Sugarcane sucrose phosphate synthase truncation and application thereof

Abstract

The invention relates to the technical field of gene engineering and enzyme engineering, and discloses a sugarcane sucrose phosphate synthase truncation and application thereof, and the amino acid sequence of the truncation is as shown in SEQ ID NO.4. The sugarcane sucrose phosphate synthase truncation provided by the invention is obtained by cutting off 37th-42nd amino acids on the basis of a wild type sugarcane sucrose synthase (ScSPSB) gene from sugarcane cinnabar 42, and compared with the wild type ScSPSB gene, the sugarcane sucrose phosphate synthase truncation has higher catalytic activity and stability. According to the invention, a wild type ScSPSB gene is subjected to truncation transformation to obtain the sugarcane sucrose phosphate synthase truncation with higher catalytic activity and higher protein stability, so that a foundation is laid for studying transcription and expression mechanisms of sugarcane sucrose phosphate synthase and further discussing an accumulation mechanism of sucrose; and a theoretical and practical basis is provided for researching the biological function of the sucrose phosphate synthase and improving crop varieties by applying the sugarcane gene.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering and enzyme engineering, and particularly relates to a sugarcane sucrose phosphate synthase truncated body and application thereof. BACKGROUND

[0002] As an important industrial raw material and food source, the market demand for sucrose continues to rise with the global population growth and consumption upgrading. Sugarcane is the main source of natural sucrose, and its economic value is directly related to the sucrose accumulation in the stem. Therefore, increasing the sucrose content of sugarcane is the core goal of agricultural production and breeding research.

[0003] The sucrose metabolism in plants presents a dynamic balance, which is jointly regulated by synthesis and decomposition pathways. The synthesis of sucrose mainly depends on the catalytic action of key enzymes such as sucrose phosphate synthase (SPS) and sucrose synthase (SuSy), among which SPS is the rate-limiting enzyme for the synthesis of sucrose-6-phosphate. The decomposition of sucrose is mainly realized through the reverse catalysis of invertase and sucrose synthase, and finally sucrose is hydrolyzed into glucose and fructose. Therefore, by regulating the activity or expression level of the above-mentioned key enzymes (such as inhibiting the decomposition pathway mediated by invertase and enhancing the synthesis pathway mediated by SPS), it is an effective technical means to increase the sucrose accumulation level of sugarcane stems.

[0004] SPS plays a decisive role in the synthesis of sucrose in higher plants, and its activity level is significantly positively correlated with the sucrose accumulation in plants. Since the 1990s, studies have shown that overexpression of SPS genes can effectively increase the sucrose content and biomass of crops: for example, the SPS activity in tomato leaves transformed with the maize SPS gene is increased by 6 times, the sucrose content is significantly increased, and the starch accumulation is reduced; the SPS activity in rice transformed with the maize SPS gene is increased by 3 times, and the plant height is also significantly increased; tobacco transformed with the Arabidopsis SPS gene shows increased sucrose accumulation in stems, elongated internodes, increased stem diameter, and increased stem weight. In addition, SPS is also widely involved in plant growth and development, yield formation, and stress resistance (such as cold resistance, drought resistance, and salt tolerance), and is a key regulatory node in the plant metabolic network.

[0005] At present, SPS genes are divided into multiple families after bioinformatics analysis of higher plant genomes. There are at least A, B, and C families in dicotyledonous plants, while monocotyledonous plants (such as wheat and sugarcane) are further divided into A, B, C, and D families, and the number of SPS genes in different species varies. As a C4 crop with extremely high photosynthetic efficiency, the strong sucrose accumulation ability of sugarcane is closely related to the high catalytic characteristics of SPS. However, the research on the SPS gene family of sugarcane is still relatively scarce, and the catalytic activity of the existing SPS protein of sugarcane still has a large space for improvement, and related research needs to be further deepened. SUMMARY

[0006] The present application aims to overcome the problem that the catalytic activity of sugarcane SPS still needs to be further improved in the prior art, and provides a sugarcane sucrose phosphate synthase truncation and application thereof.

