A method for improving drought resistance and sugar content of sugarcane through genetic transformation

Abstract

The present invention discloses a method for improving the drought resistance and sugar content of sugarcane through genetic transformation, belonging to the field of genetic engineering technology. By optimizing the AtDreb1A gene sequence and overexpressing the AtDreb1Asc gene in sugarcane, the present invention demonstrated that overexpression of the AtDreb1Asc gene improved the drought resistance of sugarcane compared to the control, while also significantly increasing the sugar content of the control and ensuring stable inheritance in the next generation. Through screening, the present invention cultivated transgenic sugarcane materials with superior drought tolerance and sugar content compared to the control, while maintaining unaffected growth and yield. This method lays the foundation for cultivating superior sugarcane varieties that are both drought-resistant and high in sugar content, without affecting growth.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, in particular to a method for improving the drought resistance and sugar content of sugarcane through genetic transformation. Background Art

[0002] Sugarcane is my country's most important sugar crop, with sugarcane sugar accounting for over 90% of the country's total sugar production. However, because most sugarcane-growing areas are located on dry, sloping land, with weak water infrastructure and uneven natural rainfall, drought has become a key constraint on my country's sugarcane production. Drought stress hinders sugarcane emergence and elongation, resulting in reduced yields. The promotion of drought-tolerant sugarcane varieties is urgently needed. While some sugarcane varieties excel in other aspects such as yield and sugar content, they lack drought tolerance. Therefore, cultivating sugarcane varieties that are both drought-resistant and perform well in other areas, providing the sugarcane industry with high-yield, stable, and drought-resistant varieties, is crucial for reducing sugarcane cultivation costs and improving production efficiency. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for improving the drought resistance and sugar content of sugarcane through genetic transformation to solve the problems existing in the above-mentioned prior art. By overexpressing the optimized AtDreb1Asc gene in sugarcane, the drought resistance and sugar content of sugarcane can be significantly improved without affecting growth, and the sugarcane can be stably inherited to the next generation.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a gene that is beneficial to improving the drought resistance and sugar content of sugarcane. The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0006] The present invention also provides a recombinant vector comprising the gene.

[0007] Preferably, the recombinant vector is obtained by introducing the gene into the expression vector pUMUK.

[0008] The present invention also provides a transgenic cell line comprising the recombinant vector.

[0009] The present invention also provides a method for improving the drought resistance and sugar content of sugarcane by genetic transformation, comprising the following steps:

[0010] The AtDreb1Asc gene was introduced into the expression vector pUMUK, genetic transformation was carried out in sugarcane, and transgenic positive plants were screened to obtain transgenic sugarcane with improved drought resistance and sugar content; the nucleotide sequence of the AtDreb1Asc gene is shown in SEQ ID NO: 1.

[0011] Preferably, the AtDreb1Asc gene is overexpressed in the sugarcane.

[0012] Preferably, the drought resistance and increased sugar content can be stably inherited to the next generation of transgenic sugarcane.

[0013] The present invention also provides the use of the gene, the recombinant vector or the transgenic cell line in any of the following:

[0014] (1) Application in improving drought resistance and sugar content of sugarcane;

[0015] (2) Application in constructing transgenic sugarcane with drought resistance and increased sugar content.

[0016] The present invention discloses the following technical effects:

[0017] This invention optimizes the AtDreb1A gene sequence and uses host-preferred codons to improve its expression efficiency in heterologous expression systems. The invention also overexpressed the AtDreb1Asc gene in sugarcane. Results showed that overexpression of the AtDreb1Asc gene improved drought tolerance in sugarcane compared to controls, while also improving sugar content and maintaining stable inheritance in the next generation. Through screening, the invention cultivated transgenic sugarcane materials with superior drought tolerance and sugar content compared to controls, while maintaining unaffected growth and yield. This approach lays the foundation for cultivating superior sugarcane varieties that are both drought-resistant and high in sugar content, without compromising growth and yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 The results of screening for AtDreb1Asc overexpressing transformants are shown in Figure 2. Dreb: sugarcane overexpressing AtDreb1Asc. WT: untransformed sugarcane.

[0020] Figure 2 PCR identification results of transgenic plants; the upper panel shows the T1 generation, and the lower panel shows the T2 generation. M: molecular weight standard, 1: negative control, 2: plasmid, 3: untransformed sugarcane, 4-8: transformed sugarcane.

