CN108441544A - The detection of expression method and amplimer of sugar grass Sucrose phosphate synthase gene - Google Patents

Abstract

The present invention is with sugar grass LTR102 (female parent), sorghum 654 (male parent) and the F that they hybridize₅The Gao Tang groups in generation are examination material with low sugar group, in seedling stage, jointing stage, heading stage and maturity period measure different times sugar grass LTR102, sorghum 654 and F₅The activity of cane sugar content and SPS in Gao Tang groups and low sugar colony leaves, determine sugar grass SPS activity and cane sugar content correlation.Qualitative analysis is carried out to above-mentioned examination material different development stage SPS gene expressions, determines the otherness of different times SPS gene expressions.Difference of the above-mentioned examination material of quantitative analysis in different growth period SPS gene expression amounts, and SPS gene expressions relative concentration and cane sugar content are subjected to correlation analysis, the result shows that SPS gene expressions relative concentration and cane sugar content are significantly correlated, result according to the present invention establishes a kind of sugar grass SPS gene expression rapid detection methods, to achieve the purpose that by quickly detecting determining the best harvest time and the screening high sugar content new material of sugar grass.

Description

Expression Detection Method and Amplification Primer of Sweet Sorghum Sucrose Phosphate Synthase Gene

technical field

The invention belongs to the field of biotechnology, and relates to a sweet sorghum sucrose phosphate synthase (SPS) gene, in particular to an expression detection method and an amplification primer for the sweet sorghum sucrose phosphate synthase gene.

Background technique

Sucrose is the main product of photosynthesis in higher plants, the main form of carbon transport, the main form of carbohydrate accumulation and storage, and the main substrate of "sink metabolism". It has a special position in plant sugar metabolism (Farrar et al. ,2000). Especially for sweet sorghum, as a good raw material for sugar industry, production of alcohol, fiber and feed mainly depends on the rich sugar in the stalk, of which 85% is sucrose, 9% glucose, 6% fructose (Gnansounou et al.,2005 ). The use of sweet sorghum sugar to produce ethanol as an alternative energy source has aroused great attention in recent years. To explore the mechanism of sweet sorghum sugar metabolism conversion is an important part of bioenergy research.

Sweet sorghum is a "double-sink" crop, which has a grain bank and a stem bank. Sucrose is the main form of sugar in sweet sorghum stalks. The accumulation of sucrose in stalks is related to the sugar content and yield of sweet sorghum stalks. , and the process of sugar accumulation in stems is inseparable from sucrose metabolism. Sucrose metabolism is regulated by a variety of enzymes. There are three key enzymes regulating sucrose metabolism: Sucrose Phosphate Synthase (Sucrose Phosphate Synthase, E.C.2.4.1.14, SPS), Sucrose Synthase (Sucrose Synthase, E.C.2.4.1.13, SS) and Sucrose Invertase (Invertase, EC3.2.1.26, Inv). Among them, SPS catalyzes the synthesis of sucrose, Inv catalyzes the degradation of sucrose, and SS has the dual properties of decomposing and synthesizing sucrose, and the three jointly catalyze the entry of sucrose into various metabolic pathways. Three enzymes play a pivotal role in the process of plant growth and development, therefore, it is very important to study these enzymes related to sucrose metabolism.

The activity of SPS directly reflects the ability of sucrose synthesis in plants. Analyzing the expression of SPS gene is an effective way to find out the law of sugar accumulation in stems, explain the differences in sugar content among different varieties, and establish a molecular regulation system. However, there is no comprehensive report on the detection methods of SPS gene expression in sweet sorghum.

Contents of the invention

In order to solve the above-mentioned technical problems, the present invention provides a method for detecting the expression of sweet sorghum sucrose phosphate synthase gene and amplification primers, using sweet sorghum LTR102 (female parent), sorghum 654 (male parent) and their hybridized F ₅ generation medium sorghum The sugar population and the F ₅ low sugar population were used as test materials, and the sucrose content and the gene expression of sucrose phosphate synthase (SPS) were detected by high-performance liquid chromatography and fluorescent quantitative PCR technology, and a rapid detection method for screening high-sugar varieties of sorghum was established. method.

