A method for genetically engineering to synergistically increase biomass and sugar yield of sugarcane

Abstract

The application discloses a kind of gene engineering methods for synergistically improving the biomass and sugar yield of sugarcane, belonging to the field of biotechnology.The application identifies the GRAS gene (HB gene) specific to PACMAD group by comparative genomics method, and overexpresses it in sugarcane.The HB gene has the nucleotide sequence shown in SEQ ID NO.1, and encodes the amino acid sequence shown in SEQ ID NO.2.The experimental results show that the vascular development of HB gene overexpressed sugarcane plants is more perfect, and the organic matter transport capacity is enhanced.At the growth period of 8 months, the plant height, single node length and stem diameter are increased by 11.85%, 11.83% and 8.20% respectively compared with the wild type.At the growth period of 12 months, the plant height and hammer value are increased by 8.11% and 9.66% respectively compared with the wild type, and the sugar yield per plant is increased by 27.88%.The application realizes the synergistic improvement of the biomass, yield and sugar yield of sugarcane, solves the problem that high yield and high sugar of sugarcane are difficult to be considered in the prior art, and has important significance for improving the comprehensive economic benefit and market competitiveness of sugarcane industry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant genetic engineering, and specifically relates to a genetic engineering method for synergistically improving the biomass and sugar yield of sugarcane. BACKGROUND

[0002] Sugarcane is the most important sugar crop and energy crop in the world, and occupies an irreplaceable position in the national economy and energy strategy. According to statistics, about 75% of the world's sugar comes from sugarcane, and its high-fiber stems are also one of the core raw materials for biomass energy power generation and fuel ethanol production. As a major sugarcane planting country in the world, China's sugarcane planting area is concentrated in the tropical and subtropical regions of the south. The sugarcane industry is not only the economic backbone of millions of farmers in the south, but also the key support for ensuring national sugar security and promoting the development of renewable energy industries.

[0003] With the expansion of sugar demand caused by population growth and the rapid development of the biomass energy industry, the demand for sugarcane is continuously rising, which puts higher requirements on the production capacity of sugarcane - not only the yield of sugarcane stems per unit area needs to be improved, but also the biomass and sugar yield need to be improved simultaneously. However, the current production of sugarcane in China and even the world is facing multiple bottlenecks, which restricts the synergistic improvement of the above indicators.

[0004] From the perspective of cultivation environment and resource constraints, sugarcane producing areas often face problems such as drought, soil infertility, and continuous cropping obstacles. Drought stress can lead to a decrease in photosynthetic efficiency and an imbalance in transpiration, which in turn inhibits stem elongation and sucrose accumulation; while long-term continuous cropping can cause soil nutrient imbalance and pathogen enrichment, leading to a decline in root activity and hindered biomass accumulation. At the same time, under the traditional planting mode, the unreasonable use of fertilizers is a common problem, which not only leads to an imbalance in the vegetative growth of sugarcane and a decrease in sugar content, but also causes soil acidification and environmental pollution, further exacerbating production problems.

[0005] In terms of varieties and technology, although the existing main sugarcane varieties have certain advantages in stress resistance or yield, they generally have the defect of insufficient synergistic improvement of biomass, yield, and sugar yield: some high-biomass varieties have low sucrose conversion rate, making it difficult to meet the sugar production demand; while some high-sugar varieties have the problems of weak stems, poor resistance to lodging, and low biomass, limiting their comprehensive utilization value. In addition, the existing technologies for improving the production performance of sugarcane mainly focus on the optimization of a single indicator, such as increasing the sugar yield by increasing the application of potassium fertilizer, or increasing the biomass by dense planting, but such technologies often ignore the overall nature of sugarcane growth and development - the improvement of a single indicator may come at the expense of other indicators, and cannot achieve the synergistic improvement of the three, and the stability of the technology is poor and is easily affected by environmental factors.

[0006] In the application of molecular biology and physiological regulation technologies, genetic engineering has become an effective way to overcome the inherent defects of sugarcane varieties. For example, introducing improved stress-resistance genes into major sugarcane varieties has achieved a synergistic improvement in drought resistance and sucrose content. Although existing research has identified some genes related to sugarcane photosynthetic efficiency and sucrose synthesis, most studies remain at the stage of gene function verification, lacking the discovery and application of key regulatory genes for growth and development. Of particular interest is the GRAS family of transcription factors, which play a central role in the regulation of plant growth and development. The HB gene, derived from maize, belongs to this family and regulates the expression of downstream target genes through a specific DNA-binding domain, deeply participating in key biological processes such as plant architecture, nutrient allocation, and responses to abiotic stresses.

