CN113186213A

CN113186213A - Mulberry spermidine synthetase gene MnSPDS2 and application thereof

Info

Publication number: CN113186213A
Application number: CN202110585152.2A
Authority: CN
Inventors: 刘丹; 邱长玉; 林强; 韦伟; 李韬; 曾燕蓉; 朱方容; 张朝华; 唐燕梅; 崔秋英
Original assignee: Guangxi Zhuang Autonomous Region Sericulture Technology Promotion Master Station
Current assignee: Guangxi Zhuang Autonomous Region Sericulture Technology Promotion Master Station
Priority date: 2021-03-11
Filing date: 2021-05-27
Publication date: 2021-07-30

Abstract

The invention discloses an application of a mulberry spermidine synthase gene MnSPDS2 in detecting drought resistance, which comprises the following steps: the nucleotide sequence of a mulberry spermidine synthetase gene MnSPDS 2; the invention further claims a preparation method of the mulberry spermidine synthase gene MnSPDS2, application of the mulberry spermidine synthase gene MnSPDS2, a fluorescent quantitative detection primer group of the mulberry spermidine synthase gene MnSPDS2, a mulberry spermidine synthase MnSPDS2 and a method for detecting drought resistance of mulberries. The invention clones the mulberry spermidine synthase gene MnSPDS2 and preliminarily learns the functions of the mulberry spermidine synthase gene MnSPDS2 through an adversity stress experiment, thereby laying a foundation for the improvement of the stress resistance of the mulberry.

Description

Mulberry spermidine synthetase gene MnSPDS2 and application thereof

Technical Field

The invention relates to the technical field of molecular biology. More specifically, the invention relates to application of a mulberry spermidine synthase gene MnSPDS2 in detecting drought resistance.

Background

Mulberry is an important economic crop, belongs to Morus of Moraceae, and is planted in different places of China. The mulberry leaves are not only used for silkworm breeding, but also contain a plurality of beneficial components, such as phenolic substances, alkaloid, polysaccharide and the like. DNJ and polysaccharide in mulberry have the function of reducing blood sugar. Moreover, the mulberry is also an important ecological forest tree species, can be planted in arid and saline-alkali areas, can be used for treating stony desertification and saline-alkali lands, and can also be used for repairing heavy metal polluted soil, so that the mulberry can more fully exert the value by improving the tolerance of the mulberry to adversity stress. Spermidine (SPD) can be involved as a signaling molecule in plant response to stress. In the biosynthesis of SPD, spermidine synthase (SPDS) is a key enzyme catalyzing putrescine to synthesize SPD. So far, no relevant research and report on whether spermidine can induce mulberry to generate resistance to adversity stress is found. The invention clones and identifies the SPDS gene of the mulberry, preliminarily knows the SPDS function of the mulberry through an adversity stress treatment experiment, and lays a foundation for the improvement of the stress resistance of the mulberry.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

Still another object of the present invention is to provide a mulberry spermidine synthase gene MnSPDS2, which can screen out mulberry seedlings with stress resistance, especially can screen out mulberry seedlings with drought resistance.

To achieve these objects and other advantages in accordance with the present invention, there is provided a mulberry spermidine synthase gene mnsspds 2 characterized in that the nucleotide sequence of the mulberry spermidine synthase gene mnsspds 2 is shown as SEQ ID No. 1.

The invention further claims a preparation method of the mulberry spermidine synthetase gene MnSPDS2, which comprises the following steps:

a. extracting total RNA from mulberry leaves;

b. the total RNA is reversely transcribed by utilizing an upstream primer MnSPDS2-F and a downstream primer MnSPDS2-R to obtain a mulberry spermidine synthetase gene MnSPDS 2.

The invention further claims the application of the mulberry spermidine synthetase gene MnSPDS2, and the mulberry spermidine synthetase gene MnSPDS2 is used for detecting the drought resistance of mulberries.