Object one of the present invention: use high-performance liquid chromatography and spectrophotometry to measure the activity of sucrose content and SPS in the leaves of sweet sorghum LTR102 (female parent), sorghum 654 (male parent) and _F5 high-sugar population and low-sugar population in different periods , to determine the correlation between sweet sorghum SPS activity and sucrose content.

The second object of the present invention is to qualitatively analyze the expression of SPS gene in different development stages of male parent, female parent and _F5 high-sugar group and low-sugar group by PCR technology, and preliminarily determine the difference of SPS gene expression in different periods.

Object three of the present invention: apply fluorescence quantitative PCR technology, quantitatively analyze sweet sorghum LTR102 (female parent), sorghum 654 (male parent) and F ₅ high-sugar population and low-sugar population the quantitative expression situation of SPS gene in different growth stages, and The correlation analysis between the relative concentration of SPS gene expression and sucrose content was carried out, and the results showed that the relative concentration of SPS gene expression was significantly correlated with sucrose content (R=0.972, P<0.05). According to the experimental results, a reaction system for rapid detection of SPS gene expression was established. and conditions, establish a rapid identification standard for screening high-sugar varieties of sweet sorghum, achieve the purpose of determining the best harvest time and screening high-sugar varieties, and provide technical support for practicing assisted breeding and shortening the breeding cycle.

(1) Construction of F ₅ population:

In the present invention, sweet sorghum LTR102 is used as the female parent, and sorghum 654 is used as the male parent, and ₅ F generations are obtained by crossing, including 87 high-sugar plants and 33 low-sugar plants.

The _F5 generation was divided into a high-sugar group and a low-sugar group. The high-sugar group and the low-sugar group consisted of 87 leaves of high-sugar plants and 33 leaves of low-sugar plants in the _F5 generation, and the enzyme solution and mRNA were extracted.

(2) Determination of sucrose content by high performance liquid chromatography; determination of SPS activity by spectrophotometry, and correlation analysis between sucrose content and SPS activity.

(3) The extraction method of sweet sorghum RNA was established, and the RNA extracted by the improved Trizol method had good integrity and no pollution, and could be used as a template in the next experiment.

(4) Design of primers for sucrose phosphate synthase (SPS) gene: According to the known cDNA sequence of the coding region of sweet sorghum SPS3-1 gene, primers were designed using Oligo6 software and Primer primer software. Named SPS-1, β-Actin is a housekeeping gene.

(5) Qualitative analysis of sweet sorghum sucrose phosphate synthase (SPS) gene expression by PCR technology, the results show that the ideal target fragment can be amplified by using β-Actin and SPS-1 as primers, with clear bands and no bands, The SPS gene can be detected at the seedling stage. With the extension of the growth period, the expression level of the SPS gene increases gradually, and the expression level reaches the maximum at the mature stage. This is consistent with the change rule of sucrose content, which can preliminarily prove that the expression of SPS gene affects the accumulation of sucrose. Fluorescent quantitative PCR detection is performed on the basis of qualitative detection to more accurately understand the change law of the SPS gene.

(6) Quantitative analysis of SPS gene expression was carried out by fluorescent quantitative PCR technology, and the results showed that the sequence of SPS gene expression from high to low was high-sugar group, female parent, low-sugar group and male parent. The change law of SPS gene is basically consistent with the change law of sweet sorghum sucrose content and SPS enzyme activity, and they all reach the maximum at the mature stage, which provides a theory for the selection of sweet sorghum high-sugar varieties and the determination of the best harvest time of sweet sorghum in accordance with.

The present invention is achieved in this way, on the one hand provides sweet sorghum sucrose phosphate synthase gene amplification primers, including primer SPS-1 and primer β-Actin, primer SPS-1 includes SPS-1 forward primer and SPS-1 reverse Primers, nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2; primer β-Actin includes β-Actin forward primer and β-Actin reverse primer, nucleotide sequences are shown in SEQ ID NO : 3 and SEQ ID NO: 4.

Further, the length of the target fragment amplified by the primer SPS-1 is 121bp; the length of the target fragment amplified by the primer β-Actin is 210bp.