[0007] Currently, in the field of sugarcane molecular breeding, there are no studies on applying maize GRAS family HB genes to improve yield and sugar production. Existing technologies still lack effective solutions for simultaneously optimizing sugarcane plant architecture, stress resistance, biomass allocation, and sucrose accumulation through heterologous expression of these genes, resulting in the inability to achieve synergistic improvement of biomass, yield, and sugar production through targeted regulation of the "source-sink-flow" system.

[0008] Therefore, in response to the urgent needs of the sugarcane industry and the shortcomings of existing technologies, and taking advantage of the clear functional advantages of the HB gene in the growth regulation of grass plants, a genetic engineering technique for introducing the maize HB gene into sugarcane has been developed. By precisely regulating sugarcane plant architecture, biomass accumulation, and sucrose synthesis, this technique can overcome environmental and varietal limitations, achieve a synergistic improvement in sugarcane biomass, yield, and sugar production, and is simple to operate, highly adaptable, and environmentally friendly. It has become a core task for the current upgrading and technological innovation of the sugarcane industry, and has significant economic and ecological value. Summary of the Invention

[0009] The purpose of this invention is to provide a genetic engineering method for synergistically improving sugarcane biomass and sugar production. By overexpressing the HB gene in sugarcane, the method achieves a synergistic improvement in sugarcane biomass, yield, and sugar production, thus solving the problem in the prior art that it is difficult to achieve both high yield and high sugar content in sugarcane.

[0010] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides a genetic engineering method for synergistically increasing sugarcane biomass and sugar production, comprising overexpressing the HB gene in sugarcane, wherein the HB gene has a nucleotide sequence as shown in SEQ ID NO.1 and encodes an amino acid sequence as shown in SEQ ID NO.2, thereby enabling simultaneous increase in sugarcane biomass and sugar production.

[0011] Furthermore, overexpression of the HB gene in sugarcane includes the following steps: S1. Obtain the HB gene; S2. Insert the HB gene into a plant expression vector to construct an overexpression vector; S3. The overexpression vector obtained in step S2 is introduced into Agrobacterium EHA105 strain; S4. The Agrobacterium obtained in step S3 is used to infect sugarcane embryonic callus and co-cultured. S5. The co-cultured callus tissue was screened, differentiated, and rooted to obtain transgenic sugarcane regenerated plants. S6. Stable sugarcane plants overexpressing the HB gene were obtained by PCR amplification and screening. PCR amplification was performed using primer pairs 43300-F and 43300-R. The sequence of primer 43300-F is shown in SEQ ID NO.3, and the sequence of primer 43300-R is shown in SEQ ID NO.4.

[0012] Further, in step S1, the method for obtaining the HB gene includes: extracting total RNA from maize KN5585 immature embryos, reverse transcribing to obtain cDNA, and using the cDNA as a template, performing PCR amplification using primer pairs G_0210_4_F and G_0210_4_R, wherein the primer sequence of G_0210_4_F is shown in SEQ ID NO.5, and the primer sequence of G_0210_4_R is shown in SEQ ID NO.6.

[0013] Further, in step S2, the plant expression vector is a 3300 vector that has been double-digested with BamHI and SacI, and the HB gene is ligated to the digested vector through homologous recombination.

[0014] Furthermore, it also includes step S7: planting the HB gene overexpressing sugarcane plants obtained in step S6 under isolation conditions and periodically measuring plant growth indicators, including plant height, single node length, stem diameter and stem thickness.

[0015] Further, in step S7, the measurement includes: measuring the height of 8 nodes, the length of a single node, and the stem diameter at 8 months of the plant's growth period, and measuring the plant height and stem diameter at 12 months of the plant's growth period.

[0016] Furthermore, step S7 also includes making a transverse cut on the sugarcane leaf 30cm from the leaf tip and calculating the ratio of the vascular area to the mesophyll area of ​​five consecutive leaves.