The invention further claims a fluorescent quantitative detection primer group of the mulberry spermidine synthetase gene MnSPDS2, which comprises an upstream primer MnSPDS2-F and a downstream primer MnSPDS2-R, and the sequences of the primers are respectively shown as SEQIDNO.7 and SEQIDNO.8.

The invention further claims a mulberry spermidine synthetase MnSPDS2, wherein the amino acid sequence of the mulberry spermidine synthetase MnSPDS2 is shown as SEQ ID NO. 2.

The invention further claims a method for detecting the drought resistance of the mulberry, which comprises the following steps:

selecting mulberry with consistent growth vigor, and immersing the root of the mulberry in Hoagland nutrient solution containing 5% of PEG6000 for a certain time;

taking the 3 rd to 5 th leaves of the mulberry which are completely unfolded, and mixing the leaves taken by each mulberry into a sample;

extracting a cDNA solution containing a mulberry spermidine synthetase gene MnSPDS2 through leaves;

preparing a real-time fluorescent quantitative PCR reaction system containing a cDNA solution, placing the system in a fluorescent quantitative PCR instrument, and setting a reaction program as follows: after 30 cycles, the temperature was controlled to 72 ℃ and the extension was carried out for 7min, wherein one cycle was: annealing at 55 deg.C for 30s, and extending at 72 deg.C for 2 min; before circulation, the temperature is controlled to be 95 ℃ for pre-denaturation for 5 min;

obtaining the Ct value of the mulberry spermidine synthetase gene MnSPDS2 after the reaction is finished;

configuring a real-time fluorescent quantitative PCR reaction system containing a mulberry A3 gene as a control to obtain the Ct value of a control gene A3 and the Ct values of a target gene mulberry spermidine synthetase gene MnSPDS2 and a control gene A3, and using 2^-ΔΔCtThe method calculates the relative expression quantity of the gene MnSPDS 1.Δ Ct ═ (Ct)_{Target gene}-Ct_A3)；ΔΔCt＝(ΔCt_{Treatment of}-ΔCt_Untreated). Let "2^{Control of-. DELTA.Ct}1 ", if" 2^{-Delta Ct target gene}>1', the expression of the gene is up-regulated, and the detected mulberry plant has stronger drought resistance; otherwise, the expression of the gene is reduced, and the drought disease resistance of the mulberry plant is weak.

Preferably, the real-time fluorescent quantitative PCR reaction system is prepared by mixing 2. mu.l of 25-50 ng/. mu.l cDNA solution, 10. mu.l of 2 XSSYBR Premix, 2. mu.l of Rox Reference Dye with a concentration of 25. mu.M, 1. mu.l of MnSPDS1-F with a concentration of 10. mu.M, 1. mu.l of MnSPDS1-R with a concentration of 10. mu.M,4 μ l of ddH₂And (4) mixing the materials.

The invention at least comprises the following beneficial effects:

firstly, the invention clones the mulberry spermidine synthetase genes MnSPDS1 and MnSPDS2 for the first time.

Secondly, the invention discovers that the mulberry spermidine synthetase gene MnSPDS2 gene may play a main role in the drought stress process of the mulberry for the first time.

Thirdly, the invention lays a foundation for stress resistance research of the mulberry and stress resistance genetic breeding of the mulberry.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a gel diagram of electrophoresis detection of mulberry SPDS gene clone in one embodiment of the present invention;

FIG. 2 is a diagram of the alignment analysis of MnSPDS protein sequences in another embodiment of the present invention;

FIG. 3 is a diagram of the evolutionary analysis of mulberry and other species SPDS according to another embodiment of the present invention;

FIG. 4 is a schematic diagram of the structure of SPDS gene of mulberry in another embodiment of the present invention;

FIG. 5 is an analysis chart of the expression patterns of mulberry spermidine synthase gene MnSPDS1 and mulberry spermidine synthase gene MnSPDS2 in Sichuan mulberry leaves after treatment of pathogenic bacteria in another technical scheme of the invention.

