A gene SSP1 for promoting growth of sugarcane and application thereof

Abstract

The present application relates to the technical field of biology, in particular to a gene SSP1 for promoting the growth of sugarcane and application thereof, the nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of a protein encoded by the gene is shown as SEQ ID NO. 2. By transferring the gene into sugarcane to perform overexpression, compared with wild-type sugarcane, the growth rate of the sugarcane overexpressing the SSP1 gene is obviously accelerated, and the plant height is significantly increased. The overexpression of the SSP1 gene proposed in the present application can significantly promote the growth of sugarcane, and in the future, can be applied to the production and molecular breeding of sugarcane through the transgenic means, and has very important application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a gene SSP1 that promotes sugarcane growth and its application. Background Technology

[0002] Sugarcane, as a core global sugar crop and an important energy crop, possesses irreplaceable economic value and strategic significance. According to statistics from the Food and Agriculture Organization of the United Nations (FAO), over 80% of the world's sugar and approximately 40% of its bioethanol originate from sugarcane. Sugarcane is a key pillar supporting sugar supply, contributing nearly 90% of sugar production. As a strategic agricultural product related to people's livelihoods and industrial security, a stable supply of sugar is crucial to the overall national economy.

[0003] Sugarcane biomass is a core and fundamental factor that determines sugar production. An increase in biomass directly means an increase in the total weight of stalks per unit planting area. Under the premise of maintaining a stable sugar content, it can not only directly promote the increase in total sugar production, but also reduce the planting, harvesting and processing costs per unit of sugarcane through economies of scale, thereby improving the production efficiency of sugar companies and the economic benefits of growers. It plays a key role in promoting the high-quality development of the sugarcane industry.

[0004] Therefore, improving sugarcane growth rate and increasing biomass have become core objectives urgently needing breakthroughs in sugarcane breeding and cultivation. However, current sugarcane growth regulation technologies still face multiple bottlenecks: traditional cultivation methods can only optimize the external growth environment and cannot overcome the limitations of growth potential at the genetic level; conventional breeding is limited by the complex genetic background of sugarcane, resulting in lengthy cycles and poor directionality; and the field of molecular breeding lacks core gene resources that can be industrially applied due to insufficient discovery and verification of key functional genes that "efficiently promote growth." Against this backdrop, there is an urgent need to systematically discover and verify key genes that can clearly promote sugarcane growth, providing precise targets for sugarcane molecular breeding, and ultimately solving the core problems of "slow growth rate and insufficient biomass" in the industry. Summary of the Invention

[0005] In view of this, the present invention provides a gene SSP1 that promotes sugarcane growth and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The present invention provides a gene SSP1 that promotes sugarcane growth. The nucleotide sequence of the gene SSP1 is shown in SEQ ID NO.1, or a nucleotide sequence of the nucleotide sequence shown in SEQ ID NO.1 that has been substituted, deleted and / or added with one or more nucleotides and expresses the same functional protein.

[0008] The present invention also provides the SSP1 protein encoded by the gene SSP1, wherein the SSP1 protein is a protein as follows (A1), (A2), or (A3):

[0009] A1) The amino acid sequence is that of the protein shown in SEQ ID NO.2;

[0010] A2) Proteins derived from A1) or proteins with more than 80% identity and function to the amino acid sequence shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues.

[0011] A3) is a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0012] The present invention also provides a biomaterial, said biomaterial being any one of B1) to B4) below:

[0013] B1) Contains an expression cassette containing the SSP1 gene, which promotes sugarcane growth;

[0014] B2) A recombinant vector containing the sugarcane growth-promoting gene SSP1 or the expression cassette described in B1);

[0015] B3) Recombinant microorganisms containing the sugarcane growth-promoting gene SSP1, the expression cassette described in B1), or the recombinant vector described in B2;

[0016] B4) A transgenic plant cell line containing the sugarcane growth-promoting gene SSP1, the expression cassette described in B1), the recombinant vector described in B2), or the recombinant microorganism described in B3).

[0017] Preferably, the recombinant vector in step B2) is obtained by effectively ligating the gene SSP1 to an expression vector.

