A method for increasing amylose content and stress resistance of corn by overexpressing sucrose synthase gene

Abstract

The present application relates to a method for increasing the amylose content and stress resistance of corn by overexpressing sucrose synthase gene, the method is to overexpress sucrose synthase gene ZmSUS1 in corn to obtain transgenic corn with high amylose content and high stress resistance, the nucleotide sequence of the sucrose synthase gene ZmSUS1 is shown as SEQ ID NO. 1. The present application overexpresses sucrose synthase gene ZmSUS1 in corn, effectively provides precursor substances for the biosynthesis of amylose, so that the corn overexpressing sucrose synthase gene ZmSUS1 has 41.1-69.2% higher amylose content than normal corn kernels, significantly increasing the amylose content in corn kernels. And the transgenic corn obtained by the method of the present application shows obvious drought resistance, confirming that the method of the present application is a green, efficient and sustainable effective way to solve the stable yield or less yield of corn under drought and water stress, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for increasing the amylose content and stress resistance of corn by overexpressing sucrose synthase genes, belonging to the field of genetic engineering technology. BACKGROUND

[0002] Corn is an important food, feed and industrial raw material. Starch is the most abundant carbohydrate in corn kernels, containing 25% amylose and about 75% amylopectin. The content and ratio of amylose and amylopectin determine the difference in processing performance in food raw materials and industrial applications. Amylose is a dietary fiber that helps improve intestinal health and function, lower blood sugar and cholesterol, and reduce the incidence of diseases such as diabetes and gallstones. Amylose has strong tensile strength and can form solid gels and films, and is widely used in industrial and health food fields. In the field of environmental protection, starch with high amylose content is the best choice for manufacturing photolysis plastics. Although high-amylose foods have been accepted by some consumers, crops with high amylose content have not been widely planted. Therefore, it is necessary to speed up the breeding of high-amylose crops to meet the rapidly growing demand in human and industrial, environmental protection and other fields.

[0003] However, the increase in amylose content of cereal crops is often accompanied by a significant decrease in total starch content and yield, and it is difficult to break the linkage between undesirable grain traits and high amylose traits using conventional breeding methods. Transgenic technology provides a new way to solve this problem. Studies have found that increasing amylose content can be achieved by inhibiting the enzyme activity related to amylopectin synthesis, increasing the expression level of starch granule binding enzyme genes, increasing the expression level of adenosine diphosphate-glucose pyrophosphorylase, and using RNAi to inhibit starch branching enzyme activity. However, there is a common problem with the above studies, that is, the high amylose trait produced by mutation of key genes is often accompanied by high moisture content and a decrease in total starch content and yield. Since the synthesis of amylose is affected by complex gene-environment interactions, increasing amylose content may reduce yield and other agronomic traits.

[0004] It is known that the starch synthesis pathway in corn endosperm mainly includes the following steps: sucrose is converted to fructose and UDP-glucose (UDPG) by sucrose synthase (SUSY), and then UDPG is converted to ADP-glucose (ADPG) by AGPase, which serves as an activated glucose donor to synthesize amylose and amylopectin. SUSY is a glycosyltransferase belonging to the glycosyltransferase-4 subfamily, and each protein exists in the form of a tetramer. Each subunit has a molecular weight of about 90 kDa. The gene size is generally 5.9 kb, the cDNA size is about 2.7 kb, and the encoded amino acid sequence is about 800 amino acid residues in length.

[0005] Many studies have shown that the activity of SUSY is well correlated with the absorption intensity of various starch storage organs. Zrenner et al. found that inhibiting sucrose synthase activity resulted in no change in sucrose content in potato tubers, but a large accumulation of reducing sugars and inhibited starch accumulation in the development of potato tubers. Studies have also found that, ZmSH1 After gene mutation, the starch content in the mutant endosperm is reduced compared with the normal endosperm.

[0006] As described above, a large number of studies have shown that SUSY plays an important role in grain starch biosynthesis, but the relationship between SUSY and amylose synthesis and stress resistance of corn has not been reported. SUMMARY

[0007] In view of the deficiencies in the prior art, the present application provides a method for increasing the content of corn amylose and stress resistance by overexpressing sucrose synthase gene.

