious insecticidal or growth-inhibiting effect on corn borer and beet armyworm at each culture time. Western analysis further showed that Z1-2 was not significantly expressed at each culture time.

Embodiment 2

[0025] Embodiment 2, cloning and expression of synthetic nucleotide sequence:

[0026] The nucleotide sequence encoding Z1-2 was obtained from Bt isolate HJC-1 by PCR. According to all codon frequencies of maize, the nucleotide sequence SEQ ID No: 1 was obtained through artificial design; Shanghai Sangong (Shanghai, China) synthesized SEQ ID No: 1. The above-mentioned nucleotide sequence was further cloned between the BamHI and SacI sites of the expression vector pET28a by general standard methods to obtain the expression vector pET28a-VEsyn. This expression vector was then transformed into E. coli BL21star. Inoculate a single colony into 100 ml of LB bacterial culture medium, shake culture at 37°C to OD 600 = 0.6, then add IPTG (lsopropyl-β-D-thiogalactoside) to 0.5 mM, and continue to cultivate under the same conditions for 4 hours. The culture solution was centrifuged at 5000 g for 10 minutes to pellet E. coli cells, and then the supernatant was discarded to collect the ...

Embodiment 3

[0027] Embodiment 3, the generation of antibody:

[0028] The Z1-2 protein expressed in Example 2 was cut out from the gel after SDS-PAGE separation. Using 100 μg of Z1-2 protein plus complete Freund's adjuvant, multi-point subcutaneous injection for the first immunization, and then for the second and third immunizations at intervals of 2 weeks. When the titer result of Western analysis was 5000 times, the serum was collected, centrifuged and stored in a -80°C refrigerator.

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PUM

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Abstract

The invention discloses an artificially synthesized nucleotide sequence, which is shown by SEQ ID NO: 1. The amino acid sequence for the disinsection protein of the nucleotide sequence coding is as shown by SEQ ID NO: 2. The invention also discloses a plasmid of the nucleotide sequence. The invention also disclose the use of the nucleotide sequence, which is a method of getting transgenic plant: the transgenic plant is stably guided. The invention can be used for detecting whether the nucleotide sequence is contained in the transgenic plant.

Description

technical field [0001] The invention relates to an artificially synthesized nucleotide sequence and its use, that is, a method for obtaining insect-resistant transgenic plants through plant transformation. Background technique [0002] Insecticidal toxin is a protein with important application value. It can be used to modify insecticidal microbial pesticides and transgenic insect-resistant crops. At present, transgenic insect-resistant technology has been widely used commercially (James C.2004 ISAAA Briefs No.32.Ithaca, NY:ISAAA.43pp), which provides a low-cost new technology for pest control. At present, plant transgenic technology is already a mature technology. [0003] At present, there are many kinds of insecticidal proteins. More commonly used is Bacillus thuringiensis (Bt) crystal protein, such as Cry1Ab, Cry1C, Cry2, Cry3 etc. (Crickmore, N., Zeigler, D.R., Feitelson, J., Schnepf, E., Van Rie, J., Lereclus, D ., Baum, J. & Dean, D.H. 1998 Microbiol. Mol. Biol. Rev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/32C07K14/325C12N15/82C12Q1/68G01N33/53A01H1/00
Inventor 沈志成李春雨徐晓丽方军
Owner ZHEJIANG UNIV
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