[0018] Preferably, the expression vector is a plant overexpression vector.

[0019] Preferably, the recombinant microorganism includes Escherichia coli or Agrobacterium.

[0020] The present invention also provides the application of the gene SSP1, the SSP1 protein, or the biomaterial in promoting sugarcane growth.

[0021] Preferably, the application involves introducing the SSP1 gene into sugarcane for expression to promote sugarcane growth.

[0022] Expression in sugarcane to promote sugarcane growth.

[0023] The present invention also provides a method for promoting sugarcane growth, wherein the method involves introducing the SSP1 gene into sugarcane for expression to promote sugarcane growth.

[0024] It contains at least the following beneficial technical effects:

[0025] This invention, through transgenic verification, shows that sugarcane lines overexpressing the SSP1 gene have an average height increase of 11.6% compared to wild-type sugarcane, significantly increased biomass, and no significant negative impact on sucrose content.

[0026] The SSP1 gene described in this invention can be directly used as the core target of molecular breeding. Through transgenic and gene editing technologies, sugarcane growth traits can be improved in a targeted manner, shortening the breeding cycle (from the traditional 8-10 years to 2-3 years) and significantly improving breeding efficiency.

[0027] Improving the growth potential of sugarcane through genetic modification can reduce the amount of chemical fertilizers used, reduce agricultural non-point source pollution, and meet the needs of green agricultural development. Attached Figure Description

[0028] Figure 1 A comparison of shoot germination between sugarcane overexpressing the SSP1 gene and wild-type sugarcane.

[0029] Figure 2 A comparison of plant height between sugarcane overexpressing the SSP1 gene and wild-type sugarcane.

[0030] Figure 3 A statistical analysis of plant height in sugarcane overexpressing the SSP1 gene and wild-type sugarcane. Detailed Implementation

[0031] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. The invention will now be described with reference to specific embodiments; however, these embodiments are merely descriptive and do not limit the invention in any way.

[0032] In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] The culture medium preparation method in the following examples is as follows (taking 1L as an example):

[0035] Co-culture medium: 25 g sucrose, 2 mg 2,4-dichlorophenoxyacetic acid, 250 µmol / L acetylsyl syringone and 8 g plant gel were added to each liter of B5 medium.

[0036] Recovery medium: Add 25 g sucrose, 300 mg termethin, 2 mg 2,4-dichlorophenoxyacetic acid, 0.5 mg 6-benzylaminopurine and 8 g plant gel per liter of B5 medium, and adjust the pH to 5.8.

[0037] Selection medium for embryogenic callus containing glufosinate: 25 g sucrose, 300 mg termethin, 2 mg 2,4-dichlorophenoxyacetic acid, 0.5 mg 6-benzylaminopurine, 8 g plant gel and 2.0 mg glufosinate per liter of B5 medium.

[0038] Differentiation screening medium: 25 g sucrose, 300 mg termethin, 1 mg 6-benzylaminopurine, 5 g plant gel and 2.0 mg glufosinate were added to each liter of B5 medium.

[0039] Rooting selection medium: Add 25 g sucrose, 300 mg termethin, 1 mg naphthaleneacetic acid and 5 g plant gel per liter of B5 medium.

[0040] Example 1

[0041] Construction and infection of SSP1 gene overexpression vector

[0042] (I) Construction of overexpression vectors

[0043] 1. The SSP1 gene nucleotide sequence was synthesized by adding the attB1 adapter sequence (SEQ ID NO.5: GGGGACAAGTTTGTACAAAAAAGCAGGCT) to the 5' end and the attB2 adapter sequence (SEQ ID NO.6: GGGGACCACTTTGTACAAGAAAGCTGGGT) to the 3' end. The gene sequence was synthesized artificially using the pUC57 vector.

[0044] 2. The vector pUC57 containing the SSP1 gene was subjected to a backpropagation (BP) reaction with the primary vector pDONR207 to construct the recombinant introductory vector pDONR207-SSP1. After incubation at 25°C for 1 h, 1 µL of proteinase K solution was added to each sample to terminate the reaction, and the samples were incubated at 37°C for 10 min.