The reference numerals are specifically:

FIG. 1: m: 5000 DNA Marker; 1: MnSPDS 1; 2: MnSPDS 2;

FIG. 4: 4: 5' UTR; 5: 3' UTR; 6: a transcription initiation site; 7: an AdoMet-MTase domain.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

Obtaining of ribosomal RNA gene of spermidine synthetase from mulberry

Randomly picking mulberry leaves in a mulberry field, cleaning the surfaces of the mulberry leaves by using sterile water, airing, adding liquid nitrogen into a mortar, quickly grinding the mulberry leaves into powder, and separating and purifying RNA according to Omniplant RNA Kit (DNase I) instructions, wherein the method comprises the following specific steps:

1. plant tissue was rapidly ground to a powder in liquid nitrogen, 600. mu.l buffer RL was added and vortexed to lyse it thoroughly.

2. Transferring the liquid obtained in the step 1 into a filter column filled with a collecting tube, centrifuging at 12000rpm for 2 minutes, and transferring the liquid in the collecting tube into a new centrifugal tube.

3. And (3) adding 0.5 time volume of absolute ethyl alcohol into the lysate obtained in the step (2), and quickly and uniformly mixing.

4. Transferring the solution obtained in the previous step into an adsorption column filled into a collecting tube, centrifuging at 12000rpm for 15 seconds, discarding waste liquid, and replacing the adsorption column into the collecting tube again.

5. Mu.l buffer RW1 was added to the adsorption column, centrifuged at 12000rpm for 1 min, the waste liquid was discarded, and the adsorption column was replaced in the collection tube.

6. Preparing DNase I mixed solution: mu.l of RNase-Free Water was taken, and 8. mu.l of 10 × Reaction Buffer and 20. mu.l of DNase I (1U/. mu.l) were added thereto, and mixed well to prepare a Reaction solution having a final volume of 80. mu.l.

7. Add 80. mu.l DNase I mixture directly to the adsorption column and incubate for 15 min at 20-30 ℃.

8. Mu.l Buffer RW1,12000rpm was added to the adsorption column and centrifuged for 1 min, the waste liquid was discarded, and the adsorption column was replaced in the collection tube.

9. To the adsorption column, 500. mu.l of Buffer RW2 was added, centrifuged at 12000rpm for 15 seconds, and the waste liquid was discarded.

10. Step 9 is repeated.

11. The column was returned to the collection tube, centrifuged at 12000rpm for 1 minute, and the column was left at room temperature for several minutes.

12. The adsorption column was placed in a new centrifuge tube, 30-50. mu.l RNase-Free Water was added to the middle of the adsorption membrane, and the mixture was left at room temperature for 1 minute and centrifuged at 12000rpm for 1 minute, and the resulting RNA solution was stored at-70 ℃ to prevent degradation.

Synthesis of mulberry spermidine synthetase cDNA

The HiFiScript cDNA Synthesis Kit is used for reverse transcription of RNA into cDNA, and the reaction system is as follows:

TABLE 1 reaction System parameters for reverse transcription of RNA into cDNA

Reaction conditions are as follows: incubating at 42 deg.C for 30-50 min, incubating at 85 deg.C for 5min, cooling on ice, and storing at-20 deg.C for a long time.

Obtaining of target genes of mulberry spermidine synthetase gene MnSPDS1 and mulberry spermidine synthetase gene MnSPDS2