[0007] In order to achieve the above-mentioned purpose, the present application provides a sugarcane sucrose phosphate synthase truncation, and the amino acid sequence of the truncation is shown in SEQ ID NO. 4.

[0008] The second aspect of the present application provides a gene encoding the above-mentioned sugarcane sucrose phosphate synthase truncation, and the nucleotide sequence of the gene is shown in SEQ ID NO. 5.

[0009] The third aspect of the present application provides a recombinant expression vector comprising the above-mentioned gene.

[0010] The fourth aspect of the present application provides a recombinant expression strain comprising the above-mentioned gene.

[0011] Preferably, the host bacteria of the recombinant expression strain is Agrobacterium.

[0012] The fifth aspect of the present application provides a method for constructing a recombinant expression strain, which comprises the following steps: (1) cloning the above-mentioned gene into an expression plasmid to obtain a recombinant expression vector; (2) transforming the recombinant expression vector into host bacteria.

[0013] The sixth aspect of the present application provides application of the above-mentioned sugarcane sucrose phosphate synthase truncation, the above-mentioned gene, the above-mentioned recombinant expression vector or the above-mentioned recombinant expression strain in sugarcane breeding technology.

[0014] The seventh aspect of the present application provides application of the above-mentioned sugarcane sucrose phosphate synthase truncation, the above-mentioned gene, the above-mentioned recombinant expression vector or the above-mentioned recombinant expression strain in research and regulation of sugarcane sugar accumulation.

[0015] The eighth aspect of the present application provides application of the above-mentioned sugarcane sucrose phosphate synthase truncation, the above-mentioned gene, the above-mentioned recombinant expression vector or the above-mentioned recombinant expression strain in improving the sugar content of sugarcane.

[0016] The ninth aspect of the present application provides application of the above-mentioned sugarcane sucrose phosphate synthase truncation, the above-mentioned gene, the above-mentioned recombinant expression vector or the above-mentioned recombinant expression strain in research of the biological function of sucrose phosphate synthase.

[0017] The sugarcane sucrose phosphate synthase truncation provided by the present application is obtained by cutting off the 37-42 amino acids based on the wild-type sugarcane sucrose synthase (ScSPSB) gene from sugarcane Gui sugar 42, and has higher catalytic activity and stability compared with the wild-type ScSPSB gene. The present application obtains the sugarcane sucrose phosphate synthase truncation with higher catalytic activity and stronger protein stability by truncation modification of the wild-type ScSPSB gene, so as to lay a foundation for further exploring the accumulation mechanism of sucrose by studying the transcription and expression mechanism of the sugarcane sucrose phosphate synthase, and provide a theoretical and practical basis for studying the biological function of the sucrose phosphate synthase and applying the sugarcane gene to improve crop varieties. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is the electropherogram of the ScSPSB gene in the embodiment 1 of the present application; Figure 2 It is the ScSPSB gene coding sequence alignment chart in the embodiment 2 of the present application; Figure 3 It is the phylogenetic tree analysis chart of the ScSPSB gene in the embodiment 2 of the present application; Figure 4 It is the schematic diagram of the construction of each sugarcane ScSPSB truncation in the embodiment 3 of the present application; Figure 5 It is the influence of each sugarcane ScSPSB truncation on protein accumulation in the embodiment 4 of the present application; Figure 6 It is the influence of the sugarcane ScSPSB truncation (ScSPSB-△2) on enzyme activity in the embodiment 4 of the present application. DETAILED DESCRIPTION

[0019] The specific embodiments of the present application are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0020] The endpoints of the ranges and any values in the ranges disclosed herein are not limited to the precise values stated. The ranges and values should be construed to be approximations that are used in a suitable, effective range or value. The endpoints of the ranges of values, the endpoints of the ranges of values and individual values between the endpoints of the ranges of values, and the individual values between the endpoints of the ranges of values can be combined with each other to form one or more new ranges of values, which should be considered to be specifically disclosed herein.

[0021] The present application provides a sugarcane sucrose phosphate synthase truncation, and the amino acid sequence of the truncation is shown in SEQ ID NO. 4.