On the other hand, the present invention also provides a rapid PCR reagent for detecting the expression of sweet sorghum sucrose phosphate synthase gene, said PCR reagent comprising 10×Buffer 2.5 μl, 25mg/ml MgCl _2.5 μl, 100 μmol/l dNTP 0.5 μl, 5U /μl Taq enzyme 0.5μl, 0.2μmol/l Forward Primer 1.0μl, 0.2μmol/l Reverse Primer 1.0μl, cDNA template 1.0μl, make up 25μl with ddH ₂ O;

The primers in the PCR reagent are primer SPS-1 or primer β-Actin according to claim 1.

Further, the method for performing PCR reaction using the above-mentioned rapid PCR reagent includes the following reaction procedure: pre-denaturation at 94°C for 2 minutes → 32 amplification cycles → extension at 72°C for 10 minutes → storage at 4°C, and the amplification cycle includes the following steps: denaturation at 94°C for 30s →Annealing at 53°C for 20s→Extension at 72°C for 1min 20s.

In another aspect, the present invention also provides a rapid fluorescent quantitative PCR reagent for detecting the expression of sucrose phosphate synthase gene. The volume of the fluorescent quantitative PCR reagent is 20 μl, including: 2×SYBR Premix ExTaq II 10.0 μl, ddH ₂ O 6.0 μl, 0.2μmol/l Forward Primer 0.8μl, 0.2μmol/l Reverse Primer 0.8μl, ROXReference DYE 0.4μl, cDNA template 2.0μl;

The primer in the fluorescent quantitative PCR reagent is the primer SPS-1 or the primer β-Actin according to claim 1.

Further, the method for performing rapid fluorescent quantitative PCR using the above reagents includes the following reaction procedure: pre-denaturation at 95°C for 15 minutes → 35 amplification cycles → melting curve program at 53-95°C; the amplification cycle includes the following steps: denaturation at 94°C for 20s → Anneal at 53°C for 20s → extend at 72°C for 1min 20s.

Compared with the prior art, the present invention has the advantages of:

The primers and expression detection method are applied to the rapid analysis of sorghum sugar content, SPS enzyme activity, and SPS gene expression. In the selection of high sugar content varieties, the elimination of low sugar variety genotypes, the acceleration of breeding process, and the determination of the best harvest time, It has great practical value in terms of saving the experimental land and cost required for breeding, and then overcomes the difficulties of phenotypic identification and the huge workload of assisted selection by common methods, and provides a technology for shortening the breeding cycle. Assure. At the same time, it fills the domestic and foreign gaps in the study of sweet sorghum SPS gene expression-assisted selection.

Description of drawings

Fig. 1 is the detection band (PCR method) of SPS gene expression in the seedling stage of the present invention.

Fig. 2 is the detection band (PCR method) of SPS gene expression in the jointing stage of the present invention.

Fig. 3 is the detection band (PCR method) of SPS gene expression at heading stage of the present invention.

Fig. 4 is the detection band (PCR method) of the mature SPS gene expression of the present invention.

Detailed ways

Referring to Figure 1, Figure 2, Figure 3 and Figure 4, as shown in the figure, Sorghum 654 (male parent) (1,5 band), sweet sorghum LTR102 (female parent) (2,6 band), F ₅ high sugar Gene pool (3,7 bands), F ₅ low sugar gene pool (4,8 bands), Marker (M bands). Band 1-4 is the PCR product amplified with the primer pair β-Actin as the primer, and the fragment length is about 210bp; Band 5-8 is the PCR product amplified with the primer pair SPS-1 as the primer, the fragment length is 121bp .

1. Materials and Methods

1.1 Test material

Sweet sorghum LTR102 (female parent), sorghum 654 (male parent) and their crossed F ₅ individuals.

1.2 Drugs, instruments and equipment

100mmol/L PH7.8 phosphate buffer solution 1%PVP, 0.1mmol/LEDTA, 0.1% resorcinol, UDPG, 100mmol/L fructose hexaphosphate, 100mmol/L fructose, agarose, chloroform, EDTA are domestic analytically pure , Trizol kit, diethyl pyrocarbonate (DEPC), Tris, isopropanol, and QuantiTect Reverse Transcription Kid are all products of Baobio (Dalian) Co., Ltd.