[0021] Figure 3Phenotypic differences between transgenic sugarcane (D55) and wild type (CK) at 4 months (A), 6 months (B), and 8 months (C) under normal water supply and drought stress conditions; CK: untransformed sugarcane under normal water supply; CK-DS: untransformed sugarcane under drought stress; D55: transgenic sugarcane under normal water supply; D55-DS: transgenic sugarcane under drought stress;

[0022] Figure 4 Figure 5. Drought tolerance-related physiological indicators, yield, and sugar content differences between transgenic sugarcane (D55) and wild type (CK); A: phenylalanine ammonia lyase (PAL) activity; B: superoxide dismutase (SOD) activity; C: peroxidase (POD) activity; D: superoxide anion scavenging rate (SASR); E: proline (PRO) content; F: malondialdehyde (MDA) content; G: H2O2 content; H: polyphenol oxidase (PPO) activity; I: catalase (CAT) activity; J: yield; K: sugar content. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Example 1

[0029] 1. Construction of vector

[0030] The AtDreb1A gene sequence was optimized according to the codon preference of sugarcane (see SEQ ID NO: 1 for the optimized sequence), ligated into the multiple cloning site of the plant expression vector pUMUK (Patent No.: ZL202210026231.4), driven by the ubi promoter, and used for subsequent sugarcane transformation after sequencing.

[0031] 2. Sugarcane genetic transformation

[0032] The above vector was introduced into sugarcane by gene gun method to overexpress AtDreb1Asc in sugarcane (the specific steps are the same as those in patent "ZL202210026231.4"):

[0033] 2.1 Plasmid DNA extraction

[0034] Extract the constructed AtDreb1Asc OE vector plasmid DNA.

[0035] 2.2 Transformation of sugarcane by gene bombardment

[0036] (1) Callus preparation: Sugarcane callus was selected and 0.2 mol·L -1 Mannitol + 0.2 mol·L -1 The cells were pretreated on MS medium containing sorbitol.

[0037] (2) DNA encapsulation: Take 50 μL of gold powder suspension in an Eppendorf tube, and add the extracted plasmid DNA, 50 μL of 2.5 M CaCl2 and 20 μL of 0.1 M spermidine in sequence.

[0038] (3) Vortex the mixed sample for 1 min, place it on ice for 1 min, and centrifuge to remove the supernatant.

[0039] (4) Add 75% ethanol, centrifuge and remove the supernatant. Repeat this step 3 times and finally resuspend the pellet in anhydrous ethanol.

[0040] (5) Callus transformation: Callus tissue was transformed using gene gun method.

[0041] 2.3 Screening and culture

[0042] After the transformed callus tissue was restored to culture, it was transferred to MS subculture medium containing 40 mg / L Geneticin (G418) and cultured for 4 weeks. It was then transferred to MS differentiation medium containing G418 for differentiation. Successfully transformed callus could grow normally on screening medium (MS subculture medium + 40 mg / L G418), while untransformed callus could not grow (see Figure 1 ).

[0043] 2.4 PCR detection of resistant seedlings

[0044] The positive plants screened out were tested by PCR to obtain transgenic strains, and the transgenic seedlings were transplanted into the substrate for further cultivation.

[0045] The test results of T1 and T2 generations showed that the gene can be stably inherited. Figure 2 The PCR test conditions are:

[0046] The upstream primer (SEQ ID NO: 2): 5'-AAGAAGACCCGCATCTGGCT-3', and the downstream primer (SEQ ID NO: 3): 5'-GTTGTAAAACGACGGCCAGTG-3'. The reaction system consisted of 50.00 μL of 2× Es Taq Master Mix, 2.00 μL each of the upstream and downstream primers, 2.00 μL of DNA template, and 19.00 μL of double-distilled water. The reaction conditions were: 95°C for 3 min, followed by 35 cycles of 95°C for 30 s, 58°C for 30 s, 72°C for 1 min, and 72°C for 5 min.