[0022] The present application has found that the existing full-length SPS protein may contain an unnecessary domain affecting its catalytic efficiency or stability, resulting in low catalytic activity of the sugarcane SPS protein. In the present application, the catalytic activity of the full-length ScSPSB gene is improved by truncating the SPSB protein to obtain a sugarcane sucrose phosphate synthase truncation body with an amino acid sequence as shown in SEQ ID NO. 4.

[0023] The present application also provides a gene encoding the sugarcane sucrose phosphate synthase truncation body as described above, and the nucleotide sequence of the gene is shown as SEQ ID NO. 5.

[0024] In the present application, the wild-type sucrose phosphate synthase (ScSPSB) gene is cloned from sugarcane Gui sugar 42. Based on the full-length sequence of the ScSPSB gene (amino acid 1-1081), a corresponding truncation body is constructed according to the difference site (SITE2 (37-42 amino acids)), and the sugarcane sucrose phosphate synthase truncation body described in the present application is obtained, which is denoted as ScSPSB-△2. The truncation body ScSPSB-△2 lacks the amino acids (VAGASP) at positions 37-42 of the ScSPSB gene. Compared with the wild-type ScSPSB gene, the catalytic activity of the truncation body ScSPSB-△2 is significantly improved. Therefore, by truncating the wild-type ScSPSB gene, the catalytic activity of the wild-type ScSPSB gene is significantly improved.

[0025] In the present application, the base sequence of the sugarcane ScSPSB gene is shown as SEQ ID NO. 1, the cDNA sequence is shown as SEQ ID NO. 2, and the encoded amino acid sequence is shown as SEQ ID NO. 3.

[0026] The base sequence of the sugarcane ScSPSB gene is shown as follows (SEQ ID NO. 1):

[0027] The amino acid sequence of the protein encoded by the sugarcane ScSPSB gene is shown below (SEQ ID NO. 3):

[0028] The amino acid sequence of the sucrose phosphate synthase truncation (sugar cane scSPSB-Δ2) according to the present application is shown below (SEQ ID NO. 4):

[0029] The nucleotide sequence of the sucrose phosphate synthase truncated body (sugarcane scSPSB-Δ2) described in the present application is shown below (SEQ ID NO. 5):

[0030] The present application also provides a recombinant expression vector comprising the gene as described above, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 5.

[0031] The present application also provides a recombinant expression strain comprising the gene as described above, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 5. The recombinant expression strain expresses the sugarcane sucrose phosphate synthase truncated body.

[0032] In some embodiments, the host bacteria of the recombinant expression strain is Agrobacterium.

[0033] The present application also provides a method for constructing a recombinant expression strain, which comprises the following steps: (1) cloning the gene as described above (the nucleotide sequence is shown as SEQ ID NO. 5) into an expression plasmid to obtain a recombinant expression vector; (2) transforming the recombinant expression vector into a host bacteria.

[0034] The present application also provides the use of the sugarcane sucrose phosphate synthase truncated body as described above, the encoding gene as described above, the recombinant expression vector as described above or the recombinant expression strain as described above in sugarcane breeding technology.

[0035] The catalytic activity of the sugarcane sucrose phosphate synthase truncated body obtained by truncation is obviously improved compared with the sugarcane sucrose phosphate synthase before modification, and therefore, the sugarcane sucrose phosphate synthase truncated body, the gene encoding the truncated body, the recombinant expression vector comprising the gene and the recombinant expression strain can be applied to cultivate sugarcane genetically modified crop varieties.

[0036] The present application also provides the use of the sugarcane sucrose phosphate synthase truncated body as described above, the encoding gene as described above, the recombinant expression vector as described above or the recombinant expression strain as described above in studying and regulating the accumulation of sucrose in sugarcane.

[0037] The present application also provides the use of the sugarcane sucrose phosphate synthase truncated body as described above, the encoding gene as described above, the recombinant expression vector as described above or the recombinant expression strain as described above in improving the sucrose content in sugarcane.