Fluorescence quantitative PCR, temperature gradient PCR, low temperature refrigerated centrifuge, electronic balance, electrophoresis instrument, refrigerator, ultra-low temperature refrigerator microwave oven, micro pipette, ultrapure water instrument.

1.3 The overall design of the experiment

Take the leaves of LTR102 (female parent), 654 (male parent) and F _5th generation individuals at the seedling stage, jointing stage, heading stage, and mature stage respectively → extract mRNA (improved Trizol method) → reverse transcribe into cDNA → sucrose phosphate synthase (SPS) Gene Amplification Primer Design→Application of PCR technology for qualitative analysis of SPS gene expression→Application of fluorescent quantitative PCR technology for quantitative analysis of SPS gene expression→Analysis of the correlation between sugar content and SPS expression→Establishment of rapid detection of SPS gene expression The method → determine the identification method → provide a theoretical basis for the selection of sweet sorghum high sugar varieties and determine the best harvest time.

1.4 Determination of sucrose phosphate synthase (SPS) activity in sweet sorghum

Weigh 1g of the sample, wash it and chop it into pieces, put it in a pre-cooled mortar, add 2.5ml of phosphate buffer, and grind in an ice bath. Grind into a homogenate and pour it into a centrifuge tube, rinse with 2.5ml phosphate buffer, and then pour it into a centrifuge tube together. Centrifuge at 10000xg for 15min, and take the supernatant for later use. Add 50 μl of crude enzyme solution to a test tube of the corresponding number, add 50 μl of buffer, 20 μl of MgCl2, 20 μl of UDPG, 20 μl of fructose-6-phosphate, and shake. 30°C, water bath for 30min. Add 200μl NaOH, cook in boiling water for 10min, and cool down. Add 1.5ml 30% hydrochloric acid, 0.5ml resorcinol. Cool in a water bath at 80°C for 10 minutes. Colorimetric at 480nm.

1.5 Sweet sorghum RNA extraction and cDNA synthesis

1.5.1 Improved Trizol method to extract total RNA

(1) Grind the leaf tissue into powder in liquid nitrogen, add 1ml Trizol reagent to every 50-100mg of tissue, the sample volume should not exceed 100μl and stand at room temperature for 5min.

(2) Add 0.2ml of chloroform to each tube sample, cap the tube tightly, shake the sample vigorously for 15 seconds, and stabilize at room temperature for 5 minutes.

(3) Centrifuge the sample at 12000xg, 4°C for 15min

(4) Take the supernatant into a new tube, add 0.5ml of isopropanol to each tube, mix well, and place at room temperature for 5 minutes.

(5) The sample was centrifuged at 12000xg at 4°C for 10 minutes.

(6) Discard the supernatant, wash the RNA pellet with 1 ml of 75% ethanol, and vortex to mix.

(7) Centrifuge at 12000xg for 5 min at 4°C. (If it is used for reverse transcription PCR, repeat step 6 twice).

(8) Discard the surface matter, dry at room temperature for 5-10 minutes (do not completely dry the precipitate), and dissolve with 30-60 μl of TE.

1.5.2 RNA purity analysis

(1) Take 10 to 20 μl RNA sample from the original mixture, dissolve it in 1.5 ml microcentrifuge tube with 990 or 980 μl RNase-free water, and obtain a 100- or 200-fold diluted RNA sample.

(2) Pipette 1ml of RNase-free water into a clean cuvette and read the absorbance of the blank.

(3) Add the diluted RNA sample into a clean cuvette and read the absorbance at 260, 280 and 230 nm. Use the following formula to determine the RNA concentration of the original sample: RNA μg/μl=A260×33×dilution factor/1000.

1.5.3 RNA integrity analysis

1.0% agarose gel was electrophoresed at 90V for 45 min, and the clarity and integrity of the RNA bands were observed on a gel imager.

1.6 Design of sucrose phosphate synthase (SPS) primers

According to the known cDNA sequence of the coding region of sweet sorghum SPS3-1 gene, primers were designed by using Oligo6 software and Primerprimier software.

