阅读说明：本技术 班氏跳小蜂葡糖脱氢酶gld基因及其扩增用引物组合和克隆方法 (Brachychiton rupestris glucose dehydrogenase GLD gene and primer combination and cloning method for amplification thereof ) 是由 张娟 黄�俊 吕要斌 唐娅媛 李明江 于 2019-09-29 设计创作，主要内容包括：本发明提供了班氏跳小蜂葡糖脱氢酶GLD基因及其扩增用引物组合和克隆方法,属于分子生物学以及基因克隆技术领域。所述GLD基因的核苷酸序列如SEQ ID No.6所示。该基因的获得可以进一步研究该基因在班氏跳小蜂细胞中的组成及基因结构,并为其在班氏跳小蜂生殖生理中的作用和功能区域位置,以及与其他物种的区别提供理论依据。(The invention provides a bombesi hopping small bee glucose dehydrogenase GLD gene and a primer combination and a cloning method for amplification thereof, belonging to the technical field of molecular biology and gene cloning. The nucleotide sequence of the GLD gene is shown as SEQ ID No. 6. The acquisition of the gene can further research the composition and the gene structure of the gene in the Brachypodium semifasciatus cell and provide a theoretical basis for the function and the functional region position of the gene in the reproduction physiology of the Brachypodium semifasciatus and the difference with other species.)

1. A bumblebee glucose dehydrogenase GLD gene is characterized in that the nucleotide sequence of the GLD gene is shown as SEQ ID No. 6.

2. The primer combination for amplification of the bombesi bracteata glucose dehydrogenase GLD gene comprises an upstream primer and a downstream primer, and is characterized in that the upstream primer comprises A-Gld-P1F, and the nucleotide sequence of the upstream primer is shown as SEQ ID No. 1; the downstream primer comprises A-Gld-P2R, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2.

3. The primer combination of claim 2, wherein the forward primer further comprises a-Gld-P2F, the nucleotide sequence of which is shown in SEQ ID No. 3; the downstream primer also comprises A-Gld-P1R, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 4.

4. The primer combination of claim 2, wherein the forward primer further comprises a-Gld-P2F, the nucleotide sequence of which is shown in SEQ ID No. 3; the downstream primer also comprises A-Gld-P1.1R, and the nucleotide sequence of the downstream primer is shown as SEQID No. 5.

5. A method for cloning a bumblebee glucose dehydrogenase GLD gene is characterized by comprising the following steps:

(1) extracting genome DNA of the Brachypodus baeri to obtain template DNA for amplification;

(2) performing PCR amplification on the template DNA for amplification by using the primer combination according to any one of claims 2 to 4 to obtain an amplification product;

(3) sequencing the amplification product to obtain a bumblebee glucose dehydrogenase GLD gene sequence.

6. The method of claim 5, wherein the PCR amplification of step (2) comprises reaction A and reaction B;

the reaction A is as follows: performing PCR amplification on the template DNA for amplification by using an upstream primer A-Gld-P1F and a downstream primer A-Gld-P1R;

the reaction B is as follows: and carrying out PCR amplification on the template DNA for amplification by using an upstream primer A-Gld-P2F and a downstream primer A-Gld-P2R.

7. The method of claim 6, wherein the enzymes used in reaction A and reaction B are independently high fidelity DNA polymerases.

8. The method of claim 5, wherein the PCR amplification of step (2) comprises reaction A' and reaction B;

the reaction A' is as follows: performing PCR amplification on the template DNA for amplification by using an upstream primer A-Gld-P1F and a downstream primer A-Gld-P1.1R;

the reaction B is as follows: and carrying out PCR amplification on the template DNA for amplification by using an upstream primer A-Gld-P2F and a downstream primer A-Gld-P2R.

9. The method of claim 8, wherein the enzyme used in reaction A' is Taq enzyme.

Technical Field

The invention relates to the technical field of molecular biology and gene cloning, in particular to a bumblebee glucose dehydrogenase GLD gene and a primer combination and a cloning method for amplification thereof.

Background

Glucose dehydrogenase (GLD) is a small peptide molecule with diverse structure and function, the molecular weight is about 2Kbp, and the isoelectric point is between 5 and 7. The gene is widely distributed in arthropods, in the egg and larva stage, in a plurality of epidermis derived tissues of insects, including some body wall reproductive organs; however, at the adult stage, the gene is only conserved in certain reproductive organs, such as the fertilized sac. In the case of the parasitic female bee, the number of the absorbed and released sperms is closely related to the number of the fertilized eggs. The GLD gene can improve the fertility of females by affecting sperm storage and release in the fertilized sac.