[0038] The present application also provides the use of the sugarcane sucrose phosphate synthase truncated body as described above, the encoding gene as described above, the recombinant expression vector as described above or the recombinant expression strain as described above in studying the biological function of sucrose phosphate synthase.

[0039] The application will be described in detail below by way of examples, but the scope of protection of the application is not limited thereto. The experimental methods in the following examples are all conventional methods in the art, unless otherwise specified. The experimental materials used in the following examples are all commercially available products, unless otherwise specified.

[0040] In the following examples, the experimental materials involved include: The plant of sugarcane GT42 was planted in an artificial greenhouse environment in the laboratory; Trizol reagent: manufacturer is TIANGEN (Beijing) Biotech Co., Ltd.; Maxima H Minus First Strand cDNA Synthesis Kit with dsDNase: manufacturer is Thermo Scientific; TransStart FastPfu DNA Polymerase kit: manufacturer is Beijing ZOMANBIO Technology Co., Ltd. (TRAN); Gel Mini Purification Kit: manufacturer is Beijing ZOMANBIO Technology Co., Ltd. (ZOMANBIO); pEAQ vector: can be a common commercially available product, or can refer to the literature Sainsbury F, Thuenemann EC, Lomonossoff GP. pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J. 2009; 7(7): 682-693. doi: 10.1111 / j.1467-7652.2009.00434.x; Sucrose phosphate synthase (SPS) activity detection kit: manufacturer is Beijing Solabio Technology Co., Ltd., product code is BC0605.

[0041] Example 1 This example is used to illustrate the cloning of sugarcane ScSPSB gene.

[0042] 1. Extraction of total RNA from sugarcane leaves Sugarcane GT42 leaf and stem samples were collected, cleaned and disinfected with 75% alcohol, then placed in a self-sealing bag and stored in a ultra-low temperature freezer at -80°C.

[0043] The total RNA of sugarcane leaves and stalks was extracted by Trizol reagent method.

[0044] 2. Synthesis of sugarcane cDNA The DNase-treated total RNA of sugarcane was reverse transcribed into cDNA by Maxima H Minus First Strand cDNA Synthesis Kit with dsDNase. The reaction system was 15 μL, including 1.0 μL Oligo dT Primer, 1.0 μL Random Primer, 1.0 μL dNTP Mixture (10 mM each), 2 μg total RNA. The reaction condition was 65℃, 5 min; after cooling on ice, 4.0 μL 5x RT Buffer, 1.0 μL Maxima H Minus Enzyme Mix were added to the above reaction solution. The reaction condition was 25℃, 10 min; 50℃, 30 min; 85℃, 5 min. After the reaction, the next step was directly performed or stored at -80℃ for standby.

[0045] 3. Cloning of ScSPSB gene A pair of specific primers was designed by Primer Premier 5 software for cloning ScSPSB mRNA sequence.

[0046] Forward primer: 5'-ATGGCGGGGAACGAGTGGATCAATG-3'; Reverse primer: 5'-CATGCCGCTAGAAGTCTTGGAGAC 3'; PCR amplification was performed by using TransStart FastPfu DNA Polymerase kit with 200 ng cDNA as template. The PCR reaction system was as follows: 5x TransStart FastPfu Buffer 10 μL, dNTP Mixture (2.5 mM each) 4.0 μL, ScSPSB-F (10 μM) 1.0 μL, ScSPSB-R (10 μM) 1.0 μL, TransStart FastPfu DNA Polymerase (2.5 U / μL) 1 μL, H2O 32 μL, cDNA 1.0 μL.

[0047] PCR reaction program as follows: 95℃ pre-denaturation 2 min; 95℃ denaturation 20 sec, 66℃ annealing 20 sec, 72℃ extension 3 min, a total of 35 amplification cycles; final 72℃ extension 5 min. After PCR amplification reaction, 5 μL of PCR reaction product was subjected to 1% agarose gel electrophoresis detection, and the results are shown in Figure 1 . Figure 1 The results show that the fragment size is between 3000 bp to 3500 bp, which is consistent with the expected size of the target fragment.

