Application of ZmGOLS2 gene and ZmRAFS gene in improving pollen viability under heat shock and drought conditions of corn

Abstract

The application discloses application of ZmGOLS2 genes and ZmRAFS genes in improving pollen vitality of corn under heat shock and drought conditions, and belongs to the technical field of genetic engineering. The application improves the pollen vitality of corn, promotes the germination of pollen under heat shock and drought stress conditions, and reduces the empty stalk rate of corn under drought stress conditions by simultaneously improving the expression levels of a corn myo-inositol galactoside synthetase coding gene and a corn raffinose synthetase coding gene.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the ZmGOLS2 gene and the ZmRAFS gene in improving pollen viability in maize under heat shock and drought conditions. Background Technology

[0002] With global warming, heat damage has become a major factor limiting the further development of my country's maize industry. Raffinose plays an important role in maize's resistance to abiotic stress. Galactinol synthase (GOLS2) and raffinose synthase (RAFS) are key enzymes responsible for raffinose synthesis (Frydman et al., 1963, Synthesis of galactosylinositol by extracts from peas. Biochemical and biophysical research communications. 12:121-125; Peterbauer et al., 2002, Functional expression of a cDNA encoding pea (Pisum sativum L.) raffinose synthase, partial purification of the enzyme from maturing seeds, and steady-state kinetic analysis of raffinose synthesis. Planta. 215:839-846). In 2016, Gu reported that overexpression of maize ZmGOLS2 in Arabidopsis improved the drought, salt, and heat resistance of Arabidopsis seedlings (Gu et al., 2016, ZmGOLS2, a target of transcription factor ZmDREB2A, offers similar protection against abiotic stress as ZmDREB2A. Plant Molecular Biology. 90:157-170); Liu reported in 2023 that overexpression of the ZmRAFS gene in maize improved the water retention capacity of maize plants under drought stress (Liu et al., 2023, Raffinose positively regulates maize drought tolerance by reducing leaf transpiration. The Plant Journal 114:55-67). However, there are still no reports on the application of raffinose in regulating maize pollen viability and reducing the rate of barren stalks under drought stress. Summary of the Invention

[0003] The purpose of this invention is to provide the application of the ZmGOLS2 gene and the ZmRAFS gene in improving pollen viability in maize under heat shock and drought conditions, so as to solve the problems existing in the prior art.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] This invention provides the application of the ZmGOLS2 gene and the ZmRAFS gene in improving the pollen viability of maize under heat shock conditions. Overexpression of the ZmGOLS2 gene and the ZmRAFS gene increases the germination rate of maize pollen under heat shock conditions.

[0006] This invention also provides recombinant vectors, expression cassettes, and transgenic lines containing the ZmGOLS2 and ZmRAFS genes for improving pollen viability in maize under heat shock conditions.

[0007] The present invention also provides a method for improving pollen viability in maize under heat shock conditions by overexpressing the ZmGOLS2 and ZmRAFS genes in maize plants.

[0008] This invention also provides the application of the ZmGOLS2 gene and the ZmRAFS gene in improving the pollen viability of maize under drought conditions. Overexpression of the ZmGOLS2 gene and the ZmRAFS gene increases the germination rate of maize pollen under drought conditions.

[0009] This invention also provides the application of recombinant vectors, expression cassettes, and transgenic lines containing the ZmGOLS2 and ZmRAFS genes in improving pollen viability in maize under drought conditions.

[0010] The present invention also provides a method for improving pollen viability in maize under drought conditions by overexpressing the ZmGOLS2 and ZmRAFS genes in maize plants.

[0011] This invention also provides the application of the ZmGOLS2 gene and the ZmRAFS gene in improving the seed setting rate of maize under drought conditions, by overexpressing the ZmGOLS2 gene and the ZmRAFS gene to improve the seed setting rate of plants under drought conditions.

[0012] This invention also provides the application of recombinant vectors, expression cassettes, and transgenic lines containing the ZmGOLS2 and ZmRAFS genes in improving the seed setting rate of maize under drought conditions.

[0013] The present invention also provides a method for improving the seed setting rate of maize under drought conditions by overexpressing the ZmGOLS2 gene and the ZmRAFS gene in maize plants.

[0014] The present invention also provides a recombinant vector, expression cassette, and transgenic line for improving pollen viability and / or seed setting rate of maize under heat shock and drought stress conditions, and / or under drought conditions, containing the ZmGOLS2 gene and the ZmRAFS gene.