[0008] The technical scheme of the present application is as follows:

[0009] A method for increasing the content of corn amylose and stress resistance by overexpressing sucrose synthase gene, the method is to overexpress sucrose synthase gene in corn ZmSUS1 , obtain transgenic corn with high amylose content and high stress resistance; the nucleotide sequence of the sucrose synthase gene ZmSUS1 is shown in SEQ ID NO. 1.

[0010] According to the present application, preferably, the stress resistance refers to drought resistance.

[0011] The present application also provides a construction method of the above-mentioned transgenic corn, comprising the following steps:

[0012] (1) cloning sucrose synthase gene from cDNA of corn kernel ZmSUS1 , then connecting the gene ZmSUS1 to plant expression vector pU130 to obtain plasmid vector pU130-BAR-MCS;

[0013] The nucleotide sequence of the sucrose synthase gene ZmSUS1 is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2;

[0014] (2) transforming the plasmid vector pU130-BAR-MCS into Agrobacterium strain, then using the recombinant Agrobacterium strain selected to infect corn young embryo, and after recovery culture, selection culture and differentiation culture, transgenic corn is obtained.

[0015] According to the application, preferably, in step (1), the plant expression vector pU130 contains a 35S promoter, and the sucrose synthase gene is used as a selection marker. bar The gene is used as a selection marker.

[0016] According to the application, preferably, in step (1), the plant expression vector pU130 contains a 35S promoter, and the sucrose synthase gene is used as a selection marker.

[0017] The sucrose synthase gene is obtained by PCR amplification using the cDNA of a corn kernel as a template, and the sequence of the PCR primers is as follows: ZmSUS1

[0018] ZmSUS1 -F: 5'-AGCAGTACAACCTGAACGGG-3',

[0019] ZmSUS1 -R: 5'-TGTCGAAGAAGTCCACGAGC-3';

[0020] The sucrose synthase gene is then connected to the plant expression vector pU130 containing a 35S promoter, and the gene is used as a selection marker, to obtain the plasmid vector pU130-BAR-MCS. ZmSUS1 bar According to the application, preferably, in step (2), the transformation of Agrobacterium is performed as follows:

[0021] EH105 Agrobacterium is selected as the transformation strain, 3 μl of the plasmid containing the pU130-BAR-MCS expression vector is added to 50 μl of the Agrobacterium competent cells, ice bath for 30 minutes, quick freezing in liquid nitrogen for 5 minutes, and 37℃ water bath for 5 minutes; then 800-1000 μl of YEP liquid medium is added, and the mixture is cultured at 25-28℃ and 180-250 rpm for 3 hours; the bacterial solution is taken out and spread on YEP solid medium containing rifampicin and kanamycin, and then the medium is placed in the dark at 25-28℃ for 3-4 days; the colonies are taken for colony PCR verification and sequencing, and the Agrobacterium with correct sequencing is preserved for infection.

[0022] Further preferably, the YEP liquid medium has the following formulation: 10 g / L of yeast extract, 10 g / L of peptone, and 5 g / L of NaCl.

[0023] The YEP solid medium is YEP liquid medium with 7 g / L of agar added.

[0024] The YEP solid medium is YEP liquid medium with 7 g / L of agar added.

[0025] According to the application, preferably, in step (2), the infection is performed as follows:

[0026] ​​a. Select the maize inbred line KN5585 as the recipient inbred line, take its ears 10-12 days after pollination, remove the husks, and treat them with 70% alcohol for 5-6 minutes in a sterile workbench. Then rinse them with sterile water 4-5 times, peel off 1.5-2 mm immature embryos, place them on immature embryo induction medium with the scutellum facing upwards, and culture them in the dark at 28°C to induce callus. After 2-3 weeks, select the embryonic callus with faster growth, soft texture, loose and fragile texture, and bright color from the induced callus and transfer them to subculture medium for subculture, subculture every 2 weeks, and use them for Agrobacterium infection.

