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

Abstract

The invention discloses a method for improving drought resistance and sugar content of sugarcane through genetic transformation, and belongs to the technical field of genetic engineering. By optimizing the AtDreb1A gene sequence and overexpressing the AtDreb1Asc gene in the sugarcane, the result shows that compared with a control group, the overexpressed AtDreb1Asc gene can improve the drought tolerance of the sugarcane, the sugar content is obviously higher than that of the control group, and the sugarcane can be stably inherited in the next generation. According to the invention, the transgenic sugarcane material with drought tolerance and sugar content superior to those of a control group and without influence on growth and yield is screened and cultivated, and a foundation is laid for cultivation of excellent sugarcane varieties with drought resistance, high sugar content and without influence on growth.

Description

Technical Field

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

[0002] Sugarcane is the most important sugar crop in my country, and sugarcane sugar accounts for more than 90% of the country's total sugar production. However, since most sugarcane areas are located on dry slopes, basic water conservancy facilities are weak, and natural precipitation is uneven, drought has become one of the key factors restricting my country's sugarcane production. Under drought stress, sugarcane seedlings are difficult to emerge and elongation is hindered, resulting in a reduction in sugarcane production. There is an urgent need to promote drought-resistant sugarcane varieties in production. Although some sugarcane varieties perform well in other aspects such as yield and sugar content, they have poor drought tolerance. Therefore, cultivating sugarcane varieties that are both drought-resistant and excellent in other aspects, and providing the sugarcane industry with high-yield, stable-yield, and drought-resistant variety materials, is of great significance to reducing sugarcane planting 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 by 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, and the growth is not affected, and it 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 which 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 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 properties 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 the 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] The present invention optimizes the AtDreb1A gene sequence and uses codons with host preference, which is conducive to improving its expression efficiency in a heterologous expression system; the present invention overexpresses the AtDreb1Asc gene in sugarcane, and the results show that compared with the control, overexpression of the AtDreb1Asc gene can improve the drought resistance of sugarcane, while the sugar content is better than the control, and can be stably inherited in the next generation. Through screening, the present invention cultivates transgenic sugarcane materials with better drought resistance and sugar content than the control, and unaffected growth and yield, laying a foundation for cultivating excellent sugarcane varieties that are both drought-resistant and high in sugar content, and unaffected 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0019] Figure 1 The results of screening for AtDreb1Asc overexpression transformants; Dreb: sugarcane overexpressing AtDreb1Asc. WT: untransformed sugarcane;

[0020] Figure 2 The PCR identification results of transgenic plants; the upper figure is T1 generation, and the lower figure is 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 The figures show the differences in physiological indicators related to drought tolerance, yield and sugar content 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 clearance 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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 (the optimized sequence is shown in SEQ ID NO: 1), connected to the multiple cloning site of the plant expression vector pUMUK (patent number: 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 this step 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 biolistic 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 CaCl 2 and 20 μL 0.1 M spermidine.

[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 three times and finally resuspend the pellet in anhydrous ethanol.

[0040] (5) Callus transformation: Callus transformation is performed using gene gun technique.

[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 the 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 matrix 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] Upstream primer (SEQ ID NO: 2): 5'-AAGAAGACCCGCATCTGGCT-3' and downstream primer (SEQ ID NO: 3): 5'-GTTGTAAAACGACGGCCAGTG-3'. The reaction system is: 50.00 μL system contains 2×Es Taq Master Mix 25.00 μL, 2.00 μL of upstream and downstream primers, 2.00 μL of DNA template, and 19.00 μL of double distilled water. The reaction conditions are: 95℃3min; 35 cycles of 95℃30s, 58℃30s, 72℃1min; 72℃5min.

[0047] 3. Evaluation and testing of relevant indicators

[0048] The drought tolerance test was conducted on transgenic sugarcane materials, and the untransformed sugarcane was used as the control (CK). The test materials were divided into two groups, one group maintained normal water supply, and the other group was subjected to intermittent water cut-off treatment, with 30 replicates in each group. Water cut-off treatment was carried out at 4, 6, and 8 months after planting, and each water cut-off lasted about 12 days until the leaves of the control sugarcane wilted. The drought resistance-related phenotypes and physiological traits of transgenic sugarcane were observed and tested. In the 6th month, samples were taken to measure relevant physiological indicators, and the sampling site was sugarcane + 1 leaf, with a total of 12 samples. The leaf tissues after sampling were quickly frozen in liquid nitrogen and then stored at -80℃ for later use. Sugarcane grown for 10 months was taken for yield and sugar content testing. The commercial kits were used to determine the content of indicators such as peroxidase (POD) activity, superoxide dismutase (SOD), polyphenol oxidase (PPO) activity, superoxide anion scavenging rate (SAFR), and proline (PRO); and the sugar content of transgenic sugarcane and the control was tested by high-performance liquid chromatography.

[0049] 4. Experimental results

[0050] Drought stress experiments were conducted on T1 and T2 transgenic sugarcane, and the phenotypic differences between the control group and the transgenic group under normal and drought conditions were compared. Under normal conditions, CK plants showed healthy growth with green leaves. However, under drought stress, CK plants showed significant leaf wilting and yellowing, indicating that drought severely affected plant growth (see Figure 3 In contrast, the transgenic group also showed good growth under normal conditions (see Figure 3 ). Although the transgenic sugarcane showed some wilting and yellowing of leaves under drought stress, its overall performance was significantly better than that of the control plants. The transgenic plants wilted less 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-resistance-related physiological and biochemical indices 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, higher superoxide anion scavenging rate and higher proline content under drought conditions, while H 2 O 2 and malondialdehyde accumulation were significantly lower than those of the control. The antioxidant enzyme activity of transgenic sugarcane under drought conditions was significantly higher than that of the control. Their synergistic effect can remove 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, there is no significant difference in the yield of transgenic sugarcane from that of the control; under drought conditions, the yield of non-transgenic sugarcane is significantly affected, while the yield of transgenic sugarcane is significantly higher than that of the control. The sugar content of transgenic sugarcane is significantly higher than that of the control under normal water supply and drought conditions, indicating that overexpression of this gene increases the sugar content of sugarcane, see 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 resistance 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 only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A gene that is beneficial for improving the drought resistance and sugar content of sugarcane, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. A recombinant vector, characterized in that: Comprising the gene according to claim 1.

3. The recombinant vector according to claim 2, characterized in that The recombinant vector is obtained by introducing the gene into the expression vector pUMUK.

4. A transgenic cell line, characterized in that Comprising the recombinant vector according to claim 2 or 3.

5. 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, 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.

6. The method according to claim 5, characterized in that The AtDreb1Asc gene is overexpressed in the sugarcane.

7. The method according to claim 5, characterized in that The drought resistance and improved sugar content properties can be stably inherited to the next generation of the transgenic sugarcane.

8. Use of the gene according to claim 1, the recombinant vector according to any one of claims 2 to 3, or the transgenic cell line according to claim 4 in any of the following: (1) Application in improving the drought resistance and sugar content of sugarcane; (2) Application in constructing transgenic sugarcane with drought resistance and increased sugar content.

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