阅读说明：本技术 一种调控白色脂肪棕色化的基因Adra1a的应用 (Application of gene Adra1a for regulating browning of white fat ) 是由 张驹 仓明 刘东军 杨杰 董艳华 路大同 马建飞 杨楠 于 2019-10-31 设计创作，主要内容包括：本发明提供了一种调控白色脂肪棕色化的基因Adra1a的应用。所述基因Adra1a的核苷酸序列如SEQ ID NO.2所示。本发明从细胞水平和小鼠个体水平验证了Adra1a通过抑制白色脂肪细胞的增殖和分化减少白色脂肪含量,使白色脂肪明显出现“多室”的棕色样脂肪形态特征,促进白色脂肪中棕色脂肪标记基因的表达,抑制白色脂肪标记基因的表达。揭示Adra1a基因调控白色脂肪棕色化的作用机理是通过PI3K-AKT信号通路影响棕色脂肪标志基因Pgc1α基因的表达,进而影响白色脂肪的棕色化。本发明提供了Adra1a基因在调控白色脂肪棕色化的新用途,在制备有效缓解和治疗肥胖的药物方面具有实用价值。(The invention provides an application of a gene Adra1a for regulating browning of white fat, wherein the nucleotide sequence of the gene Adra1a is shown in SEQ ID No.2, the invention verifies from a cell level and a mouse individual level that Adra1a reduces the content of white fat by inhibiting proliferation and differentiation of white fat cells, so that the white fat obviously has the shape characteristic of multi-chamber brown-like fat, promotes expression of a brown fat marker gene in the white fat, and inhibits expression of the white fat marker gene.)

Use of the Adra1a gene or its encoded protein for regulating browning of white fat.

The application of the Adra1a gene or the protein coded by the Adra1a gene or the Adra1a gene expression promoter in improving or promoting the expression level of brown fat marker genes or reducing or inhibiting the expression level of white fat marker genes.

The Adra1a gene or its coded protein can improve or promote the expression of Pgc1 α gene through PI3K-AKT signal channel.

Use of an inhibitor of the expression of the Adra1a gene for inhibiting an increase in the number of brown adipocytes and an increase in volume in vivo, or for the preparation of a medicament for inhibiting proliferation and differentiation of brown adipocytes.

Use of the Adra1a gene or its encoded protein, or Adra1a gene expression promoter for inhibiting increase in white adipocytes, increase in volume, proliferation, differentiation, or for alleviating or treating obesity.

Application of the Adra1a gene, or protein coded by the Adra1a gene, or expression promoter of the Adra1a gene in preparing medicines for relieving or treating obesity.

7. The use according to any one of claims 1 to 6, wherein the protein encoded by the Adra1a gene has any one of the following amino acid sequences:

(1) an amino acid sequence shown as SEQ ID NO. 1;

(2) the amino acid sequence of the protein with the same function is obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1;

(3) an amino acid sequence having at least 80% homology with the amino acid sequence shown as SEQ ID No. 1; preferably, the homology is at least 90%; more preferably 95%;

or the CDS sequence of the Adra1a gene has any one of the following nucleotide sequences:

(1) a nucleotide sequence shown as SEQ ID NO. 2;

(2) the nucleotide sequence shown as SEQ ID NO.2 is obtained by replacing, inserting or deleting one or more nucleotides in the nucleotide sequence to encode the same functional protein.

8. A medicament for the alleviation or treatment of obesity, said medicament comprising a biological and/or chemical agent that promotes an increase in the expression of Adra1a gene.

9. An sgRNA targeting an Adra1a gene, characterized in that the nucleotide sequence is shown in SEQ ID NO.45 or SEQ ID NO. 46.

10. A CRISPR/Cas9 targeting vector containing the sgRNA of claim 9.

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to application of a gene Adra1a for regulating and controlling browning of white fat.

Background

The obesity is fat metabolism imbalance caused by multiple factors, and is characterized in that the number of white fat cells in a body is increased, the volume of the fat cells is increased, and the fat cells are excessively deposited locally, brown fat is fat with the function opposite to that of the white fat, the increase of the fat is beneficial to energy consumption, and the obesity can be effectively relieved and treated, a large number of researches prove that an effective way for increasing the brown fat is to brown the white fat, at present, more transcription factors, secreted proteins, small RNA and the like which influence the brown of the white fat are researched, but the brown of the white fat is regulated by multiple factors or multiple genes, so that a key gene for regulating the brown of the white fat is excavated, a solid foundation is laid for researching the obesity, in order to explore the mechanism of the brown of the white fat, Adra1A (Adrenoceptor Alpha 1A) is obtained through transcriptome sequencing and screening, and the key gene for regulating the brown of the white fat is used for coding an epinephrine α -1A receptor and can mediate signal conduction of heart, nerves and other tissues, but the action mechanism of the brown of the white fat is not clear at present.