[0045] 3. The constructed primary vector pDONR207-SSP1 was subjected to an LR reaction with the overexpression vector pB7WG2 to construct the overexpression vector pB7WG2-SSP1. After incubation at 25℃ for 1 h, 1 µL of proteinase K solution was added to each sample to terminate the reaction, and the samples were incubated at 37℃ for 10 min.

[0046] (II) Cloning of Recombinant Plasmids

[0047] 1. Transform the constructed recombinant plasmid into E. coli DH5α cells. The specific method is as follows:

[0048] 2. Add 5 μL of the constructed vector to 100 μL of Escherichia coli DH5α competent cells and incubate on ice for 30 min;

[0049] 3. Heat shock at 42℃ for 60 seconds, followed by an ice bath for 2 minutes;

[0050] 4. Add 800 μL of LB liquid medium and incubate at 37°C with shaking for 30 min;

[0051] 5. Centrifuge at 6000 rpm for 3 min, discard the supernatant, spread on a medium plate containing spectinomycin, and incubate upside down at 37℃ for 12–16 h;

[0052] 6. Pick 8 single colonies from each plate and place them in 1 mL of LB liquid medium containing spectinomycin, and incubate at 37°C and 200 rpm for 4 h with shaking.

[0053] 7. Take 1 μL of bacterial culture for PCR detection;

[0054] 8. After culturing the positive bacterial culture for 16 hours, extract the plasmid using a plasmid extraction kit.

[0055] (III) Transformation of Agrobacterium with recombinant plasmids

[0056] 1. Take 100 μL of Agrobacterium EHA105 competent cells that have thawed on ice, add 5 μL of plasmid, gently tap the tube wall, and incubate on ice for 30 min;

[0057] 2. Freeze in liquid nitrogen for 1 min, water bath at 37°C for 5 min, ice bath for 3 min;

[0058] 3. Add 500 μL of antibiotic-free YEB medium and incubate at 28°C with shaking at 200 rpm for 6 h;

[0059] 4. Centrifuge at 3000 rpm for 1 min, discard the supernatant, keep 100 μL of bacterial solution, gently pipette to resuspend the bacterial block, spread it on YEB solid medium containing spectinomycin, and incubate at 28℃ until single colonies grow.

[0060] 5. Pick a single colony and inoculate it into liquid YEB medium containing the corresponding antibiotic, and incubate at 28°C and 200 rpm for 12 h with shaking.

[0061] 6. Positive clones identified by PCR are added to 50% sterile glycerol (1:1) and stored at -80℃ for later use.

[0062] (iv) Agrobacterium-mediated genetic transformation of sugarcane

[0063] 1. Take the tender leaf tissue from the apical growing point of the robust sugarcane variety ROC22 after 5 months of planting, peel off the outer old leaf sheath, and leave the tightly rolled tender leaf tissue.

[0064] 2. Rinse once with sterile water, then immerse in 75% ethanol solution for 30 seconds for disinfection, then immerse in 0.1% mercuric chloride solution for 10 minutes for disinfection, and rinse twice with sterile water.

[0065] 3. After wiping off the surface moisture with sterile paper, peel off the outer layer of leaves, cut thin slices horizontally with a sterile blade, about 1 mm thick, and transfer them into embryogenic callus induction medium and incubate in the dark at 28°C.

[0066] 4. Change the embryogenic callus induction medium every 14 days until dense granular embryogenic callus is obtained.

[0067] 5. Take a single colony of the transformed Agrobacterium and incubate it overnight in 100 mL of YEP liquid medium containing spectinomycin (50 µg / mL). Then dilute it to OD600≈0.6.

[0068] 6. Take embryogenic callus and place it in a culture dish containing sterile filter paper. Air dry the surface of the embryogenic callus in a laminar flow hood. Use a sterile spatula to transfer the air-dried embryogenic callus to an Erlenmeyer flask and pour in 50 mL of the prepared Agrobacterium tumefaciens solution.