[0027] b. Pick a single colony of Agrobacterium containing the pU130-BAR-MCS expression vector, add it to 5-6 mL of YEP (Kan) medium, and culture overnight; centrifuge at 5000-6000 rpm at room temperature for 5-10 min to collect the bacteria; suspend the bacteria in an infection solution containing acetosyringone and make OD600 = 0.6-0.8, mix well, and set aside; activate the prepared infection solution at 25-28°C and 180 rpm on a shaker for 1-2 hours for infection; collect maize embryonic callus tissue and place it in a sterile Erlenmeyer flask, then pour the infection solution, break up large callus tissue pieces and shake well to ensure that the callus tissue is fully exposed to Agrobacterium and infect for 15-20 min; remove the callus tissue and inoculate it onto the co-cultivation medium and culture it in the dark at 19-22°C for 3 days to complete the infection.

[0028] Further preferably, the formula of the induction medium is: MS medium containing 1 mg / L 2,4-D, 0.69 g / L L-proline, 0.5 g / L hydrolyzed casein, 30 g / L sucrose, 7.5 g / L agar, pH 5.8;

[0029] The formula of the subculture medium is: MS medium containing 1 mg / L 2,4-D, 1.38 g / L L-proline, 0.5 g / L hydrolyzed casein, 30 g / L sucrose, and 7.5 g / L agar, pH 5.8;

[0030] The formula of the co-culture medium is: MS medium containing 1 mg / L 2,4-D, 100 mg AS, 0.5 g / L MES, 20 g / L sucrose, and 7.5 g / L agar, with a pH of 5.8.

[0031] According to the present invention, preferably, in step (2), the recovery culture is carried out as follows:

[0032] The surface of the callus is washed with sterile water containing antibiotics for 3-5 times, the liquid is discarded when the added water is no longer turbid, the callus is transferred into a flat dish with filter paper, the water on the surface of the callus is blown dry in a super-clean bench, and the callus is transferred into a recovery culture medium and cultured at 28°C in dark for 7-10 days to complete recovery culture;

[0033] The recovery culture medium is MS medium containing 1 mg / L of 2,4-D, 0.69 g / L of L-proline, 0.5 g / L of hydrolyzed casein, 20 g / L of sucrose, 250 mg / L of cephalosporin, and 7.5 g / L of agar, and the pH is 5.8.

[0034] According to the application, preferably, in step (2), the screening culture is performed according to the following method:

[0035] After the recovery culture, the transformed callus is transferred into a screening culture medium added with glufosinate, and is cultured in dark at 25-28°C for 15-20 days after the screening culture for two weeks and the screening culture medium is replaced with a new one, the callus is dispersed and transferred into the new screening culture medium for continuous screening, and the total screening is performed for two rounds, and the culture is performed for 30-40 days to complete the screening culture.

[0036] Further preferably, the screening culture medium is MS medium containing 1 mg / L of 2,4-D, 0.69 g / L of L-proline, 0.5 g / L of hydrolyzed casein, 20 g / L of sucrose, 250 mg / L of cephalosporin, 10 mg / L of glufosinate, and 7.5 g / L of agar, and the pH is 5.8.

[0037] According to the application, preferably, in step (2), the differentiation culture is performed according to the following method:

[0038] The resistant callus is transferred into a differentiation culture medium, and is cultured in dark at 25-28°C for 7-10 days, and then is transferred into a light culture box and cultured at 25-28°C until the regenerated sprouts grow to 3-5 cm, and then is transferred into a rooting culture medium, and after a large number of strong roots grow, the seedlings are cultured for 2-3 days, the roots are washed with the culture medium, and then are transplanted into sterilized nutrient soil, and are transplanted into a field after indoor seedling culture for 7 days to obtain transgenic corn with high amylose content and stress resistance.

[0039] Further preferably, the differentiation culture medium is MS medium containing 0.5 mg / L of 6-BA, 0.5 g / L of MES, 10 mg / L of glufosinate, 250-300 mg / L of cephalosporin, 20 g / L of sucrose, and 7 g / L of agar, and the pH is 5.8.

[0040] The rooting culture medium is MS medium containing 20 g / L of sucrose and 7 g / L of agar, and the pH is 5.8.