[0015] Based on the above technical solution, the present invention has the following technical effects:

[0016] This invention improves maize pollen viability by simultaneously increasing the expression levels of the maize inositol galactoside synthase encoding gene and the maize raffinose synthase encoding gene, thereby promoting pollen germination under heat shock and drought stress and reducing the rate of empty stalks in maize under drought stress. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Identification of transgenic maize Zong31 lines and the created improved Zhengdan 958 hybrid; (A) schematic diagram of the pTF-ZmGOLS2-ZmRAFS vector structure; (B) flowchart of backcrossing transgenic Zong31 into the commercial maize inbred line Chang7-2 to create the improved Zhengdan 958 hybrid; (C) genomic level identification of maize Zong31 overexpression lines, Z31: genome of maize Zong31 inbred line, GOLS2+RAFS: maize overexpression of ZmGOLS2 and ZmGOLS2+RAFS. mRAFS strain 31; (DE) Identification of ZmGOLS2 and ZmRAFS protein levels in leaves of maize overexpression lines, with the numbers on the left indicating the size of the protein bands; ZmGAPDH: glyceraldehyde-3-phosphate dehydrogenase, used as an internal control; (F) Detection of raffinose content in pollen of Zhengdan 958 (control) and modified Zhengdan 958 (modified) hybrids, with 3 biological replicates for each line. Data are expressed as mean ± standard deviation, *** indicates extremely significant difference (P<0.001), (Student's t test);

[0019] Figure 2 This study presents the pollen germination of Zhengdan 958 (control) and improved Zhengdan 958 (improved) hybrids at 31℃ and 40℃. (A, C) Images of pollen germination at 31℃ (A) and 40℃ (C); (B, D) Statistical analysis of pollen germination rates at 31℃ (B) and 40℃ (D). Nine biological replicates were performed for each line. Data are presented as mean ± standard deviation. *** indicates highly significant (P < 0.001) (Student's t test).

[0020] Figure 3 This study presents the germination of pollen from Zhengdan 958 (control) and improved Zhengdan 958 (improved) hybrids under normal and 10% PEG6000 simulated drought stress conditions. (A, C) Images of pollen germination under normal (A) and 10% PEG6000 (C) conditions; (B, D) Statistical analysis of pollen germination rates under normal (B) and 10% PEG6000 (D) conditions. Nine biological replicates were performed for each line. Data are presented as mean ± standard deviation. *** indicates highly significant (P < 0.001) (Student's t test).

[0021] Figure 4 The results show the seed setting rate of Zhengdan 958 (control) and improved Zhengdan 958 (improved) hybrid under drought conditions; (A) Photographs of half rows of ears of Zhengdan 958 (control) and improved Zhengdan 958 (improved) hybrid; (B) Comparison of the field empty stalk rate of Zhengdan 958 (control) and improved Zhengdan 958 (improved) hybrid under drought conditions. Nine biological replicates were used for each line. Data are expressed as mean ± standard deviation. ** indicates significant difference (P<0.01) (Student's test). Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0028] Example 1

[0029] The coding regions of the maize inositol galactosyl synthase gene ZmGOLS2 (as shown in SEQ ID NO:1) and the maize raffinose synthase gene ZmRAFS (as shown in SEQ ID NO:2) were cloned by PCR, and expression frameworks of the two genes were constructed on the same vector.

[0030] SEQ ID NO:1:

[0031]

[0032] SEQ ID NO:2:

[0033]

[0034] The vector construction method is as follows: Using plasmid vector PHP69219 (Lowe et al., 2016, Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation. Plant Cell. 28: 1998-2015.) DNA as a template, maize Ubiquitin promoter (Ubi) was amplified by PCR using primers (pF: 5'-TTCGAGCTCGGTACCAGCTTGCATGCCTGC-3' and pR: 5'-TGGGAGCCAGGATCCCGAGTTGAGCCATGGTCTAGAGTCGACCTGCAG-3'). The PCR product was then ligated into the pCSGFPBT-ZmRAFS vector (Li et al., 2017, Regulation of Seed Vigor by Manipulation of Raffinose Family Oligosaccharides in Maize and Arabidopsis). The 35S promoter was replaced with that of the Arabidopsis thaliana Histone Gene AtHTA1, obtained by pCSGFPBT-Ubi:ZmRAFS vector. Then, using pCSGFPBT-Ubi:ZmRAFS vector DNA as a template, the Ubi-ZmRAFS-PolyA expression framework was amplified and ligated into the pTF101.1 vector by PCR using primers 5'-GACTCTAGAGGATCCGCATGCCTGCAGTGCAGCGT-3' and 5'-GGTACCCGGGGATCCGTCACTGGATTTTGGTTTTAG-3' (Zheng et al., 2009, Expression of the Arabidopsis thaliana Histone Gene AtHTA1 Enhances Rice Transformation Efficiency. Molecular Plant. 2:832-837). The Ubi-ZmGOLS2-PolyA amplification (primers: 5'-GACTCTAGAGGATCCGCATGCCTGCAGTGCAGCGT-3' and 5'-GGTACCCGGGGATCCGTCACTGGATTTTGGTTTTAG-3') was ligated into the pTF101.1-Ubi-ZmRAFS vector using the same strategy, ultimately obtaining the pTF101.1-Ubi-ZmGOLS2+Ubi-ZmRAFS vector, the structure of which is shown below. Figure 1 As shown in Figure A.