[0069] 7. Incubate in the dark at 28℃ and 100 rpm for 10 min, then transfer to an ultrasonic cleaner with a power of 150 W and treat with ultrasonic waves at maximum power for 2 min.

[0070] 8. Replace with 50 mL of Agrobacterium tumefaciens culture, place in a vacuum urn, treat under vacuum at 0.1 MPa for 5 min, then incubate in the dark at 28℃ and 100 rpm with low shaking for 10 min. Filter out the embryogenic callus tissue with a sterile sieve, blot dry the culture on sterile filter paper, and transfer to co-culture medium. Incubate in the dark at 21℃ for 3 days.

[0071] 9. Transfer the embryogenic callus tissues to recovery medium and culture them in the dark at 28°C for 7 days. Then transfer them to embryogenic callus screening medium containing glufosinate and culture them in the dark at 28°C for 30 days.

[0072] 10. The obtained resistant callus tissue was then transferred to the corresponding differentiation screening medium and cultured for differentiation screening for 40 days. The culture conditions were 30℃, 14 h / 10 h light-dark alternation, and daytime light intensity of 2000 Lux. The differentiation screening medium was replaced once during the differentiation screening culture.

[0073] 11. After the differentiation and selection culture is completed, select the best-growing shoots and transfer them to the rooting selection medium for rooting selection culture for 40 days. The culture conditions are 30℃, 14 h / 10 h light and dark alternation, and daytime light intensity of 2000 Lux. The rooting selection medium is changed in the middle of the rooting selection culture, and the stronger plants are selected for rooting culture.

[0074] 12. Select the seedlings that have taken root and transfer them to sterilized nutrient soil for growth. After two weeks of growth, take sugarcane leaves for later use.

[0075] Example 2

[0076] Target gene detection in sugarcane overexpressing the SSP1 gene

[0077] Sugarcane leaves grown for 2 weeks as described in Example 1 were placed in 2 mL centrifuge tubes, ground with grinding beads, and then frozen in liquid nitrogen. The leaves were then ground using a grinder, and total DNA was extracted using a plant genomic DNA extraction kit. The SSP1 gene was amplified by PCR using primers (SSP1-F, SSP1-R) as shown in SEQ ID NO. 3 and SEQ ID NO. 4. The 25 μL PCR reaction system included 10.5 μL of sterile double-distilled water (ddH2O), 12.5 μL of 2 × Easy Taq PCR SuperMix, 0.5 μL of SSP1-F (20 μmol / L), 0.5 μL of SSP1-R (20 μmol / L), and 1.0 μL of template DNA. The PCR reaction program was: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 35 cycles; 72℃ for 10 min. After amplification, 10 µL of the PCR amplification product was subjected to electrophoresis on a 1.5% agarose gel. Plants with an amplification band of approximately 351 bp were positive for overexpression of the SSP1 gene.

[0078] The nucleotide sequence of the SSP1 gene is shown in SEQ ID NO.1:

[0079] ATGGGTCAAGTTTTTCAGAATTTAAAAAAACAAGTTCTTCAGAAAAAATATGTGATTCTTCAGAAAAAGAAATTGAAATTTTAATATTTAAAATAAATGATTTATCAAAACAAATAAATTTAATTTTACAAAACATCGATAGAATAGAAAAATCATTATTATTCTCATTTAAAAAT TCAAAGTAAAAATAATTCTAATGTTGCAGAGAGAAATTCAAGTTTTTTGAAATTTCAAATATCTTTTTTAAAACAAAAGAAATCCTTACTTAAAAAACAATTAAATGAAAATAATTTTAGAAAAAATAGATTTGGAAATTAAATTAAGAAAAATTTTGTATTCTGATCAAAAATAG.

[0080] The nucleotide sequence of protein SSP1 is shown in SEQ ID NO.2:

[0081] MGQVFSEFKKTSSSEKICDSSEKEIEILIFKINDLSKQINLILQNIDRIEKSLLSHLKIQSKNNSNVAERNSSFLKFQISFLKQKKSLLKKQLNEIILEKIDLEIKLRKILYSDQK.