[0017] The present invention also provides a kit for obtaining transgenic sugarcane by the method, comprising: an overexpression vector containing the HB gene, Agrobacterium EHA105 strain, and PCR primers for screening transgenic plants.

[0018] The beneficial effects of this invention are as follows: Sugarcane plants overexpressing the HB gene obtained through the method of this invention exhibit increased plant height, single node length, and stem diameter by 11.85%, 11.83%, and 8.20% respectively compared to the wild type at 8 months of growth; at 12 months of growth, plant height and stem diameter are increased by 8.11% and 9.66% respectively compared to the wild type, and sugar yield per plant is increased by 27.88%. This invention achieves a coordinated improvement in biomass, yield, and sugar production, which can both expand sugarcane biomass to meet the needs of biomass energy development and ensure the core production benefits of this sugar crop. It provides an efficient and precise technical path for the cultivation of superior sugarcane varieties and is of great significance for improving the overall economic benefits and market competitiveness of the sugarcane industry. Attached Figure Description

[0019] Figure 1 To construct a phylogenetic tree of Gramineae GRAS genes using MAFFT and FastTree.

[0020] Figure 2 The figures include statistics on various physiological indicators of sugarcane and photographs of the plants. Figure A is a bar chart of plant height at 8 months of growth, Figure B is a bar chart of single-internode length at 8 months of growth, Figure C is a violin-shaped bar chart of stem diameter at 8 months of growth, Figure D is a bar chart of plant height at 12 months of growth, Figure E is a bar chart of helix length at 12 months of growth, Figure F is a box plot of the ratio of five consecutive vascular areas to leaf mesophyll area at 8 months of growth, and Figure G is a photograph of sugarcane at 12 months of growth.

[0021] Figure 3 This is a graph showing the sugar yield of a single sugarcane plant. Detailed Implementation

[0022] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0023] Example 1: Evolutionary analysis of the ancestral HB gene of PACMAD To screen for dominant genes in the evolution of the PACMAD taxa, 46 high-quality genomic genomes of the Poaceae family were collected from plant genome databases such as Gramene and Phytozome. Comparative genomic analysis was then performed to identify GRAS genes specific to the PACMAD taxa. According to literature reports, the screened GRAS gene (named High Biomass, HB, based on its biomass-increasing properties) may be involved in the development of vascular bundles in the PACMAD taxa, influencing plant size and nutrient transport.

[0024] To further determine the classification and function of HB in the GRAS transcription factor family (Figure 1 The study found that the HB gene belongs to the LISCL subfamily, and the two GRAS genes with similar evolutionary positions do not exhibit branch-specific distributions in all Poaceae families, suggesting that the HB gene likely arose after the divergence of PACMAD and BOP. Further analysis revealed that the HB gene's sister GRAS gene is located in a proximal position within the genome of the same species, indicating that the HB gene's origin may be a result of tandem duplication, and that it subsequently acquired new functions during evolution.

[0025] Currently, the HB genes identified using comparative genomics include Pgl_GLEAN_10021578, Pgl_GLEAN_10021577, CsA702231, CsA702236.1, Cl032106, Cl032106, rna-gnl|WGS_LWDX|Do010510.1, rna-gnl|WGS_LWDX|Do010510.1, rna-gnl|WGS_LWDX|Do010510.1, De010331g0300.1, De011001g0189.1, De011001g0189.1, and De011001g0189.1. , De011001g0189.1, AH05.717, BH05.779, BT05.1099, TVU18330, evm.model.ctg4981, evm.model.ctg4982, Eru01G003650, Eru07G006240, Eru07G0 06241, rna-NCGR_LOCUS3508, rna-NCGR_LOCUS3508, rna-NCGR_LOCUS9015, rna-NCGR_LOCUS9016, rna-NCGR_LOCUS56537, rna-NCGR_LOCUS56538, rn a-NCGR_LOCUS56538, rna-NCGR_LOCUS59015, rna-NCGR_LOCUS59016, Oropetium_20150105_27740A.v1.0, rna-XM_015836963.2, KN538901.1_FGT0 01, rna-XM_025949443.1, longmi025112, longmi028927, longmi028928, rna-XM_039980348.1, rna-XM_039980349.1, rna-XM_039942950.1, rna-XM _039942949.1, rna-XM_039942948.1, Pavag09G010600.1.v3.1, Pavag09G010700.1.v3.1, Pau_c03263_0020., Pau_c03263_0020, Pau_c03263_002 0. Pau_c03464_0020.07G0011460-3C, SSpon.07G0011460-4D, Sh09_t001300, Sh09_t001310, rna-XM_0228 25063.1, rna-XM_022824953.1, rna-XM_034732865.1, rna-XM_034732861, KXG38103 The gene sequenced as follows: KXG21079, KXG21080, Sp2s00032_12171, Sp2s00032_12171, Sp2s00032_12171, Zm00001eb266930, Zla01G013220.1, Zjn_sc00016.1.g06200.1.am.mk. In subsequent functional validation, the maize HB gene was used for experiments. Its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0026] Example 2: Preparation and identification of sugarcane plants overexpressing the HB gene 1. Construction of overexpression vectors (1) Total RNA was extracted from the embryos of maize KN5585 (a maize inbred line name) and reverse transcribed to obtain cDNA.