1.7 PCR reaction system, reaction procedure and detection method

1.7.1 Establishment of PCR reaction system and reaction program

Use QuantiTect Reverse Transcription Kid for reverse transcription, take 0.2ml Eppendorf tubes, add the following components gDNA Wipeout Buffer 2μl to each tube according to the sample number, the total amount of Template RNA is between 10pg and 1μg, add RNase-free water to make the total reaction volume As large as 14 μl. After adding the sample, put it into a water bath to remove the genomic DNA in the RNA and incubate at 42°C for 10min. Add 1ul of QuantiTect Reverse Transcriptase, 4μl of QuantiTect RT Buffrt, and 1μl of RT Primer Mix to the tube, and the reaction volume reaches 20μl. 42°C water bath for 15 minutes, 95°C for 3 minutes.

The establishment of the reaction system

After the RNA has been reverse-transcribed into cDNA, take a 0.2ml Eppendorf tube, and add the following components to each tube according to the sample number: the reaction system is 25μl, including 10×Buffer 2.5μl, 25mg/ml MgCl ₂ 2.5μl, 100μmol/ldNTP 0.5 μl, 5U/μl Taq enzyme 0.5μl, 0.2μmol/l Forward Primer 1.0μl, 0.2μmol/l ReversePrimer 1.0μl, cDNA template 1.0μl, add 25μl with ddH ₂ O;

Reaction procedure:

Put the above-mentioned Eppendorf tubes loaded with samples into the EN61010-1 PCR amplification instrument for PCR reaction. The reaction program is: pre-denaturation at 94°C for 2 minutes→(denaturation at 94°C for 30s→annealing at 53°C for 20s→extension at 72°C for 1min 20s)32 cycle → extend at 72°C for 10 min → store at 4°C.

1.7.2 Analysis of PCR products

The PCR product was detected by 1% agarose gel electrophoresis, and the amplified product was further analyzed to determine product specificity.

1.8 Real-time quantitative PCR detection of the expression level of sweet sorghum sucrose phosphate synthase (SPS)

1.8.1 Establishment of standard curve

The cDNA was diluted into 5 gradients (22, 24, 25, 26, 27), and the SYBR Green I real-time quantitative PCR reaction of the target gene SPS and the internal reference gene β-actin were carried out respectively, and the standard curve of the two genes was prepared and the amplification efficiency was detected.

1.8.2 Reaction system and conditions of fluorescent quantitative PCR

SYBR fluorescent quantitative PCR reaction system: 20 μl: 2×SYBR Premix ExTaq II 10.0 μl, ddH2O 6.0 μl, Forward Primer 0.8 μl, Reverse Primer 0.8 μl, ROX Reference DYE 0.4 μl, cDNA template 2.0 μl.

Reaction program: pre-denaturation at 95°C for 15min→(denaturation at 94°C for 20s→annealing at 53°C for 20s→extension at 72°C for 1min and 20s) for 35 cycles→melting curve program at 53-95°C. After the reaction, the instrument automatically generates CT (thresholdcycles) values and melting curves picture. Three replicate tubes were set up for each sample, and a template-free negative control was set up at the same time.

Example 1: Correlation analysis between sweet sorghum sucrose phosphate synthase (SPS) activity and sucrose content

In the present invention, by measuring the SPS activity in the leaves of male parents, female parents and their crossed _F5 high-sugar populations and low-sugar populations in different periods, the variation rule of SPS among different populations in different periods is known. The activity of SPS in leaves of parents, F ₅ high-sugar group and low-sugar group increased gradually. At the maturity stage, the activities of the three sweet sorghum varieties, the female parent, the high-sugar population and the low-sugar population, all reached the maximum values of 14.86, 17.63, and 13.76 μmol/hgFW; decline. There was no significant difference in the activities of SPS among the four varieties at the seedling stage, but there was a difference in the activities of SPS among the four varieties from the jointing stage. The SPS activity of the high-sugar group and the low-sugar group showed a linear increase, and the activity growth rate was faster than that of the parents, and the SPS enzyme activity of the high-sugar variety was the highest at the mature stage. The correlation analysis between sucrose and SPS activity in sorghum and sweet sorghum leaves showed that there was a significant positive correlation between sucrose and SPS activity (R=0.763, P<0.01).

Implementation example 2: Analysis of extraction results of sweet sorghum RNA

Agarose gel electrophoresis detection showed that the RNA extracted by the modified Trizol method had three bands, and the brightness of the 28S band in the total RNA was about twice that of the 18S band, indicating that it was less degraded during the extraction process. The integrity of the extracted RNA is good. The 5S band is relatively light, indicating that there is no contamination of RNA, which can be used as a template in the next experiment.