The Apis gracilis Afenasii bambawalei is a dominant species parasitic wasp of an important exotic invasive pest, namely Phenacoccus solenopsis of Hibiscus solenopsis. At present, a plurality of technical bottlenecks exist in the indoor mass breeding and reproduction of the small bombesi bangesii population, wherein population degeneration and partial male offspring are important factors for restricting the research and application of the wasp. And the fertilization capability of the female bees is improved, and the problems can be effectively solved.

At present, no relevant research report about the GLD gene of the Brachypodium banseri exists, the sequence about the GLD genes of other parasitic wasps in the NCBI database is also a predicted sequence, and no accurate sequence uploading record and relevant literature report exist.

Disclosure of Invention

In view of the problems of the background art, the present invention aims to provide a bumblebee glucose dehydrogenase GLD gene, a primer combination for amplification thereof, and a cloning method.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a bumblebee glucose dehydrogenase GLD gene, and the nucleotide sequence of the GLD gene is shown in SEQ ID No. 6.

The invention also provides a primer combination for amplification of the bumblebee glucose dehydrogenase GLD gene, which comprises an upstream primer and a downstream primer, wherein the upstream primer comprises A-Gld-P1F, and the nucleotide sequence of the upstream primer is shown as SEQ ID No. 1; the downstream primer comprises A-Gld-P2R, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2.

Preferably, the upstream primer also comprises A-Gld-P2F, and the nucleotide sequence of the upstream primer is shown as SEQ ID No. 3; the downstream primer also comprises A-Gld-P1R, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 4.

Preferably, the upstream primer also comprises A-Gld-P2F, and the nucleotide sequence of the upstream primer is shown as SEQ ID No. 3; the downstream primer also comprises A-Gld-P1.1R, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 5.

The invention provides a method for cloning a bumblebee glucose dehydrogenase GLD gene, which comprises the following steps:

(1) extracting genome DNA of the Brachypodus baeri to obtain template DNA for amplification;

(2) carrying out PCR amplification on the template DNA for amplification by using the primer combination to obtain an amplification product;

(3) sequencing the amplification product to obtain a bumblebee glucose dehydrogenase GLD gene sequence.

Preferably, the PCR amplification in step (2) comprises a reaction A and a reaction B;

the reaction A is as follows: performing PCR amplification on the template DNA for amplification by using an upstream primer A-Gld-P1F and a downstream primer A-Gld-P1R;

the reaction B is as follows: and carrying out PCR amplification on the template DNA for amplification by using an upstream primer A-Gld-P2F and a downstream primer A-Gld-P2R.

Preferably, the enzymes used in reaction A and reaction B are independently high fidelity DNA polymerases.

Preferably, the PCR amplification in step (2) comprises reaction A' and reaction B;

the reaction A' is as follows: performing PCR amplification on the template DNA for amplification by using an upstream primer A-Gld-P1F and a downstream primer A-Gld-P1.1R;

the reaction B is as follows: and carrying out PCR amplification on the template DNA for amplification by using an upstream primer A-Gld-P2F and a downstream primer A-Gld-P2R.

Preferably, the enzyme used in reaction A' is Taq enzyme.

Has the advantages that: the invention provides a bombesi saccharum glucose dehydrogenase GLD gene, a primer combination for amplification and a cloning method thereof. The nucleotide sequence of the GLD gene is shown as SEQ ID No. 6. The acquisition of the gene can further research the composition and the gene structure of the gene in the Brachypodium semifasciatus cell and provide a theoretical basis for the function and the functional region position of the gene in the reproduction physiology of the Brachypodium semifasciatus and the difference with other species.

Drawings

FIG. 1 is a 1% gel electrophoresis of the CDS region PCR product of the GLD gene of P.bambusae of example 1.

Detailed Description

The invention provides a bumblebee glucose dehydrogenase GLD gene, and the nucleotide sequence of the GLD gene is shown in SEQ ID No. 6.

According to the invention, the CDS region of the bumblebee GLD gene is predicted, then a primer is designed according to the predicted sequence, and the expression sequence of the bumblebee GLD gene (including the complete CDS region sequence) is obtained through PCR amplification cloning. The acquisition of the gene can further research the composition and the gene structure of the gene in the Brachypodium semifasciatus cell and provide a theoretical basis for the function and the functional region position of the gene in the reproduction physiology of the Brachypodium semifasciatus and the difference with other species.

The invention provides a primer combination for amplification of bumblebee glucose dehydrogenase GLD gene, which comprises an upstream primer and a downstream primer, wherein the upstream primer comprises A-Gld-P1F, and the nucleotide sequence of the upstream primer is shown as SEQ ID No. 1; the downstream primer comprises A-Gld-P2R, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 2. In the invention, the A-Gld-P1F is designed based on a part within 130bp of the front section of the initiation codon; the A-Gld-P2R is designed based on the exon part 130bp after TAA.