[0041] The application also provides a bacterial transformation vector pU130-BAR-MCS capable of increasing the amylose content and stress resistance of corn.

[0042] The application also discloses a method for increasing the amylose content and stress resistance of corn by overexpressing a sucrose synthase gene ZmSUS1 The application also discloses a method for increasing the amylose content and stress resistance of corn and a corn transgenic strain obtained.

[0043] The parts not described in detail in the application can be implemented according to the prior art.

[0044] The application has the following beneficial effects:

[0045] 1. The application overexpresses a sucrose synthase gene in corn ZmSUS1 , effectively providing precursor substances for the biosynthesis of amylose, so that the corn overexpressing the sucrose synthase gene has an amylose content increased by 41.1-69.2% compared with normal corn kernels, and the amylose content in the corn kernels is significantly increased. ZmSUS1

[0046] 2. The application provides a technology and a method for increasing the amylose content of corn by overexpressing a sucrose synthase gene, and discloses a corn transgenic strain obtained by overexpressing the sucrose synthase gene ZmSUS1 , which lays a foundation for further screening of excellent corn varieties and is also beneficial to the mass production of amylose by using the transgenic corn.

[0047] 3. The transgenic corn obtained by the method of the application shows obvious drought resistance, which proves that the method of the application is an effective way to solve the problem of stable yield or less yield reduction of corn under drought and water stress, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 Fig. 1 is a schematic diagram of a plant expression vector map and a detection diagram of transgenic corn;

[0049] Fig. 1 is a schematic diagram of a plant expression vector map and a detection diagram of transgenic corn;

[0050] Figure 2 Fig. 2 is a diagram showing the changes of expression levels, sucrose synthase activities and contents of transgenic corn in different pollination days; ZmSUS1 Fig. 2 is a diagram showing the changes of expression levels, sucrose synthase activities and contents of transgenic corn in different pollination days;

[0051] Fig. 2 is a diagram showing the changes of expression levels, sucrose synthase activities and contents of transgenic corn in different pollination days; SUS1 ​b) expression levels; b) sucrose synthase activity in transgenic lines 10, 20, and 30 days after pollination; c) sucrose synthase content in transgenic lines 10, 20, and 30 days after pollination. Three samples were analyzed for each line, each from three independent experiments. Values ​​are the mean ± SD of nine replicates. * and ** indicate significant differences between transgenic maize and the control. P < 0.05 and 0.01, respectively, by t-test (n = 3).

[0052] Figure 3 The amylose content in the kernels of ZmSUS1 transgenic corn.

[0053] Figure 4 The morphology and size of starch grains in ZmSUS1 gene-transfected corn.

[0054] In the figure: ad is the morphology of starch granules in the endosperm of mature grains (bar 10µm); e is the diameter of the starch granules.

[0055] Figure 5 For transfer ZmSUS1 Results of simulated drought before flowering of genetically modified corn.

[0056] Figure 6 For transfer ZmSUS1 Results of genetically modified corn PEG simulation of drought. DETAILED DESCRIPTION

[0057] To make the technical problems, technical solutions, and advantages of the present invention more apparent, the present invention is further illustrated below by way of examples, but is not limited thereto. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0058] Example 1: Construction of plasmid vector pU130-BAR-MCS

[0059] Sucrose synthase was obtained by PCR amplification using corn kernel cDNA as template. ZmSUS1 Sequence, PCR primer sequences are as follows:

[0060] ZmSUS1 -F: 5′-AGCAGTACAACCTGAACGGG-3′,

[0061] ZmSUS1 -R: 5′-TGTCGAAGAAGTCCACGAGC-3′;

[0062] Then sucrose synthase gene ZmSUS1 The sequence was connected to the 35S promoter. barThe gene is selected as a marker in the plant expression vector pU130, and a plasmid vector pU130-BAR-MCS is obtained, specifically as shown in Figure 1 a.

[0063] The PCR amplification system is as follows: 10 μM of the upstream primer 1 μL, 10 μM of the downstream primer 1 μL, the target gene template 2 μL, Pfu enzyme (high-fidelity enzyme) 20 μL, and ddH2O is supplemented to 50 μL.