Implementation example 3: sucrose phosphate synthase (SPS) primer design

The primers in Table 1 were designed based on a known cDNA sequence of the coding region of sweet sorghum SPS3-1 gene, using Oligo6 software and Primer primer software. Named as SPS-1, β-Actin is the housekeeping gene, and the lengths of the target fragments amplified by the two pairs of primers are 121bp and 210bp respectively.

Table 1 Primers used for SPS gene amplification

Implementation Example 4: Qualitative analysis of sucrose phosphate synthase (SPS) gene expression

The expression of the SPS gene in the leaves of the seedling stage parents, the _F5 high-sugar group and the low-sugar group is shown in Figure 1, and the 1-4 bands in the figure are the cDNAs of the male parent, the female parent, the high-sugar group and the low-sugar group, with β-Actin is the PCR product amplified by primers, and the fragment length is about 210bp; bands 5-8 are the cDNAs of the paternal parent, female parent, high-sugar group and low-sugar group, amplified with SPS-1 as primer PCR product, the fragment length is 121bp. The results showed that the expression of SPS gene had already started at the seedling stage.

The expression of the SPS gene at the jointing stage is shown in Figure 2. Bands 1-4 are the cDNAs of the male parent, the female parent, the high-sugar group and the low-sugar group. The PCR product amplified with β-Actin as primers has a fragment length of 210 bp; Bands 5-8 are the cDNAs of the paternal parent, female parent, high-sugar group and low-sugar group, PCR products amplified with SPS-1 as primers, and the fragment length is about 121bp. The results showed that the expression of SPS gene in the male parent was weaker at the jointing stage, and the expression of SPS gene in the high-glucose group was stronger.

The expression of the SPS gene at the heading stage is shown in Figure 3. Bands 1-4 are the cDNAs of the father, mother, high-sugar group and low-sugar group. The PCR product amplified with β-Actin as primers has a fragment length of 210bp ; Bands 5-8 are PCR products amplified from the cDNA of the male parent, female parent, high-sugar group and low-sugar group at the heading stage, using SPS-1 as a primer, and the fragment length is about 121bp. The results showed that the brightness and thickness of the slices at the heading stage were stronger than those in the previous two stages, indicating that the expression of SPS gene was larger than that at the seedling stage and jointing stage.

The expression of the SPS gene at the mature stage is shown in Figure 4. Bands 1-4 are the cDNAs of the father, mother, high-sugar group and low-sugar group. The PCR product amplified with β-Actin as primers has a fragment length of 210bp ; Bands 5-8 are the cDNAs of the paternal parent, the female parent, the high-sugar group and the low-sugar group. The PCR product amplified with SPS-1 as the primer has a fragment length of about 121bp. The results showed that the expression level of SPS gene reached the maximum value at the mature stage.

In summary, the target fragment can be amplified with β-Actin and SPS-1 as primers, and the bands are clear and free of impurities. It can be seen from Figure 1-4 that the expression level of SPS gene is small at the seedling stage, and gradually increases with the change of growth period, and reaches the maximum at the mature stage. This is consistent with the change rule of sucrose content, which can preliminarily prove that the expression of SPS gene affects the accumulation of sucrose.

Implementation Example 5: Quantitative analysis of sucrose phosphate synthase (SPS) gene expression

5.1 Quantitative analysis of SPS gene expression at seedling stage

The results of quantitative analysis showed that, taking the relative concentration of SPS gene in the leaves of the female parent as the control, the relative concentration of the SPS gene in the high-sugar group at the seedling stage was relatively large, which was 1.59 times the expression level of the SPS gene in the female parent. The relative concentration of the SPS gene in the paternal parent was the smallest, only 1/2 of the expression level of the maternal SPS gene. (See Table 2)

Table 2 Quantitative analysis of SPS gene expression at seedling stage

5.2 Quantitative analysis of SPS gene expression at jointing stage

At the jointing stage, the expression levels of parents, F ₅ high-sugar group and low-sugar group had little difference. The expression level of SPS gene in the largest high-sugar group was 1.25 times that of the smallest low-sugar group. (See Table 3)