At present, although genes for partially regulating the browning of white fat of mice are found, the research on the browning of white fat just starts, and the browning of white fat is controlled by multiple factors or multiple genes, so that the excavation of key genes for regulating the browning of white fat is very important.

The Adra1a gene is a member of the G protein-coupled receptor family, encoding a multi-pathway transmembrane protein. Can be combined with various hormones such as adrenalin and adrenalin to regulate cardiovascular function and sympathetic nerve activity, thereby regulating blood pressure.

The protein encoded by Adra1a binds to epinephrine and norepinephrine to mediate signaling in cells of the cardiac, neural, and other organ systems α 1-adrenergic receptors are widely distributed in the central and peripheral nervous systems and play a major role in smooth muscle contraction, promoting mitotic responses and regulating the growth and proliferation of a variety of cells.

However, studies on the browning of white fat and the action mechanism of the receptor of epinephrine α -1A are not reported at present.

Disclosure of Invention

The invention aims to provide application of a gene Adra1a for regulating browning of white fat.

The invention obtains 99 genes differentially expressed by C57BL/6 mouse white fat and brown fat through RNA-seq technology, q-PCR verifies and obtains candidate genes with obvious differential expression of Adra1a, Cxcr5 and Galnt6, the candidate genes are over-expressed and interfered in white fat and brown fat cells, the influence of the candidate genes on the browning of the white fat is determined at RNA level and protein level through q-PCR and Western Blot technology, finally Adra1a is obtained through screening and is an important gene influencing the browning of the white fat, then the invention verifies that the expression of the brown marker gene in the Adra1a gene can effectively promote the browning of the white fat at cell level, the over-expression of the Adra1a gene is beneficial to the browning of the white fat cells, the proliferation and differentiation of the white fat cells are inhibited through CRISPR/Cas9 technology mediated Adra1 gene knockout inhibition, the proliferation and inhibition of the brown marker gene expression and inhibition of the proliferation and brown fat, the inhibition of the proliferation and inhibition of the proliferation of the brown fat, the Adra1 gene expression of Adra1 gene is beneficial to the increase of the white fat cells through individual expression of the fat cells, the activation of the white fat, the white fat activation of the white fat, the fat activation of the white fat, the fat activation of the white fat, the fat activation of the fat, the fat activation of the white fat, the activation of the white fat, the activation of the white fat, the activation of.

The coding protein of the Adra1a gene provided by the invention has any one of the following amino acid sequences:

(1) an amino acid sequence shown as SEQ ID NO. 1;

(2) the amino acid sequence of the protein with the same function is obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown as SEQ ID NO. 1;

(3) an amino acid sequence having at least 80% homology with the amino acid sequence shown as SEQ ID No. 1; preferably, the homology is at least 90%; more preferably 95%;

the CDS sequence of the Adra1a gene provided by the invention has any one of the following nucleotide sequences:

(1) a nucleotide sequence shown as SEQ ID NO. 2;

(2) the nucleotide sequence shown as SEQ ID NO.2 is obtained by replacing, inserting or deleting one or more nucleotides in the nucleotide sequence to encode the same functional protein.

The invention constructs an overexpression vector of Adra1a and transfects white fat cells, and Adra1a overexpression promotes the expression of brown fat marker genes such as Ucp1, Cidea, Fndc5 and the like in the white fat cells, inhibits the expression of white fat marker genes such as Serpina3k, resistin, Asc1 and the like, and simultaneously inhibits the proliferation and differentiation of the white fat cells.

The invention obtains Adra1a transgenic mice by a prokaryotic injection method, and obtains 34 Adra1a transgenic mouse offspring by propagation, under the standard feeding condition, the Adra1a transgenic mice have reduced content of white fat in vivo compared with wild mice, and the white fat obviously has the shape characteristic of 'multi-chamber' brown-like fat, molecular biology detection shows that the expression of specific genes of brown fat in white fat of Adra1a over-expressed mice is increased, the expression of specific genes of white fat is reduced, Adra1a over-expressed mice have obviously increased ratio of brown fat to white fat in vivo and increased content of brown fat compared with wild mice, molecular biology detection shows that the expression of genes such as Ucp1, Prdm16 and Pgc1 α in brown fat in vivo of Adra1a over-expressed mice, and the individual level result of mice is mutually printed with the cell level result, which shows that the Adra1a gene regulates the brown fat.