[0027] (2) Using the cDNA obtained in step (1) as a template, PCR amplification was performed using primer pairs composed of G_0210_4_F and G_0210_4_R (Table 1) to obtain the amplification product.

[0028] Table 1 Primer sequence information for amplifying the HB gene G_0210_4_R 5'-AACGATCGGGGAAATTCGCTGATTAGTATATGATTCCTTTGGTTTCCATGAGG -3' (SEQ ID NO. 6) (3) The amplification product recovered in step (2) was combined with the 3300 vector digested by BamhI and SacI to carry out homologous recombination reaction, and the recombination product was transformed into DH5α Escherichia coli competent cells.

[0029] (4) E. coli were screened on Kana-resistant LB solid medium and sequenced to confirm that the target fragment was ligated into the 3300 vector.

[0030] 2. Preparation of HB gene overexpression plants (1) The final vector plasmid 3300-UBI-HB was introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium containing recombinant plasmid 3300-UBI-HB, which was named Agrobacterium EHA105-3300-UBI-HB.

[0031] (2) Agrobacterium EHA105-3300-UBI-HB was used to soak and infect the embryogenic callus tissue of sugarcane, and then co-culture, screening culture, differentiation culture and rooting culture were carried out in sequence to obtain 22 T0 generation regenerated plants.

[0032] (3) T0 generation regenerated plants were used as test plants, and transgenic plants were screened by PCR identification.

[0033] PCR identification method: Take leaves of the test plant, extract genomic DNA, and use primer pair composed of 43300-F and 43300-R (Table 2) for PCR identification. If the amplification product of 330 bp is shown, the test plant is a transgenic plant.

[0034] Table 2 Primer sequence information for identifying transgenic plants 43300-R 5'-TGGCGTGTGAGGGTGCAGAA-3' (SEQ ID NO. 4) 3. Identification of sugarcane agronomic traits and yield Test plants: wild-type sugarcane plants (12 plants, CK) and T2 generation plants of the OEHB line (12 plants, UBI::HB).

[0035] The test plants were planted at the Wenchang Experimental Base of the Ministry of Agriculture's Transgenic Plants and Plant Microorganisms Environmental Safety Supervision and Testing Center (Haikou), Coconut Research Institute Team 4, Maihao Town, Wenchang City, Hainan Province.

[0036] At eight months of growth, the height of eight nodes, the length of a single node, and the stem diameter were measured. The results showed that at eight months of growth, the average height of eight nodes for wild-type sugarcane was 122.4 cm, while the average height of eight nodes for HB-overexpressing sugarcane was 136.9 cm. Figure 2 (A) The average length of a single segment in the wild type was 15.3 cm, and the average length of a single segment in the HB-overexpressing type was 17.11 cm. Figure 2 The average stem diameter of the wild type was 21.95 mm, and the average stem diameter of the HB-overexpressing type was 23.75 mm. Figure 2 (C) Compared to wild-type sugarcane, plants overexpressing HB (UBI::HB) have longer single nodes, taller plants, and thicker stems.

[0037] In addition, cross sections were taken of sugarcane leaves 30 cm from the leaf tip, and the ratio of five consecutive vascular areas to the leaf mesophyll area was calculated. The results showed that the ratio of five consecutive vascular areas to the leaf mesophyll area in wild-type sugarcane plants was 0.412, while that in HB-overexpressing plants was 0.4701. Figure 2 (F in the text). Compared with wild-type sugarcane, the proportion of UBI::HB bundle sheath was significantly increased, indicating that overexpression of the HB gene in sugarcane can improve the plant's ability to transport organic matter.