[0047] 3. Evaluation and testing of relevant indicators

[0048] Drought tolerance tests were conducted on transgenic sugarcane, with untransformed sugarcane serving as the control (CK). The experimental materials were divided into two groups: one maintained with normal water supply and the other subjected to intermittent water deprivation, with 30 replicates per group. Water was withheld for approximately 12 days each time, at 4, 6, and 8 months after planting, until the leaves of the control sugarcane wilted. Drought-resistant phenotypes and physiological traits of the transgenic sugarcane were observed and tested. Physiological parameters were measured at 6 months old, from the cane and one leaf, for a total of 12 samples. Leaf tissue was quickly frozen in liquid nitrogen and stored at -80°C until later use. Sugarcane samples grown for 10 months were collected for yield and sugar content testing. Commercial kits were used to measure peroxidase (POD) activity, superoxide dismutase (SOD), polyphenol oxidase (PPO) activity, superoxide anion scavenging rate (SAFR), and proline (PRO) content. High-performance liquid chromatography was also used to determine sugar content in both transgenic and control sugarcane.

[0049] 4. Experimental results

[0050] Drought stress experiments were conducted on T1 and T2 transgenic sugarcane plants, and the phenotypic differences between the control group and the transgenic group under normal and drought conditions were compared. Under normal conditions, the CK plants showed healthy growth with green leaves. However, under drought stress, the CK plants showed significant leaf wilting and yellowing, indicating that drought seriously affected plant growth (see Figure 3 In contrast, the transgenic group also showed good growth under normal conditions (see Figure 3 Under drought stress, although the transgenic sugarcane showed some wilting and yellowing of leaves, its overall performance was significantly better than that of the control plants. The transgenic plants wilted less severely and had less yellowing of leaves under drought conditions, indicating that the transgenic plants had stronger drought resistance under drought conditions (see Figure 3 ).

[0051] Analysis of drought-resistant related physiological and biochemical indicators showed that after drought treatment, compared with CK, transgenic sugarcane showed higher phenylalanine ammonia lyase activity and antioxidant enzyme activities such as peroxidase, superoxide dismutase, and polyphenol oxidase under drought conditions, higher superoxide anion scavenging rate and higher proline content, while H2O2 and malondialdehyde accumulation were significantly lower than the control. The antioxidant enzyme activity of transgenic sugarcane under drought conditions was significantly higher than that of the control. They can synergistically clear a large amount of reactive oxygen formed by drought stress, thereby inhibiting membrane lipid peroxidation, allowing metabolic activities to proceed normally, and enhancing the drought resistance of the plant. Yield and sugar content are the most important traits of sugarcane varieties. Under normal water supply conditions, the yield of transgenic sugarcane had no significant difference from that of the control; under drought conditions, the yield of non-transgenic sugarcane was significantly affected, while the yield of transgenic sugarcane was significantly higher than that of the control. The sugar content of transgenic sugarcane was significantly higher than that of the control under normal water supply and drought conditions, indicating that overexpression of this gene increased the sugar content of sugarcane. Figure 4 .

[0052] SEQ ID NO: 1

[0053] .

[0054] The above results show that after screening multiple transgenic strains in T1 and T2 generations, the present invention has cultivated transgenic sugarcane materials that are stably inherited, have better drought tolerance than the control, and have no effect on growth and yield, and have higher sugar content than the control.

[0055] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for improving the drought resistance and sugar content of sugarcane by genetic transformation, characterized in that: The following steps are involved: The AtDreb1Asc gene was introduced into the expression vector pUMUK, genetically transformed into sugarcane, and transgenic positive plants were screened to obtain transgenic sugarcane with improved drought resistance and sugar content; the nucleotide sequence of the AtDreb1Asc gene is shown in SEQ ID NO: 1; The AtDreb1Asc gene is overexpressed in the sugarcane.

2. The method according to claim 1, wherein The drought resistance and increased sugar content properties can be stably inherited to the next generation of the transgenic sugarcane.

3. Use of the AtDreb1Asc gene or a recombinant vector comprising the AtDreb1Asc gene in any of the following: (1) Application in improving drought resistance and sugar content of sugarcane; (2) Application in the construction of transgenic sugarcane with drought resistance and increased sugar content; The nucleotide sequence of the AtDreb1Asc gene is shown in SEQ ID NO: 1; The recombinant vector is constructed by introducing the AtDreb1Asc gene into the expression vector pUMUK; The AtDreb1Asc gene is overexpressed in the sugarcane to improve the drought resistance and sugar content of the sugarcane.

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