The CRISPR/Cas9 knockout vector of Adra1a is constructed and transfected into the brown fat cell, and the knockout of Adra1a inhibits the expression of genes Ucp1, Prdm16, Cidea and the like in the brown fat cell and inhibits the proliferation and differentiation of the brown fat.

In a first aspect, based on the above findings, the present invention provides the use of the Adra1a gene or its encoded protein, or an Adra1a gene expression promoter for regulating browning of white fat. The application specifically relates to the fact that the overexpression of the Adra1a gene inhibits the proliferation and differentiation of white adipocytes.

The invention provides application of an Adra1a gene or a protein coded by the Adra1a gene or an Adra1a gene expression promoter in reducing or inhibiting the expression quantity of a white fat marker gene. Specifically, the white fat marker genes include Serpina3k, Asc1, Leptin, Fad3, resistin, SCD, FASN, SREBP1, ACC, and Psat 1.

Further, the invention provides application of the expression inhibitor of the Adra1a gene in promoting increase of white fat cell number, volume, cell proliferation and differentiation in vivo.

Furthermore, the invention provides the application of the expression inhibitor of the Adra1a gene in preparing the medicines for increasing white fat cells, increasing the volume, proliferating and differentiating the cells in vivo

The application of the Adra1a gene or the protein coded by the gene or the Adra1a gene expression promoter in improving or promoting the expression amount of the brown fat marker gene also belongs to the protection scope of the invention, and concretely, the brown fat marker gene comprises Ucp1, Fndc5, Prdm16, Pgc1 α, Ppar gamma, Fabp4, Adipoq and Cidea.

The invention provides application of an expression inhibitor of Adra1a gene in inhibiting increase of the number and volume of brown adipocytes in vivo or in preparing a medicament for inhibiting proliferation and differentiation of the brown adipocytes.

The invention provides application of Adra1a gene or protein coded by the Adra1a gene in improving or promoting Pgc1 α gene expression quantity through a PI3K-AKT signal pathway.

In a second aspect, the invention also provides the use of the Adra1a gene or its encoded protein, or an Adra1a gene expression promoter for the alleviation or treatment of obesity.

The invention provides application of an Adra1a gene, or a protein coded by the gene, or an expression promoter of an Adra1a gene in preparation of a medicament for relieving or treating obesity.

The Adra1a gene expression promoter refers to any biological or chemical product capable of increasing or over-expressing Adra1a gene expression level. The Adra1a gene expression inhibitor refers to any biological or chemical product capable of reducing or interfering the expression of Adra1a gene.

In a third aspect, the present invention provides a medicament for alleviating or treating obesity, the medicament comprising a biological and/or chemical product capable of promoting increased expression of the Adra1a gene.

In a fourth aspect, the invention provides sgRNA targeting an Adra1a gene, the nucleotide sequence of which is shown in SEQ ID No.45 or SEQ ID No. 46. Experiments of the invention find that after the sgRNA1 with the nucleotide sequence shown in SEQ ID No.44 targets the Adra1a gene, the expression level of the Adra1a gene is reduced to 72% of that of a control, the sgRNA2 with the nucleotide sequence shown in SEQ ID No.45 reduces the expression level of the Adra1a gene to 59% of that of the control, and the sgRNA3 with the nucleotide sequence shown in SEQ ID No.46 reduces the expression level of the Adra1a gene to 55% of that of the control, so that the targeting efficiency of the sgRNA2 and the sgRNA3 is higher than that of other sgRNAs.

The CRISPR/Cas9 targeting vector containing the sgRNA belongs to the protection scope of the invention.

The invention has the excellent effects that a novel gene Adra1a for regulating the browning of white fat is found for the first time in the field, an overexpression vector and a knockout vector of the gene are constructed at the cellular level, the browning of the white fat of a mouse regulated by the Adra1a gene is verified from the positive and negative aspects, an Adra1a overexpression mouse model is prepared, the conversion of the white fat of the mouse to the brown fat can be regulated and controlled by the Adra1a gene verified at the individual level, the invention further discloses an action mechanism of regulating the browning of the white fat by the Adra1a gene, and the gene influences the expression of the brown fat marker gene Pgc1 α gene through a PI3K-AKT signal channel and further influences the browning of the white fat, so that the invention provides a novel application of the Adra1a gene in regulating the browning of the white fat, and has practical value in the aspect of preparing medicaments for effectively relieving and treating obesity.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, experimental materials, reagents, instruments and the like used in the examples of the present invention are commercially available, and unless otherwise specified, technical means used in the examples are conventional means well known to those skilled in the art.