The upstream primer of the invention preferably also comprises A-Gld-P2F, and the nucleotide sequence of the upstream primer is shown as SEQ ID No. 3; the downstream primer preferably also comprises A-Gld-P1R or A-Gld-P1.1R, and the nucleotide sequence of the A-Gld-P1R is shown as SEQ ID No. 4; the nucleotide sequence of the A-Gld-P1.1R is shown as SEQ ID No. 5. In the invention, the A-Gld-P2F and the A-Gld-P2R can form a new primer pair; the A-Gld-P1R or the A-Gld-P1.1R can form a new primer pair with A-Gld-P1F. Because the GLD gene segment is longer, the addition of the intermediate primer (A-Gld-P2F, A-Gld-P1R or A-Gld-P1.1R) can carry out segmented amplification on the GLD gene, thereby being beneficial to amplification to obtain a more complete and accurate GLD gene sequence.

The invention also provides a method for cloning the bumblebee glucose dehydrogenase GLD gene, which comprises the following steps:

(1) extracting genome DNA of the Brachypodus baeri to obtain template DNA for amplification;

(2) carrying out PCR amplification on the template DNA for amplification by using the primer combination to obtain an amplification product;

(3) sequencing the amplification product to obtain a bumblebee glucose dehydrogenase GLD gene sequence.

The invention firstly extracts the genome DNA of the Brachypodium bambusicola to obtain the template DNA for amplification. The method for extracting genome DNA of the Brachypodium bambusicola is not particularly limited and can be performed by conventional operations in the field.

After obtaining the template DNA for amplification, the invention uses the primer combination provided above to perform PCR amplification on the template DNA for amplification, and obtains an amplification product.

In the present invention, the PCR amplification preferably comprises reaction a and reaction B; the reaction A is as follows: performing PCR amplification on the template DNA for amplification by using an upstream primer A-Gld-P1F and a downstream primer A-Gld-P1R; the reaction B is as follows: and carrying out PCR amplification on the template DNA for amplification by using an upstream primer A-Gld-P2F and a downstream primer A-Gld-P2R.

In a preferred embodiment of the present invention, the enzymes used in reaction A and reaction B are independently preferably high fidelity DNA polymerases. In the present invention, the reaction system of the reaction a is preferably: mu.L of template, 2. mu.L each of 10. mu. mol/L of upstream primer A-Gld-p1F and downstream primer A-Gld-p1R, 2X Phata Max Master Mix 25. mu.L, and the reaction system was made up to 50. mu.L with PCR water; the procedure for the reaction a is preferably: 1 cycle, denaturation at 95 ℃ for 2 min; 30 cycles of denaturation at 94 ℃ for 15s, annealing at 59 ℃ for 15s, and extension at 72 ℃ for 35 s; 1 cycle, extension at 72 ℃ for 5 min; keeping the temperature at 4 ℃. The reaction system of the reaction B is preferably: mu.L of template, 2. mu.L each of 10. mu. mol/L of forward primer A-Gld-p2F and reverse primer A-Gld-p2R, 2X Phata Max MasterMix 25. mu.L, and the reaction system was made up to 50. mu.L with PCR water. The procedure for the reaction B is preferably: 1 cycle, denaturation at 95 ℃ for 2 min; 30 cycles of denaturation at 94 ℃ for 15s, annealing at 58 ℃ for 15s, and extension at 72 ℃ for 35 s; 1 cycle, extension at 72 ℃ for 5 min; keeping the temperature at 4 ℃.

In another preferred embodiment of the present invention, the PCR amplification comprises reaction a' and reaction B; the reaction A' is as follows: performing PCR amplification on the template DNA for amplification by using an upstream primer A-Gld-P1F and a downstream primer A-Gld-P1.1R; the reaction B is as follows: and carrying out PCR amplification on the template DNA for amplification by using an upstream primer A-Gld-P2F and a downstream primer A-Gld-P2R.

In the present invention, the enzyme used in the reaction A' is preferably ordinary Taq enzyme. In the present invention, the reaction system of the reaction a' is preferably: mu.L of template, 2. mu.L each of 10. mu. mol/L of upstream primer A-Gld-p1F and downstream primer A-Gld-p1.1R, and 2X Phata Max Master Mix 25. mu.L, and the reaction system was supplemented to 50. mu.L with PCR water; the procedure for the reaction a is preferably: 1 cycle, denaturation at 95 ℃ for 2 min; 30 cycles of denaturation at 94 ℃ for 15s, annealing at 59 ℃ for 15s, and extension at 72 ℃ for 35 s; 1 cycle, extension at 72 ℃ for 5 min; keeping the temperature at 4 ℃.

After the amplification product is obtained, the amplification product is sequenced to obtain the bumblebee glucose dehydrogenase GLD gene sequence. The present invention is not particularly limited to specific sequencing methods, and any method can be used as is conventional in the art.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.