[0038] Plant height and height were measured at 12 months of the plant's growth period.

[0039] The statistical results are shown in Figures E and F below.

[0040] The results showed that, at twelve months of growth, the average plant height of wild-type sugarcane was 3.587 m, while the average plant height of HB-overexpressing sugarcane was 3.878 m. Figure 2 The average hammer angle of wild-type sugarcane was 20.91°Bx, and the average hammer angle of HB-overexpressing sugarcane was 22.93°Bx. Figure 2 F in the text). Overexpression of HB (UBI::HB) resulted in a plant height that was still higher than that of wild-type sugarcane. Figure 2 The expression of the HB gene in sugarcane, which is higher than that of the wild type, indicates that overexpression of the HB gene in sugarcane can increase sugar production in addition to increasing biomass and yield, thus achieving a coordinated improvement in biomass, yield and sugar production.

[0041] Based on statistical calculations of various physiological values, overexpression of the HB gene in sugarcane can increase sugar yield by 27.88% per plant. Figure 3 A single wild-type sugarcane plant can produce 173.949g of sugar, while after overexpression of the HB gene in sugarcane, a single sugarcane plant can produce approximately 222.438g of sugar.

Claims

1. A genetic engineering method for synergistically enhancing sugarcane biomass and sugar yield, characterized in that, This includes overexpressing the HB gene in sugarcane, the HB gene having the nucleotide sequence shown in SEQ ID NO.1 and encoding the amino acid sequence shown in SEQ ID NO.2, thereby enabling simultaneous increase in sugarcane biomass and sugar production.

2. The method according to claim 1, characterized in that, Overexpression of the HB gene in sugarcane includes the following steps: S1. Obtain the HB gene as described in claim 1; S2. Insert the HB gene into a plant expression vector to construct an overexpression vector; S3. The overexpression vector obtained in step S2 is introduced into Agrobacterium EHA105 strain; S4. The Agrobacterium obtained in step S3 is used to infect sugarcane embryonic callus and co-cultured. S5. The co-cultured callus tissue was screened, differentiated, and rooted to obtain transgenic sugarcane regenerated plants. S6. Stable sugarcane plants overexpressing the HB gene were obtained by PCR amplification and screening. PCR amplification was performed using primer pairs 43300-F and 43300-R. The sequence of primer 43300-F is shown in SEQ ID NO.3, and the sequence of primer 43300-R is shown in SEQ ID NO.

4.

3. The genetic engineering method according to claim 2, characterized in that, In step S1, the method for obtaining the HB gene includes: extracting total RNA from maize KN5585 immature embryos, reverse transcribing to obtain cDNA, and using the cDNA as a template, performing PCR amplification using primer pairs G_0210_4_F and G_0210_4_R, wherein the primer sequence of G_0210_4_F is shown in SEQ ID NO.5, and the primer sequence of G_0210_4_R is shown in SEQ ID NO.

6.

4. The genetic engineering method according to claim 2, characterized in that, In step S2, the plant expression vector is a 3300 vector that has been double-digested with BamHI and SacI, and the HB gene is ligated to the digested vector through homologous recombination.

5. The genetic engineering method according to claim 2, characterized in that, The method also includes step S7: planting the HB gene overexpressing sugarcane plants obtained in step S6 under isolation conditions and periodically measuring plant growth indicators, including plant height, single node length, stem diameter and stem thickness.

6. The genetic engineering method according to claim 5, characterized in that, In step S7, the measurement includes: measuring the height of 8 nodes, the length of a single node, and the stem diameter at 8 months of the plant's growth period, and measuring the plant height and stem diameter at 12 months of the plant's growth period.

7. The genetic engineering method according to claim 5, characterized in that, Step S7 also includes making a transverse cut on the sugarcane leaf 30cm from the leaf tip and calculating the ratio of the vascular area to the leaf mesophyll area of ​​five consecutive leaves.

8. A kit for obtaining transgenic sugarcane by the method according to any one of claims 1-7, characterized in that, include: An overexpression vector containing the HB gene, Agrobacterium EHA105 strain, and PCR primers for screening transgenic plants.

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