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Bombyx mori tRNA prenyltransferase gene and its recombinant vector and application

A technology of isopentenyl and recombinant vectors, applied in transferase, application, genetic engineering, etc.

Active Publication Date: 2021-06-08
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bombyx mori is an organism with high-efficiency protein synthesis ability, whether its high-efficiency protein synthesis is regulated by the 37-position adenine base prenylation modification of tRNA has not been reported so far

Method used

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  • Bombyx mori tRNA prenyltransferase gene and its recombinant vector and application
  • Bombyx mori tRNA prenyltransferase gene and its recombinant vector and application
  • Bombyx mori tRNA prenyltransferase gene and its recombinant vector and application

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1, the acquisition of BmIPT gene sequence of silkworm

[0034] Design the following amplification primers, BmIPT-F1: 5'-atggctttgcgtactgtgat-3' (SEQ ID NO.1); BmIPT-R1: 5'-ttaatcttgtttttgttccttgtt-3' (SEQ ID NO.2); As a template, PCR amplification was carried out, and the PCR amplification product was detected by agarose gel electrophoresis, and the results were as follows: figure 1 shown. The results showed that three bands of different sizes were amplified.

[0035] Bands of different sizes were recovered and cloned and sequenced. After sequence analysis and splicing, the results were as follows: figure 2 shown. It was found that the three bands were all obtained by primer-specific amplification, came from the same position on the genome, and belonged to different alternative splicing forms of the same gene, named BmIPT1, BmIPT2 and BmIPT3 respectively, and their nucleotide sequences are shown in SEQ ID NO .3. SEQ ID NO.4 and SEQ ID NO.5. The recovere...

Embodiment 2

[0037] Embodiment 2, the expression feature analysis of BmIPT

[0038] Based on the gene chip expression profile data of silkworm, the expression characteristics of BmIPT in silkworm were analyzed. First, the expression characteristics of BmIPT in the third day of the fifth instar silkworm tissue samples were analyzed, and the tissue samples included testis, ovary, head, epidermis, fat body, midgut, blood cells, Malpighian tubules, anterior / middle silk gland, posterior silk glands, results such as Figure 4 shown. The results showed that tRNA prenyltransferase gene was expressed in various tissues and organs of silkworm, among which the expression level was the highest in the testis, followed by the ovary; the expression level of BmIPT was also higher in blood cells and silk glands. Secondly, analyze the period expression characteristics of BmIPT in silkworm, such as Figure 5 shown. The results showed that BmIPT was significantly expressed at a high level during the fifth...

Embodiment 3

[0046] Example 3, BmIPT detection of functional complementation of tRNA isopentenyl transferase-deficient yeast

[0047] The genotype of the yeast strain MT-8 used in this study is: MATa SUP7 ura3-1his5-2 leu2-3, 112ade2-1 trp1 lys1-1 lys2-1 can1-100 MOD5::TRP1. The mod5 gene is a tRNA prenyltransferase gene in yeast that is mutated in the MT-8 strain. In addition, the ade2-1 gene in the de novo adenine synthesis pathway in MT-8 strain contained a nonsense mutation, and the tRNA SUP7 nonsense mutation suppressor could not suppress its nonsense mutation due to the lack of prenylation modification. Therefore, when MT-8 is grown on nutrient-rich media, intermediates in the adenine pathway accumulate and become oxidized, resulting in a red color of the yeast strain. The prenylated tRNA SUP7 repressor could repress the ade2-1 gene only when the homologous functional tRNA prenyltransferase gene transferred into the mod5 gene in MT-8 restored its prenylation ability The nonsense mu...

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Abstract

The present invention relates to a silkworm tRNA prenyl transferase gene and its recombinant vector and application, the nucleotides of the silkworm tRNA prenyl transferase gene such as SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO As shown in .5, there are three different alternative splicing forms. The tissue expression shows that the expression level is the highest in the testis, followed by the ovary; the yeast function test shows that BmIPT1 has the function of tRNA prenyl transferase and can be used as a prenylation modification It can also be used as a catalyst for silkworm breeding and as a target gene for lepidopteran pest control.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a silkworm tRNA prenyl transferase gene, and also relates to a recombinant expression vector containing the gene and its application. Background technique [0002] The main value of silkworm (Bombyx mori) domesticated and utilized by humans lies in its efficient protein synthesis ability and ability to produce silk protein fibers. Fast growth, large individual, and high cocoon layer rate are the main traits of silkworm domestication and selection for thousands of years. The research on the high-efficiency protein synthesis ability of silkworm has important theoretical and application value. [0003] The silkworm feeds on mulberry leaves, and its feeding period is only more than 20 days, but its weight increases rapidly, from 0.5mg at the beginning of the first instar to 5g at the end of the fifth instar, an increase of about 10,000 times, and the silk gland, the silk secr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/85A01K67/04
CPCA01K67/04A01K2267/02C12N9/1085C12N15/85
Inventor 王根洪夏庆友陈燕飞赵萍柏冰川
Owner SOUTHWEST UNIV
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