TABLE 2 reaction systems obtained with MnSPDS1 and MnSPDS2 genes of interest

The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; annealing at 55 deg.C for 30s, extending at 72 deg.C for 2min, and performing 30 cycles; extension at 72 ℃ for 7 min. The gene of interest was subjected to 1% agarose gel electrophoresis as shown in FIG. 1. Inserting the SPDS gene into a peasy-blunt vector to construct a recombinant vector, transferring into escherichia coli, picking out positive clones, sequencing by Shanghai's chemical company, wherein the length of Mn002704 is 1188bp, the length of Mn027798 is 1005bp, the length of Mn027798 is 334 amino acids (figure 2) is consistent with the length of the presumed fragment and the sequence is consistent, which indicates that the coding regions of 2 mulberry SPDS genes have been successfully cloned and are respectively named as MnSPDS1 and MnSPDS 2. The nucleotide sequence of the mulberry spermidine synthetase gene MnSPDS1 is shown as SEQ ID NO.1, and the amino acid sequence of the mulberry spermidine synthetase MnSPDS2 is shown as SEQ ID NO. 2; the nucleotide sequence of the mulberry spermidine synthetase gene MnSPDS1 is shown as SEQ ID NO.3, and the amino acid sequence of the mulberry spermidine synthetase MnSPDS2 is shown as SEQ ID NO. 4.

Bioinformatics analysis of Mulberry spermidine synthetase MnSPDS2

The mulberry SPDS protein sequence is compared and analyzed with the reported SPDS protein sequences of other species by using ClustalX2.1 local software, and the software MEGA5 is used to obtain the evolutionary map of the mulberry SPDS protein sequence and the SPDS protein sequences of other species (figure 3), and in figure 3, the sequences are shown as follows: triangles show the SPED of mulberry, AtSPDS1(AEE30436.2), AtSPDS2 (NP-973900.2), AtSPDS3(BAB08415.1), AtSPDS4(AAM64782.1), OsSPDS3(ACD87085.1), OsSPDS1(BAD30581.1), SlSPDS (NP-001234493.1), StOsSPDS (CAC51027.1), PsSPDS (APO15246.1), OeSPDS (ACZ73829.1), ShSPDS (AUT32283.1), MdSPDS (AIZ03432.1), TaSPDS (AEL33692.1), NtSPDS (AIZ70319.1), MnSPDS1(EXB95030.1), MnSPDS1 (EXB95. 1. the mulberry spermidine synthase MnSPDS1 and the Arabidopsis thaliana SPDS2 are clustered with one protein, indicating that these two proteins are closely related to Arabidopsis thaliana.

Prediction and analysis of related properties of amino acid sequence of mulberry spermidine synthetase MnSPDS2

The amino acid sequence of the mulberry SPDS is subjected to conservative domain online prediction through NCBI (national center for Biotechnology information) conservative domain analysis, the result shows that the mulberry SPDS1 protein and the mulberry SPDS2 protein both belong to AdoMet-MTase superfamily, Class I is subjected to signal peptide, chloroplast transit peptide and subcellular localization online prediction respectively, the result shows that the mulberry MnSPDS family protein does not contain signal peptide and chloroplast transit peptide, the subcellular localization online prediction result shows that the mulberry spermidine synthase MnSPDS2 is localized in Golgi, the structural schematic diagram of the mulberry spermidine synthase gene M nSPDS (figure 4) is drawn according to bioinformatics analysis, and the marker 4 in the diagram is 5' UTR; marker 5 is 3' UTR; marker 6 is the transcription start site; marker 7 is the AdoMet-MTase domain.

Expression level of mulberry spermidine synthase gene MnSPDS2 under adversity stress

Selecting mulberry seedlings with consistent morphological growth vigor, immersing the roots of the mulberry seedlings in Hoagland nutrient solution of 5% PEG6000 for drought treatment, and sampling after 0, 8, 24, 32 and 48 hours. And (3) taking the completely stretched 3-5 th leaves, mixing the leaves taken by each plant into a sample, and setting 3 repeats. Leaves of treated seedlings were received directly into liquid nitrogen for total RNA extraction. Relative expression of genes was obtained by qPCR and experimental results are shown as mean ± SD.