[0048] After the PCR reaction product was purified by Gel Mini Purification Kit kit, it was connected to pJET1.2 cloning vector and transformed into E. coli host bacteria TOP10 competent cells. 100 μL of the transformed bacteria were spread on LB solid medium containing ampicillin (50 μg / mL), and after incubation at 37℃ in the dark overnight, several white single colonies were picked and inoculated into LB liquid medium containing ampicillin (50 μg / mL), and incubated at 37℃ in the dark with shaking for 6-8 h. 0.5 μL of the bacterial liquid was subjected to bacterial liquid PCR detection, and the PCR reaction system was as follows: 5 x TransStart FastPfu Buffer 10 μL, dNTP Mixture (2.5 mM each) 4.0 μL, ScSPSB-F (10 μM) 1.0 μL, ScSPSB-R (10 μM) 1.0 μL, TransStart FastPfu DNA Polymerase (2.5 U / μL) 1 μL, H2O 32 μL, cDNA 1.0 μL. PCR reaction program as follows: 95℃ pre-denaturation 2 min; 95℃ denaturation 20 sec, 66℃ annealing 20 sec, 72℃ extension 3 min, a total of 35 amplification cycles; final 72℃ extension 5 min. After bacterial liquid PCR detection, three positive clones were selected for sequencing verification. After sequencing, the ligation product had nucleotides as shown in SEQ ID NO: 1, which was named ScSPSB, and the amino acid sequence of the encoded transcription factor ScSPSB was as shown in SEQ ID NO: 3.

[0049] Example 2 This example is used to illustrate the bioinformatics analysis of the sequence of sugarcane ScSPSB gene.

[0050] The sugarcane ScSPSB gene obtained in Example 1 was subjected to bioinformatics analysis, and the results are shown in Figure 2 .

[0051] Figure 2The results show that the ScSPSB protein contains a typical glycosyltransferase domain. Through homologous sequence comparison, it is found that the cloned SPSB gene contains two serine conserved domains of SPS protein family, one is a 14-3-3 protein specific binding site, and the other is an osmotic stress activation site. They can be phosphorylated or dephosphorylated by the corresponding protein kinase or phosphatase to change the enzyme activity, and can also specifically bind to 14-3-3 protein to change the enzyme activity. The physical and chemical properties, protein structure and other molecular characteristics of the gene coding protein are predicted by bioinformatics software and online tools.

[0052] The protein sequences of the B subfamily members of SPS genes in rice and corn are downloaded from the NCBI website respectively, the Muscle operation method in MEGA6.0 software is used for alignment with the ScSPS protein sequence, the neighbor-joining method is used for constructing the phylogenetic tree, and the phylogenetic tree analysis is shown in Figure 3 .

[0053] Example 3 This example is used to illustrate the construction of the recombinant expression vector and the recombinant expression strain of sugarcane ScSPSB gene, the truncated body of sugarcane sucrose phosphate synthase (ScSPSB-△2) and other truncated bodies according to the present application.

[0054] The target fragment cloned in Example 1 is transformed into the pEAQ vector by homologous recombination.

[0055] On this basis, according to the three difference sites SITE2 (37-42aa), SITE4 (607-627aa) and SITE5 (628-640aa) identified in the full-length sequence (amino acid 1-1081) of ScSPSB gene, we constructed the related truncated bodies (scSPSB-△2, scSPSB-△4, scSPSB-△5, scSPSB-△2△4△5, scSPSB-△4△5) according to the difference sites, and the construction form is shown in Figure 4 . Figure 4 In the figure, ScSPSB-FL refers to the full-length sequence of ScSPSB gene; scSPSB-△2 is the sequence after deleting SITE2 from the full-length sequence of ScSPSB (the amino acid sequence is shown as SEQ ID NO. 4, and the nucleotide sequence is shown as SEQ ID NO. 5); scSPSB-△4 is the sequence after deleting SITE4; scSPSB-△5 is the sequence after deleting SITE5; ScSPSB-△4△5 refers to the sequence after deleting SITE4 and SITE5, and ScSPSB-△2△4△5 refers to the sequence after deleting SITE2, SITE4 and SITE5.