[0035] Example 2

[0036] Genetic transformation of maize

[0037] Genetic transformation of maize was performed according to the published method (Ishida et al., 2007, Agrobacterium-mediated transformation of maize. Nat Protocol. 2: 1614-1621.). The recipient material was the immature embryo of maize inbred line 31 (Zong31), and transgenic lines that simultaneously overexpressed ZmGOLS2 and ZmRAFS were obtained.

[0038] Example 3

[0039] Backcrossing, molecular identification, and hybridization of transgenic inbred line 31 to commercial maize inbred line Chang 7-2

[0040] See Figure 1 As shown in (B), in this embodiment, seeds of the transgenic Zong 31 inbred line overexpressing ZmGOLS2 and ZmRAFS and the maize Chang 7-2 inbred line were simultaneously sown in the field. During the flowering period, the female and male ears were bagged separately, and pollen from the transgenic Zong 31 was sprinkled onto the silks of the female ear of Chang 7-2 before bagging. After maturity, the seeds were harvested. Then, five generations of backcrossing were performed, followed by two generations of self-crossing. The homozygous transgenic improved Chang 7-2 was selected as the male parent and crossed with the non-transgenic Zheng 58 as the female parent to obtain the improved Zhengdan 958 hybrid (improved). The control was the unimproved Zhengdan 958 hybrid.

[0041] Example 4

[0042] Molecular identification of transgenic lines

[0043] First, the transgenic plants obtained in Example 2, the improved plants obtained in Example 3, and the unimproved plants were identified at the genomic level by PCR. Then, the expression levels of ZmGOLS2 and ZmRAFS proteins in the transgenic plants obtained in Example 2, the improved plants obtained in Example 3, and the unimproved plants were identified by Western blot.

[0044] The PCR amplification program used for genome-level identification was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 8 min. Primers 5'-ATGGAACAAGGGCAGAAGATT-3' and 5'-GCACCATCGTCAACCACTAC-3' were used to identify the Bar gene, primers 5'-TTTTAGCCCTGCCTCATAC-3' and 5'-GCGAAGGGGGTGGGCGGAGTC-3' were used to identify the ZmGOLS2 gene, and primers 5'-TTTTAGCCCTGCCTTCATAC-3' and 5'-CCCTGCTTGTACTCCCTGAAC-3' were used to identify the ZmRAFS gene.

[0045] The results are as follows Figure 1 As shown in Figure C, transgenic maize lines that simultaneously overexpress the ZmGOLS2 and ZmRAFS genes were successfully obtained.

[0046] The Western blot procedure followed the existing technique described below (Gu et al., 2016, ZmGOLS2, a target of transcription factor ZmDREB2A, offers similar protection against abiotic stress as ZmDREB2A. Plant Molecular Biology. 90:157-70.). The primary antibodies ZmGOLS2 and ZmRAFS were prepared by our research group using immunization of rabbits at a dilution of 1:5000; the secondary antibody (goat anti-rabbit) was purchased from Kangwei Company and diluted 1:10000.

[0047] The results are as follows Figure 1 As shown in D and E, the obtained transgenic lines simultaneously increased the protein levels of ZmGOLS2 and ZmRAFS.

[0048] Example 5

[0049] Identification of free sugar components in pollen of transgenic lines

[0050] The soluble sugars in the pollen obtained in Example 3 were determined using the following method: Collected pollen was placed in a 10 mL centrifuge tube and 6 mL of 80% ethanol was added and mixed thoroughly. The sample was heated in an 80°C water bath for 30 min, then centrifuged at 12000×g for 20 min at room temperature. The supernatant was transferred to another clean 10 mL centrifuge tube and heated at 90°C to evaporate the ethanol. The sample was then frozen at -80°C for 24 hours and freeze-dried under vacuum. The sample was resuspended in double-distilled water and centrifuged at 12000×g for 10 min. The supernatant was stored at -20°C and filtered through a 0.22 μm aqueous filter before analysis. The HPLC detector was a Waters 2424 ELSD, and the column was a Waters Xbrige amino column. The sample loading volume was 10 μL.