[0064] The PCR amplification procedure is as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 sec, 58°C annealing for 30 sec, 72°C extension for 2.5 min, 36 cycles; 72°C final extension for 5 min, and 4°C storage.

[0065] Example 2, obtaining transgenic corn

[0066] 1. Selecting EH105 Agrobacterium as the transformation strain, 3 μl of the plasmid containing the pU130-BAR-MCS expression vector constructed in Example 1 is added to 50 μl of the Agrobacterium competent cells, ice bath for 30 min, liquid nitrogen freezing for 5 min, 37°C water bath for 5 min; then 800-1000 μl of YEP liquid medium is added, and the culture is incubated at 25-28°C, 180-250 rpm for 3 hours; the bacterial solution is taken out and spread on solid YEP medium containing rifampicin and kanamycin, and incubated at 25-28°C in the dark for 3-4 days; the colonies are taken for colony PCR verification and sequencing, and the Agrobacterium with correct sequencing is preserved for infection.

[0067] 2. Selecting corn inbred line KN5585 as the receptor inbred line, taking the ear 10 days after pollination, removing the bracts, first treating with 70% alcohol for 5 min, then washing with sterile water for 5 times, peeling off the 2 mm young embryos, placing the scutes upwards on the young embryo induction medium, and incubating at 28°C in the dark to induce callus; after 3 weeks, the embryogenic callus with faster growth, soft texture, loose and easy to break, and bright color is selected and transferred to the subculture medium for subculture, and subcultured every 2 weeks for Agrobacterium infection;

[0068] The agrobacterium single colony containing the pU130-BAR-MCS expression vector was picked and added into 5 mL of YEP (Kan) medium for overnight culture; the bacteria were collected by centrifugation at 6000 rpm for 8 min at room temperature; the bacteria were suspended in the infection liquid containing acetosyringone, and the OD600 was adjusted to 0.6-0.8; the bacteria were mixed and used; the prepared infection liquid was activated at 28°C for 2 hours at 180 rpm, and was used for infection; the embryogenic callus of corn was collected in a sterile flask, and then was poured into the infection liquid; the large callus pieces were shaken to make the callus fully contact with the agrobacterium, and the infection was performed for 20 min; the callus was taken out, and was inoculated on the co-culture medium for dark culture at 19-22°C for 3 days, and the infection was completed.

[0069] 3. The callus surface was washed 5 times with sterile water containing an antibiotic, and the liquid was discarded when the added water was no longer turbid; the callus was transferred to a flat dish with filter paper, and the callus surface was blown dry in the clean bench; the callus was transferred to the recovery medium, and was cultured at 28°C in the dark for 10 days, and the recovery culture was completed.

[0070] After the recovery culture, the transformed callus was transferred to the selection medium added with glufosinate, and was selected for two weeks; then, the new selection medium was replaced, and the callus was cultured at 25°C in the dark for 20 days; the callus was shaken and was transferred to the new selection medium for continuous selection, and the total selection was performed for 2 rounds, and the culture was performed for 40 days, and the selection culture was completed.

[0071] The resistant callus was transferred to the differentiation medium, and was cultured at 28°C in the dark for 7 days; then, the callus was transferred to the light culture box, and was cultured at 28°C until the regenerated sprouts grew to 5 cm; then, the callus was transferred to the rooting medium, and after a large number of strong roots grew, the callus was hardened for 2 days; after the medium on the roots was washed, the callus was transplanted into the sterilized nutrient soil; after the callus was hardened in the room for 7 days, the callus was transplanted into the field, and the transgenic corn was obtained.

[0072] In this embodiment, 6 transgenic corn lines were obtained, and were named as L1-L6.

[0073] The medium used in this embodiment is as follows:

[0074] The formula of the YEP liquid medium is: 10 g / L of yeast extract, 10 g / L of peptone, and 5 g / L of NaCl.

[0075] The YEP solid medium is the YEP liquid medium added with 7 g / L of agar.