[0082] The nucleotide sequence of the SSP1-F primer is shown in SEQ ID NO.3:

[0083] ATGGGTCAAGTTTTTTCAGAATTT.

[0084] The nucleotide sequence of the SSP1-R primer is shown in SEQ ID NO.4:

[0085] CTATTTTTGATCAGAATACAAAAT.

[0086] Example 3

[0087] Sugarcane growth phenotypic observation

[0088] By comparing the germination rate and plant height of sugarcane overexpressing the SSP1 gene with wild-type sugarcane grown in pots and in the field, the specific experimental procedures and data are as follows.

[0089] Sugarcane overexpressing the SSP1 gene and wild-type sugarcane were cut into single bud segments and placed in trays. Water was added to cover the sugarcane stems, and the germination was observed in an incubator with 16℃, 60% relative humidity, and alternating light and dark conditions for 12 h / 12 ​​h. The bud germination rate of sugarcane overexpressing the SSP1 gene was significantly faster than that of wild-type sugarcane. Figure 1 This is a comparison of the germination of sugarcane overexpressing the SSP1 gene and wild-type sugarcane after 30 days of experimentation.

[0090] Sugarcane overexpressing the SSP1 gene and wild-type sugarcane were cut into single-bud segments and planted in flowerpots, with one single-bud segment per pot, and placed outdoors for growth. After 6 months, the sugarcane plant height was measured. The average plant height of the sugarcane overexpressing the SSP1 gene was 119 cm, and the average plant height of the wild-type sugarcane was 101 cm. Figure 2 This is a comparison of the plant height of sugarcane line 8 overexpressing the SSP1 gene and wild-type sugarcane after 3 months of potted cultivation.

[0091] Sugarcane overexpressing the SSP1 gene and wild-type sugarcane were cut into double-bud segments and planted in the field. After 6 months, the sugarcane plant height was measured. The average plant height of sugarcane overexpressing the SSP1 gene was 236 cm, and the average plant height of wild-type sugarcane was 212 cm. Figure 3 This is a statistical analysis chart of plant height after 6 months of field planting.

[0092] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A gene SSP1 that promotes sugarcane growth, characterized in that, The nucleotide sequence of the gene SSP1 is shown in SEQ ID NO.

1.

2. The SSP1 protein encoded by the gene SSP1 according to claim 1, characterized in that: The SSP1 protein is a protein whose amino acid sequence is shown in SEQ ID NO.

2.

3. A fusion protein, characterized in that, The fusion protein is obtained by attaching a protein tag to the N-terminus and / or C-terminus of the SSP1 protein according to claim 2.

4. A biomaterial, characterized in that: The biomaterial is any one of B1) to B4) below: B1) An expression cassette containing the sugarcane growth-promoting gene SSP1 as described in claim 1; B2) A recombinant vector containing the sugarcane growth promoting gene SSP1 as described in claim 1 or the expression cassette as described in B1); B3) A recombinant microorganism containing the sugarcane growth promoting gene SSP1 as described in claim 1, the expression cassette as described in B1), or the recombinant vector as described in B2); B4) A transgenic plant cell line containing the sugarcane growth promoting gene SSP1 as described in claim 1, the expression cassette as described in B1), the recombinant vector as described in B2), or the recombinant microorganism as described in B3).

5. The biomaterial according to claim 4, characterized in that, B2) The recombinant vector is obtained by effectively ligating the gene SSP1 into an expression vector.

6. The biomaterial according to claim 5, characterized in that, The expression vector is a plant overexpression vector.

7. The biomaterial according to claim 4, characterized in that, The recombinant microorganism is Escherichia coli or Agrobacterium.

8. The application of the gene SSP1 of claim 1, the SSP1 protein of claim 2, or the biomaterial of claim 4 in promoting sugarcane growth.

9. The application according to claim 8, characterized in that, The application involves introducing the SSP1 gene into sugarcane for expression to promote sugarcane growth.

10. A method for promoting sugarcane growth, characterized in that, The method involves introducing the SSP1 gene described in claim 1 into sugarcane for expression to promote sugarcane growth.

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