[0056] The reaction conditions are as follows: after mixing the reaction system, incubate at 50°C for 2 hours, add 5 μL of the reaction solution to 50 μL of E. coli TOP10 competent cells, mix, and stand in an ice bath for 30 minutes, gently take out, heat shock at 42°C for 90 seconds, immediately ice bath for 2 minutes, add 500 μL of LB medium, and incubate at 37°C for 1 hour; take 100 μL of the bacterial solution and spread on an LB plate containing Kan (kanamycin) resistance, and incubate overnight. Pick the positive colonies obtained by antibiotic screening, extract the plasmid to obtain a prokaryotic expression vector. The prokaryotic expression vector is transferred into an Agrobacterium strain, and cultured at a temperature of 28°C and a rotation speed of 200 rpm, and when the culture reaches OD 600 0.5.

[0057] Example 4 This example is used to illustrate the protein accumulation amount of the sugarcane ScSPSB gene, the sugarcane sucrose phosphate synthase truncation (ScSPSB-△2) and other truncations according to the present application.

[0058] I. Experimental materials and methods 1. Tobacco planting First, prepare the tobacco planting substrate, with a ratio of peat soil: perlite: vermiculite: wood ash = 5:1:1:1; then sow Nicotiana benthamiana seeds on the substrate, and incubate in the dark at 25°C for 1 week; after the seedlings break through the soil, move them to light, with a light: dark = 16h:8h, and a humidity of 75%; when the tobacco grows 6-8 true leaves, it can be used for transient expression analysis.

[0059] 2. Construction of tobacco expression vector Construct a transient expression vector of ScSPSB gene. Refer to the method shown in Example 3 to construct a prokaryotic expression vector, and the specific process includes: amplify the vector pEAQ by inverse PCR, and recover the large plasmid fragment; due to the method of homologous recombination, introduce homologous arms on the forward and reverse primers for amplifying the ScSPSB cDNA fragment; link the PCR product with the recovered vector fragment to obtain the ScSPSB-pEAQ expression vector and each truncation expression vector. Enzymatically cut the constructed recombinant expression vector for verification.

[0060] 3. Introduction of recombinant expression vector into Agrobacterium The Agrobacterium competent cells were thawed on ice, 200 ng of the recombinant expression vector constructed above was added, the centrifuge tube was mixed gently, and then electroporation was performed. The tube was placed on ice for about 2 min, 1 mL of LB medium without antibiotics was added, and the mixture was incubated at 28°C for 3 h. Then, 200 μL of the mixture was spread on LB medium containing kanamycin (100 mg / L) and rifampicin (25 mg / L), and the mixture was incubated at 28°C for 2 days. The positive clones were detected by colony PCR.

[0061] 4. Transient expression of ScSPSB by Agrobacterium infiltration Preparation of the infiltration solution: The Agrobacterium strain containing pEAQ-ScSPSB or pEAQ-ScSPSB truncation prepared above was inoculated into 2 mL of medium containing kanamycin and rifampicin, and the mixture was incubated at 28°C overnight. On the second day, 1 mL of the culture was transferred into 5 mL of LB medium containing MES and antibiotics, and the mixture was incubated at 28°C overnight. On the third day, the OD of the culture was detected by spectrophotometry. 600 The mixture was centrifuged at 4°C and at a centrifugal force of 4000 g for 15 min, the supernatant was removed, the bacterial pellet was resuspended in infiltration buffer (10 mM MgCL2, 10 mM MES, 150 μM acetosyringone), and the concentration of the bacterial solution was adjusted (OD 600 =0.5).

[0062] The lower two leaves were selected for infiltration, and the leaves were marked in advance with a marker pen. The infiltration was performed as follows: 3 mL of the infiltration solution was taken with a disposable syringe, the syringe was pressed gently, and the fingers were used to support the front of the leaf to allow the infiltration solution to penetrate into the back of the leaf. The operation was repeated several times until the entire leaf surface was wet. The empty vector pEAQ and the infiltration buffer were used as controls. Two days after the injection, 3 samples were taken from each injected leaf with a puncher with an inner diameter of 6 mm; and the proteins were extracted.