Taking the cDNA of the Sichuan mulberry as a template, and adopting a qPCR system as follows:

TABLE 3 qPCR System parameters

The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; annealing at 55 deg.C for 30s, extending at 72 deg.C for 2min, and performing 30 cycles; extension at 72 ℃ for 7 min. Ct values of the objective mulberry spermidine synthetase gene MnSPDS2 and the control gene A3 were obtained after the reaction was completed, and 2 was used^-ΔΔCtThe method calculates the relative expression quantity of the mulberry spermidine synthetase gene MnSPDS 2.Δ Ct ═ (Ct)_{Target gene}-Ct_A3)；ΔΔCt＝(ΔCt_{Treatment of}-ΔCt_Untreated). Let "2^{Control of-. DELTA.Ct}1 ", if" 2^{-Delta Ct target gene}>1', the expression of the gene is up-regulated, whereas the expression of the gene is down-regulated.

The transcript levels of 2 SPDS genes in the leaves of morus tinctorius were analyzed by qPCR. When plants are in drought stress, the expression quantity of 2 mulberry spermidine synthase genes MnSPDS is changed. The expression level of the mulberry spermidine synthetase gene MnSPDS2 has a peak value when the mulberry spermidine synthetase gene is subjected to drought stress for 32h, and then the expression level is reduced, which shows that the mulberry spermidine synthetase gene MnSPDS2 has a certain effect in the drought stress process of the mulberry. And after drought stress, the peak value of the expression quantity of the mulberry spermidine synthetase gene MnSPDS1 is 3.2 times of that of the mulberry spermidine synthetase gene MnSPDS2 (figure 5), which shows that the mulberry spermidine synthetase gene MnSPDS2 plays a main role in the mulberry drought stress process compared with the mulberry spermidine synthetase gene MnSPDS1, and the mulberry spermidine synthetase gene MnSPDS2 and primers thereof can be used for detecting the drought resistance after the mulberry is treated with 5% PEG6000 Hoagland nutrient solution for 32 hours.

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor for which the invention may be embodied with additional modifications as would be readily apparent to those skilled in the art, and the invention is therefore not limited to the details given herein and to the embodiments shown and described without departing from the generic concept as defined by the claims and their equivalents.

SEQUENCE LISTING

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Application of mulberry spermidine synthase gene MnSPDS in detection of gray mold resistance

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Claims

1. A mulberry spermidine synthase gene MnSPDS2 is characterized in that the nucleotide sequence of the mulberry spermidine synthase gene MnSPDS2 is shown as SEQ ID No. 1.

2. The method for preparing the mulberry spermidine synthase gene MnSPDS2 according to claim 1, comprising the steps of:

a. extracting total RNA from mulberry leaves;

3. The use of the mulberry spermidine synthase gene MnSPDS2 according to claim 1, wherein the mulberry spermidine synthase gene MnSPDS2 is used for detecting the drought resistance of mulberries.

4. A fluorescent quantitative detection primer group of a mulberry spermidine synthetase gene MnSPDS2 is characterized by comprising an upstream primer MnSPDS2-F and a downstream primer MnSPDS2-R, wherein the sequences of the upstream primer MnSPDS2-F and the downstream primer MnSPDS2-R are respectively shown as SEQ ID No.7 and SEQ ID No. 8.

5. A mulberry spermidine synthase MnSPDS2 is characterized in that the amino acid sequence of the mulberry spermidine synthase MnSPDS2 is shown as SEQ ID No. 2.

6. A method for detecting drought resistance of mulberry is characterized by comprising the following steps:

7. The method for detecting drought resistance of mulberry trees as claimed in claim 6, wherein the real-time fluorescence quantitative PCR reaction system is prepared by mixing 2. mu.l of 25-50 ng/μ l cDNA solution, 10. mu.l of 2 XSSYBR Premix, 2. mu.l of Rox Reference Dye with 25 μ M concentration, 1. mu.l of 10 μ M concentrationMnSPDS1-F, 1. mu.l of MnSPDS1-R with a concentration of 10. mu.M, 4. mu.l of ddH₂And (4) mixing the materials.