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Application and method of bombyx mori amylase gene BmAmy1

An amylase, silkworm technology, applied in genetic engineering, biochemical equipment and methods, enzymes, etc., can solve problems such as functions to be studied, and achieve the effect of improving the weight of the whole cocoon and important production and application potential

Active Publication Date: 2021-02-02
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the functions of more silkworm amylase genes need to be studied, laying the foundation for breeding better silkworm varieties

Method used

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  • Application and method of bombyx mori amylase gene BmAmy1
  • Application and method of bombyx mori amylase gene BmAmy1
  • Application and method of bombyx mori amylase gene BmAmy1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1, Identification, cloning and expression profile analysis of the silkworm BmAmy1 gene

[0033] 1. Identification of silkworm α-amylase gene (BmAmy1)

[0034] Download the reported amino acid sequences of silkworm α-amylase genes and other species α-amylase genes from NCBI as search seed sequences, and use the silkworm genome database (SilkDB) to carry out Blast comparison and identification (Blast parameter is set to Blastp) to obtain candidates sequence. Then the candidate sequences were manually screened, and finally the domains were predicted on the SMART website (http: / / smart.embl-heidelberg.de / ) to obtain the nucleic acid and protein sequences of BmAmy1.

[0035] 2. Multiple sequence alignment and phylogenetic analysis of BmAmy1

[0036] According to the reported literature and NCBI database search, the protein sequences of α-amylase genes of multiple species were downloaded, multiple sequence alignments were performed using Clustalx, and the files were ...

Embodiment 2

[0044] Embodiment 2, the expression characteristic analysis of BmAmy1 gene

[0045] The expression characteristics analysis of BmAmy1 was carried out by fluorescent quantitative PCR method, and the fluorescent quantitative PCR primers of BmAmy1 were BmAmy1-qRT-F: 5'-ccatcatccgtcctgctctat-3' (SEQ ID NO.4) and BmAmy1-qRT-R: 5'-ggcaagttgtgattcaagtcct -3' (SEQ ID NO. 5). The internal reference gene fluorescent quantitative PCR primers were sw22934-F: 5'-ttcgtactggctcttctcgt-3' (SEQ ID NO.6) and sw22934-R: 5'-caaagttgatagcaattccct-3' (SEQ ID NO.7). The qPCR experiment used the SYBR Premix Ex Taq II kit from Takara, and the fluorescent quantitative PCR instrument was qTOWER in Jena, Germany 3 touch quantitative PCR instrument. The PCR program was as follows: pre-denaturation at 95°C for 30s, followed by denaturation at 95°C for 3s, annealing at 60°C for 30s, and 40 cycles. Three experiments were carried out for each sample, and the Ct value was collected for data analysis. thro...

Embodiment 3

[0046]Example 3, Eukaryotic expression, purification and activity detection of the silkworm BmAmy1 gene

[0047] For eukaryotic expression, Pichia pastoris (Pichia pastoris) X-33 strain was selected. X-33 Pichia pastoris contains the AOX1 gene, and the resulting transformant is Mut + genotype. The expression vector is pPICZαA vector containing Zeocin resistance.

[0048] (1) Yeast eukaryotic expression vector construction and transformation

[0049] According to the sequence characteristics of BmAmy1 and the structural characteristics of the pPICZαA vector, a gene-specific primer EcoRI-BmAmy1-F with EcoR I and Not I restriction sites was designed: 5’-cg gaattc aatcatcataaaaggacgaacc-3' (SEQ ID NO.8) and NotI-BmAmy1-R: 5'-attt gcggccgc tatcttctgcttgatctggag-3' (SEQ ID NO. 9). Using the previously constructed T5-BmAmy1 vector as a template, the gene fragment with EcoRI and Not I restriction sites was amplified by PCR with TransTaq HiFi DNA polymerase. The gel was cut and ...

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Abstract

The invention discloses an application and method of a bombyx mori amylase gene BmAmy1. A nucleotide sequence of the bombyx mori amylase gene BmAmy1 is as shown in SEQ ID NO.3. By analyzing the sequence characteristics of the bombyx mori amylase gene BmAmy1 and detecting the tissue and period expression characteristics of the bombyx mori amylase gene BmAmy1, BmAmy1 protein is expressed and purified through a yeast eukaryotic expression system, and the amylase activity of the BmAmy1 is verified through an in-vitro enzyme activity experiment; and then a bombyx mori transgenic vector is constructed, transgenic bombyx mori is prepared, the economic traits of a transgenic line are investigated and analyzed, a result shows that the BmAmy1 transgenic line is superior to a control in the aspects of individual size, whole cocoon weight, cocoon layer rate and the like, and the amount of residual starch in silkworm excrement is also obviously smaller than that of the non-transgenic control. Therefore, the purpose of improving the economic traits such as the whole cocoon weight and the cocoon layer rate can be achieved by overexpressing the BmAmy1 in the bombyx mori through a biotechnologicalmeans, and a brand-new direction is opened up for cultivation of more excellent bombyx mori varieties.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of the silkworm amylase gene BmAmy1, and also to a specific application method. Background technique [0002] The utilization efficiency of animal feed directly determines the economic benefits of animal breeding. Insects use their digestive system to extract different nutrients and other substances needed for life activities from the food they get. Substances in food mostly exist in the form of macromolecules and other complex substances such as proteins, polysaccharides, fats and nucleic acids. These large molecules must be broken down by catabolic reactions into smaller molecules such as amino acids and simple sugars, which are then taken up by body cells for growth, development and reproduction. This breakdown process is called digestion. The digestive tract of insects can be divided into foregut, midgut, and hindgut. Most digestion occurs in the midgut, which...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N9/26A01K67/033C12P19/14
CPCC12N15/8509C12N9/2414C12Y302/01001A01K67/0339C12P19/14C12N2800/105A01K2227/706A01K2267/02Y02P60/87
Inventor 王根洪颜颢文凤赵萍夏庆友
Owner SOUTHWEST UNIVERSITY
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