[0063] 5. Western Blot The tobacco leaves were collected and placed in liquid nitrogen, and then ground with a grinding rod. 200-500 μL of prepared cell protein lysis solution was added, the mixture was vortexed for 15 s, and then centrifuged at 4°C and at a speed of 12000 rpm for 10 min. The supernatant was transferred to a new 1.5 mL EP tube, the protein concentration was detected using a nano, the protein solution was vortexed, denatured at 95°C for 10 min, and then stored in a refrigerator at -80°C for later use.

[0064] Select 15% SDS-PAGE precast gel, add 50 μg denatured protein solution and 5 μL protein marker to each well, run the gel at 120 V for 2 h, transfer the membrane at 200 mA for 1 h, use PVDF membrane during the transfer process, block the PVDF membrane with skimmed milk for 1 h, after the blocking, cut the membrane according to the size of the target protein and the position of the marker, and make a mark, put the cut strip into the primary antibody, and incubate at 4°C overnight, after the primary antibody, wash the membrane with PBST for three times, incubate the secondary antibody for 1 h, after the secondary antibody, wash the membrane with PBST for three times, add the color developing solution and expose.

[0065] The effect of each sugarcane ScSPSB truncated body on the protein accumulation amount is shown in Figure 5 , wherein, Figure 5 , the A and B graphs in , are respectively the Western Blot graph and the gray scale analysis graph (the gray scale value of the target protein divided by the gray scale value of the internal reference, and the result represents the relative content of the target protein of a sample) after the transient overexpression of ScSPSB different truncated bodies in tobacco leaves for 48 h, Figure 5 , the C and D graphs are respectively the Western Blot graph and the gray scale analysis graph after 6 days of expression in tobacco leaves.

[0066] Figure 5 The results of the A and B graphs in show that the protein accumulation amount of each truncated body of ScSPSB different truncated bodies in tobacco leaves is significantly improved relative to the full-length sequence of ScSPSB after the transient overexpression of ScSPSB different truncated bodies in tobacco leaves for 48 h; Figure 5 The results of the C and D graphs in show that at the 6th day, the ScSPSB-△2 protein still has obvious accumulation, while other truncated bodies are basically degraded, indicating that the ScSPSB-△2 truncated body protein is more stable compared with other truncated bodies.

[0067] 6. Enzyme activity detection of leaf sucrose synthase after transient injection: Sucrose is not only an important photosynthetic product, but also a main material for transportation in plants, and one of the storage forms of carbohydrates. Sucrose phosphate synthase (SPS) takes fructose-6-phosphate as the acceptor, and the formed sucrose phosphate forms sucrose under the action of sucrose phosphatase. Generally, the sucrose phosphate synthase-sucrose phosphatase system is regarded as the main pathway of sucrose synthesis.

[0068] Sucrose phosphate synthase catalyzes the formation of sucrose phosphate from fructose-6-phosphate, and sucrose reacts with resorcinol to show color change, with a characteristic absorption peak at 480 nm, and the enzyme activity is proportional to the color depth.

[0069] The enzyme activity detection of sucrose synthase is carried out by using a sucrose phosphate synthase (SPS) activity detection kit.

[0070] 6.1 Sample Processing Homogenize the tissue in an ice bath at a ratio of 1:5~10 (approximately 0.1 g of tissue to 1 mL of extraction solution). Centrifuge at 8000 g and 4°C for 10 min, collect the supernatant, and place it on ice for analysis.

[0071] 6.2 Measurement Procedure (1) Preheat the spectrophotometer / ELISA reader for more than 30 minutes, adjust the wavelength to 480 nm, and zero the spectrophotometer with distilled water.

[0072] (2) Sample determination Add the reagents shown in Table 1 below to a 1.5 mL EP tube. The reagents are from a sucrose phosphate synthase (SPS) activity assay kit.