[0076] The formula of the induction medium is: MS medium containing 1 mg / L of 2,4-D, 0.69 g / L of L-proline, 0.5 g / L of hydrolyzed casein, 30 g / L of sucrose, and 7.5 g / L of agar, and the pH is 5.8;

[0077] The formula of the subculture medium is: MS medium containing 1 mg / L 2,4-D, 1.38 g / L L-proline, 0.5 g / L hydrolyzed casein, 30 g / L sucrose, and 7.5 g / L agar, pH 5.8;

[0078] The formula of the co-culture medium is: MS medium containing 1 mg / L 2,4-D, 100 mg AS, 0.5 g / L MES, 20 g / L sucrose, and 7.5 g / L agar, with a pH of 5.8.

[0079] The recovery culture medium has a formula of: MS culture medium containing 1 mg / L 2,4-D, 0.69 g / L L-proline, 0.5 g / L hydrolyzed casein, 20 g / L sucrose, 250 mg / L cephalosporin, and 7.5 g / L agar, with a pH of 5.8.

[0080] The screening culture medium formula is: MS culture medium containing 1 mg / L 2,4-D, 0.69 g / L L-proline, 0.5 g / L hydrolyzed casein, 20 g / L sucrose, 250 mg / L cephalosporin, 10 mg / L glufosinate ammonium, and 7.5 g / L agar, with a pH of 5.8.

[0081] The differentiation medium is formulated as follows: MS medium containing 0.5 mg / L 6-BA, 0.5 g / L MES, 10 mg / L glufosinate, 250-300 mg / L cephalosporin, 20 g / L sucrose, and 7 g / L agar, with a pH of 5.8;

[0082] The rooting culture medium formula is: MS culture medium containing 20g / L sucrose and 7g / L agar, with a pH of 5.8.

[0083] Example 3: Sucrose synthase gene in transgenic corn ZmSUS1 Verification of expression

[0084] 1. PCR testing

[0085] PCR verification was performed on the six transgenic maize lines L1 to L6 obtained in Example 2 and the control maize using the M5 Superlight Mix kit (Polymer, Beijing, China). Primers were designed based on the herbicide screening marker gene bar sequence. The primer sequences are as follows:

[0086] bar-F: 5′-ATGAGCCCAGAACGACGCC-3′,

[0087] bar-R: 5′-TCAGATCTCGGTGACGGGC-3′.

[0088] PCR amplification products were detected by agarose gel electrophoresis, the results are shown in Figure 1 c.

[0089] 2. PAT / bar protein rapid test strip detection

[0090] The transgenic maize L1~L6 and control maize leaves were taken into a centrifuge tube, and an appropriate amount of ultrapure water was added. The juice was ground with a grinding rod, and the test strip was inserted. After about 30 seconds, the expression level of bar protein was determined qualitatively or semi-quantitatively according to the color depth of the detection line, and the results are shown in Figure 1 b.

[0091] 3. Real-Time PCR detection

[0092] The RNA of the corn kernels of the 6 transgenic maize lines L1~L6 and the control maize obtained in Example 2 after 10 days of pollination was extracted by MiniBEST Plant RNA Extraction Kit (Takara, Dalian, China). The concentration and quality of the RNA were detected by ultramicro UV spectrophotometer (Denovix, Wilmington, USA). The qualified RNA was reverse transcribed by reverse transcription kit (Takara, Dalian, China). RT-PCR was performed in Applied Biosystems real-time fluorescent quantitative PCR instrument using SYBR Green RT-PCR kit (Takara, Dalian, China). The relative gene expression level was calculated by 2-ΔΔCt method, each sample was set for three repeats, and ZmActin1 (NM_001155179.1) was used as the internal reference gene, and the results are shown in Figure 1 d.

[0093] 4. Western blot detection

[0094] The total protein of the corn kernels of the 6 transgenic maize lines L1~L6 and the control maize obtained in Example 2 after 10 days of pollination was extracted by plant protein extraction kit (Solep, Beijing, China), and was quantified by BCA and then subjected to Western blot detection. The primary antibody used was the antibody of the tag protein EYFP (Abbkine, California, USA), and the internal reference was the antibody of actin ACT11 (Polymer, Beijing, China). The Western blot detailed steps were performed according to the method of Chiaki Sakuma et al., and the results are shown in Figure 1 e.

