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Codon vegetalization-transformed PMI gene and applications thereof

A plant-based, codon-based technology, applied in the fields of biotechnology and plant genetic engineering, can solve the problems of affecting expression efficiency, adverse effects of transforming receptor genomes, and affecting use effects, etc.

Active Publication Date: 2014-09-17
RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, direct use of PMI without artificial optimization design will affect its expression efficiency in eukaryotic cells and affect its use as a screening marker
In addition, since PMI is derived from Escherichia coli, it may adversely affect the genome of the transformed recipient and may also raise concerns about its safety

Method used

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  • Codon vegetalization-transformed PMI gene and applications thereof
  • Codon vegetalization-transformed PMI gene and applications thereof
  • Codon vegetalization-transformed PMI gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1——the codon phytochemical transformation of PMI gene

[0037] According to the information in the GENBANK database, the inventors of the present application collected 92188 CDS sequences of rice, with a total of 34132283 codons, and analyzed the distribution of these codons in rice on the website http: / / www.kazusa.or.jp / codon / Usage. For the same amino acid in the same species, the frequencies of different codons are different. Some codons are used frequently, while others are used less frequently. For example, in rice, the most frequently used leucine (L) codon is CUC, reaching 28.51%, and the least frequently used is UUA, only 6.79%.

[0038] Using rice-preferred codons, the PMI gene from Escherichia coli was planted to obtain a new DNA sequence, and a rice-preferred stop codon TGA was added to the end of the DNA sequence to form a new gene, which was named It is plant PMI, the sequence is shown in SEQ ID NO: 1, see the sequence comparison with PMI figu...

Embodiment 2

[0045] Embodiment 2 - the construction of plant PMI gene plant expression vector and the transformation of Agrobacterium

[0046] From Escherichia coli JM110 containing the PUC57-AMP-plant PMI vector above, use the Axygen plasmid extraction kit to extract the plasmid, digest it with Xho I, and recover the plant PMI fragment. Utilize Xho I enzyme to carry out linearization process to pCAMBIA1381 simultaneously, reclaim pCAMBIA1381, above-mentioned plant PMI fragment and pCAMBIA1381 fragment are connected with T4 ligase (purchased from TaKaRa Company), obtain plant expression vector pCAMBIA1381-plant PMI ( image 3 ), using the freeze-thaw method to transfer the plant expression vector into Agrobacterium tumefaciens EHA105 strain (preserved by the Rice Research Institute of Anhui Academy of Agricultural Sciences) for genetic transformation.

Embodiment 3

[0047] Embodiment 3——using plant PMI as the rice genetic transformation method of screening marker gene

[0048] 1. Induction and pre-culture of mature embryo callus

[0049] The mature seeds of Nipponbare (the Paddy Rice Research Institute of Anhui Academy of Agricultural Sciences are preserved) are shelled, and the seeds with normal appearance and cleanness without mildew are selected, shaken for 90 sec with 70% alcohol, and pour off the alcohol; then use 50% sodium hypochlorite ( The concentration of available chlorine in the stock solution is greater than 4%. Add 1 drop of Tween20) solution per 100 milliliters to clean the seeds, and shake for 45 minutes (180 r / min) on a shaker. Pour off the sodium hypochlorite, wash with sterile water 5-10 times until there is no smell of sodium hypochlorite, finally add sterile water, soak overnight at 30°C. Use a scalpel to separate the embryos along the aleurone layer, put the scutellum up on the induction medium (see Table 1 for ingr...

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Abstract

The invention provides a codon vegetalization-transformed PMI (phosphomannose isomerase) gene, which is designed and synthesized by utilizing optimized rice codon. Furthermore, the invention provides an expression cassette and an expression carrier, as well as applications of the expression cassette and an expression carrier on aspect of genetic transformation. A plant expression carrier is constructed by utilizing the transformed PMI gene, and an exogenous gene is injected to rice cells by taking mannose as a screening reagent. According to the codon vegetalization-transformed PMI gene, the high-efficiency genetic transformation of rice can be realized as transformation efficiency of the plant PMI subjected to codon vegetalization transformation is obviously improved.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and plant genetic engineering. Specifically, the present invention relates to a PMI gene whose codons have undergone phytochemical transformation, and its application as a screening marker in plant genetic transformation. Background technique [0002] Transgenic technology is an effective method for directional improvement of plants developed in the early 1980s. This technology mainly introduces the target gene into the receptor through methods such as Agrobacterium-mediated and gene gun transformation, and obtains stably expressed transformants after marker screening and molecular detection, and realizes rapid and directional improvement of target traits. It has a wide range of available gene resources, improved High efficiency and other advantages. Transgenic technology has provided a new path and opened up new space for crop yield improvement, quality improvement and resistance enhance...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/82A01H5/00
Inventor 李莉杨剑波魏鹏程宋丰顺李浩杨亚春倪大虎汪秀峰马卉秦瑞英陆徐忠倪金龙
Owner RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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