[0073] Table 1

[0074] Mix well, incubate in an 80 ℃ water bath (with sealing film to prevent cap bursting) for 20 min, cool, and centrifuge at 12000 rpm for 10 min at room temperature. Take 200 mL of the supernatant and measure the absorbance of each tube at 480 nm. Calculate ΔAmeasured = Ameasured tube - Acontrol tube, ΔAstandard = Astandard tube - Ablank tube.

[0075] 6.3 Calculation of SPSB Vitality Units (1) Calculation based on sample quality Unit definition: One unit of enzyme activity is defined as the amount of sucrose produced per gram of tissue per minute.

[0076] SPSB activity (U / g mass) = (C standard tube × V1 × ΔA measured ÷ ΔA standard) ÷ (W × V1 ÷ V2) ÷ T = 50 × ΔA measured ÷ ΔA standard ÷ W; C standard tube: Standard tube concentration, 500 pg / mL; V1: The volume of sample added to the reaction system, 0.01 mL; V2: Add 1 mL of the extraction solution; W: Sample mass, g; T: Reaction time, 10 min.

[0077] The effect of sugarcane scSPSB truncated section (ScSPSB-△2) on enzyme activity is as follows: Figure 6 As shown, Figure 6 In this context, D2 refers to the second day, and D6 refers to the sixth day.

[0078] Figure 6The results show that the SPSB enzyme activity in the tobacco leaf of ScSPSB-△2 truncated body is also improved relative to the enzyme activity of SPSB full-length sequence, especially at the 6th day, the SPSB enzyme activity in the tobacco leaf of ScSPSB-△2 is significantly improved relative to the enzyme activity of SPSB full-length sequence.

[0079] As can be seen from the above, the ScSPSB-△2 provided by the present application can significantly improve the enzyme activity by cutting off the amino acids at the 37th-42nd positions based on the ScSPSB gene. Therefore, the present application not only lays a foundation for further exploring the accumulation mechanism of sucrose by studying the transcription and expression mechanism of sugarcane sucrose phosphate synthase, but also provides a basis for studying the biological function of sucrose phosphate synthase and improving crop varieties by using sugarcane genes by obtaining the purified protein with biological activity through the amino acid sequence.

[0080] The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as disclosed by the present application and belong to the protection scope of the present application.

Claims

1. A truncated form of sucrose phosphate synthase, characterized in that, The amino acid sequence of the truncated form is shown in SEQ ID NO.

4.

2. The gene encoding the truncated form of sucrose phosphate synthase as described in claim 1, characterized in that, The nucleotide sequence of this gene is shown in SEQ ID NO.

5.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the gene as described in claim 2.

4. A recombinant expression strain, characterized in that, The recombinant expression strain contains the gene described in claim 2.

5. The recombinant expression strain according to claim 4, characterized in that, The host bacterium for this recombinant expression strain is Agrobacterium.

6. A method for constructing a recombinant expression strain, characterized in that, The method includes the following steps: (1) The gene described in claim 2 is cloned into an expression plasmid to obtain a recombinant expression vector; (2) Transform the recombinant expression vector into the host bacteria.

7. The application of the truncated form of sugarcane sucrose phosphate synthase according to claim 1, the gene according to claim 2, the recombinant expression vector according to claim 3, or the recombinant expression strain according to claim 4 or 5 in sugarcane breeding technology.

8. The application of the truncated form of sucrose phosphate synthase according to claim 1, the gene according to claim 2, the recombinant expression vector according to claim 3, or the recombinant expression strain according to claim 4 or 5 in the study and regulation of sucrose accumulation.

9. The application of the truncated form of sucrose phosphate synthase according to claim 1, the gene according to claim 2, the recombinant expression vector according to claim 3, or the recombinant expression strain according to claim 4 or 5 in increasing sucrose content.

10. The application of the truncated form of sucrose phosphate synthase according to claim 1, the gene according to claim 2, the recombinant expression vector according to claim 3, or the recombinant expression strain according to claim 4 or 5 in the study of the biological function of sucrose phosphate synthase.

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