Table 3 Quantitative analysis of SPS gene expression at jointing stage

5.3 Quantitative analysis of SPS gene expression at heading stage

The relative concentration of SPS gene in the male parent leaves at the heading stage was the lowest, and the expression levels of the other three populations had little difference, indicating that the difference in the expression level of SPS genes between sweet sorghum and sorghum increased from the heading stage. The expression level of SPS gene in sweet sorghum was about 2 times that of sorghum gene. (See Table 4)

Table 4 Quantitative analysis of SPS gene expression at heading stage

5.4 Quantitative analysis of SPS gene expression in mature stage

The expression levels of the mature parent, F ₅ high-sugar group and low-sugar group were quite different at this stage, and the expression level of SPS gene in the high-sugar group was 15 times that of the smallest low-sugar group. (See Table 5)

Table 5 Quantitative analysis of SPS gene expression in mature stage

To sum up, in each period, the relative concentration of the maternal SPS gene was used as the control, and the expression level of SPS gene from high to low was high sugar group, female parent, low sugar group and male parent. In the mature stage, this change trend is consistent with the accumulation law of sucrose and the change law of SPS activity. It can be seen that the higher the expression level of SPS gene, the more sucrose content.

SEQUENCE LISTING

<110> Shenyang Normal University

<120> Expression Detection Method and Amplification Primer of Sweet Sorghum Sucrose Phosphate Synthase Gene

<130> 2018

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<170> PatentIn version 3.3

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Claims (6)

1. sweet sorghum sucrose phosphate synthase gene amplification primer is characterized in that, comprises primer SPS-1 and primer β-Actin, and primer SPS-1 comprises SPS-1 forward primer and SPS-1 reverse primer, nucleotide The sequences are respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2; the primer β-Actin includes a β-Actin forward primer and a β-Actin reverse primer, and the nucleotide sequences are respectively as SEQ ID NO: 3 and SEQ ID NO: 4. 2. sweet sorghum sucrose phosphate synthase gene amplification primer as claimed in claim 1, is characterized in that, the length of the target fragment of described primer SPS-1 amplification is 121bp; The purpose of described primer β-Actin amplification The length of the fragment is 210bp. 3. A rapid PCR reagent for detecting sweet sorghum sucrose phosphate synthase gene expression, characterized in that, said PCR reagent comprises 10× Buffer 2.5 μl, 25mg/ml MgCl _2.5 μl, 100 μmol/l dNTP 0.5 μl, 5U/ml μl Taq enzyme 0.5μl, 0.2μmol/l Forward Primer 1.0μl, 0.2μmol/l Reverse Primer 1.0μl, cDNA template 1.0μl, make up 25μl with ddH ₂ O; The primers in the PCR reagent are primer SPS-1 or primer β-Actin according to claim 1. 4. The method of using the reagent as claimed in claim 3 to carry out PCR reaction is characterized in that it comprises the following reaction procedures: pre-denaturation at 94°C for 2min → 32 amplification cycles → extension at 72°C for 10min → preservation at 4°C, amplification cycle It includes the following steps: denaturation at 94°C for 30s → annealing at 53°C for 20s → extension at 72°C for 1min 20s. 5. A rapid fluorescent quantitative PCR reagent for detecting the expression of sucrose phosphate synthase gene, characterized in that the volume of the fluorescent quantitative PCR reagent is 20 μl, including: 2×SYBR Premix ExTaq II 10.0 μl, ddH ₂ O 6.0 μl, 0.2 μmol /lForward Primer 0.8μl, 0.2μmol/l Reverse Primer 0.8μl, ROX Reference DYE 0.4μl, cDNA template 2.0μl; The primer in the fluorescent quantitative PCR reagent is the primer SPS-1 or the primer β-Actin according to claim 1. 6. The method for performing fast fluorescent quantitative PCR using the reagent as claimed in claim 5, is characterized in that it comprises the following reaction procedures: pre-denaturation at 95°C for 15min→35 amplification cycles→melting curve program at 53-95°C; amplification The cycle includes the following steps: denaturation at 94°C for 20s → annealing at 53°C for 20s → extension at 72°C for 1min 20s.