[0051] The results are as follows Figure 1 As shown in Figure F, raffinose was not detected in the control strain, while the content of raffinose was significantly increased in the improved strain.

[0052] Example 6

[0053] Normal field planting and pollen collection of genetically modified corn

[0054] Zhengdan 958 (control) and improved Zhengdan 958 (improved) were planted in the field with a planting row width of 80 / 40 cm and a plant spacing of 25 cm, and double seeds were sown. When the corn grew to the three-leaf stage, the seedlings were thinned, and when they grew normally to the flowering stage, the pollen was collected.

[0055] Example 7

[0056] In vitro germination of corn pollen

[0057] Prepare a liquid germination medium for maize pollen (10% sucrose, 0.01% boric acid, 0.1% yeast extract, 10 mM CaCl2, 50 μM KH2PO4, 15% PEG-4000). Take 3 mL of culture medium into a 6-well plate and put the pollen obtained in Example 5 into the liquid medium. The normal germination conditions are 31℃, the heat shock conditions are 40℃, and the germination time is 5 h. Observe and count the pollen germination under a microscope.

[0058] The results are as follows Figure 2 As shown, the pollen germination rate of the improved strain was significantly higher than that of the control strain under heat stress at 40℃.

[0059] Example 8

[0060] PEG6000 simulates in vitro pollen germination under drought stress

[0061] Liquid germination medium for maize pollen was prepared. The normal pollen germination medium was the same as in Example 7, and the drought stress medium (10% sucrose, 0.01% boric acid, 0.1% yeast extract, 10mM CaCl2, 50μM KH2PO4, 15% PEG-4000, 10% PEG6000) was used. 3 mL of culture medium was taken into each well of a six-well plate, and the pollen obtained in Example 5 was placed in the liquid medium and germinated at 32℃ for 5 h. The pollen germination was observed and recorded under a microscope.

[0062] The results are as follows Figure 3 As shown, the pollen germination rate of the improved strain under 10% PEG6000 stress was significantly higher than that of the control strain.

[0063] Example 9

[0064] Identification of seed setting rate of Zhengdan 958 (control) and improved Zhengdan 958 (modified) under field drought treatment

[0065] Zhengdan 958 (control) and improved Zhengdan 958 (improved) were planted in the field with a row width of 70 / 40 cm, a plant spacing of 22 cm, a row length of 5 m, and 10 rows for each variety, with double seeds sown. The corn was thinned when it reached the three-leaf stage, and drought was applied throughout the entire growth period. The empty stalk rate was counted before harvest. The corn ears were harvested and one row of ears from each variety was photographed.

[0066] The results are as follows Figure 4 As shown, compared with the control strain, the improved strain has an increased seed setting rate and a significantly reduced empty stalk rate.

[0067] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. ZmGOLS2 Genes and ZmRAFS The application of genes in improving pollen viability under heat shock conditions in maize is characterized by, Overexpression ZmGOLS2 Genes and ZmRAFS Genes that improve the germination rate of corn pollen under heat shock conditions.

2. Contains ZmGOLS2 Genes and ZmRAFS Application of gene recombinant vectors and expression cassettes in improving pollen germination rate under heat shock conditions in maize.

3. A method for improving pollen germination rate in maize under heat shock conditions, characterized in that, Overexpression in maize plants ZmGOLS2 Genes and ZmRAFS Gene.

4. ZmGOLS2 Genes and ZmRAFS The application of genes in improving pollen viability in maize under drought conditions is characterized by, Overexpression ZmGOLS2 Genes and ZmRAFS Genes that improve the germination rate of corn pollen under drought conditions.

5. Contains ZmGOLS2 Genes and ZmRAFS Application of gene recombinant vectors and expression cassettes in improving pollen germination rate in maize under drought conditions.

6. A method for improving pollen germination rate in maize under drought conditions, characterized in that, Overexpression in maize plants ZmGOLS2 Genes and ZmRAFS Gene.

7. ZmGOLS2 Genes and ZmRAFS The application of genes in improving the seed setting rate of maize under drought conditions is characterized by, Overexpression ZmGOLS2 Genes and ZmRAFS Genes that improve the fruit set rate of plants under drought conditions.

8. Contains ZmGOLS2 Genes and ZmRAFS Application of gene recombinant vectors and expression cassettes in improving maize seed setting rate under drought conditions.

9. A method for improving the seed setting rate of maize under drought conditions, characterized in that, Overexpression in maize plants ZmGOLS2 Genes and ZmRAFS Gene.

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