[0095] The above detection processes were all taken as the control (CK, CK1, CK2 and CK3) of wild type maize.

[0096] Depend on Figure 1 c and Figure 1 d It can be seen that the leaves of the six transgenic corn lines obtained in Example 2 have sucrose synthase ZmSUS1 The transcription product of Figure 1 b and Figure 1 e shows that sucrose synthase at the protein level ZmSUS1 The protein was successfully expressed in the six transgenic maize lines obtained in Example 2.

[0097] Example 4: Transfer ZmSUS1 Determination of the Content and Activity of Sucrose Synthase in Genetically Modified Corn Kernels

[0098] The conversion obtained in Example 2 ZmSUS1 Developing kernels of maize L1, L2, and L3 were ZmSUS1 The expression level, sucrose synthase activity and enzyme content were detected. The results were as follows Figure 2 Wild-type maize was used as the control (CK).

[0099] The specific detection methods are as follows:

[0100] Five corn kernels from different strains and the control corn were taken 10, 20, and 30 days after pollination, wrapped in tin foil, and quickly frozen in liquid nitrogen. Extraction buffer (50mM Hepes-NaOH pH7.5, 5mM EDTA, 1mM DTT, 2mMKCL, 1%PVP) was added and ground in a pre-cooled mortar. The homogenate was transferred to a centrifuge tube, and the mortar was rinsed twice with the extract. The rinse was poured into the centrifuge tube and centrifuged at 12000prm at 4°C for 10 minutes. The supernatant was the enzyme extract, and the sucrose synthase content was determined by the Coomassie Brilliant Blue method (Betti et al. 2021).

[0101] Depend on Figure 2 It can be seen that after 10, 20, and 30 days of pollination, the transgenic grains ZmSUS1 The expression levels were higher than those in the control group ( Figure 2 a). Ten days after pollination, the sucrose synthase activity of L1 in the transgenic line increased by 25.8% compared with the control; 20 days after pollination, the sucrose synthase activity of L2 in the transgenic line increased by 40.9% compared with the control; and 30 days after pollination, the sucrose synthase activity of L3 in the transgenic line increased by 41.8% compared with the control ( Figure 2 b); 10 days after pollination, the sucrose synthase content in L1 of the transgenic line increased by 173.0% compared with the control; 20 days after pollination, the sucrose synthase content in L2 of the transgenic line increased by 98.9% compared with the control; 30 days after pollination, the sucrose synthase content in L3 of the transgenic line increased by 226.9% compared with the control ( Figure 2c). In summary, the mRNA levels of all strains increased significantly during the first 20 days of development, and the sucrose synthase activity and content at different pollination days were higher than those of the control.

[0102] Example 5: Transfer ZmSUS1 Determination of Amylose Content in Genetically Modified Corn Kernels

[0103] 1. The conversion obtained from Example 2 ZmSUS1 Five kernels were randomly selected from each of the genetically modified corn L1, L2, and L3 lines, with wild-type corn kernels serving as the control (CK). These kernels were then boiled in boiling water for 10 minutes, the seed coat removed, the embryo removed, and the endosperm dried to a constant weight. The dried endosperm was ground into powder using a mortar and pestle and passed through a 40-mesh sieve to obtain a crude starch sample. Amylose content was determined using the starch-iodine binding method (Stawski 2008). The results are shown in Table 1. Figure 3 shown.

[0104] Depend on Figure 3 It can be seen that the amylose content in the grains of the transgenic lines L1, L2, and L3 increased by 41.1%, 51.2%, and 69.2%, respectively, compared with the control group, indicating that the overexpression of sucrose synthase led to increased accumulation of corn amylose and increased the amylose content in corn.

[0105] 2. The conversion obtained from Example 2 ZmSUS1 Mature seeds were randomly selected from genetically modified corn L1, L2, and L3, with mature wild-type corn seeds used as controls (CK). A 2mm thick slice was then cut near the top of the kernel using a scalpel. To maintain the integrity of the starch granules, the slices were allowed to break naturally. The samples were then placed on a stage, gold-sprayed, and placed in the sample chamber of a scanning electron microscope (SU8010, ZEISS, Germany). Scanning was performed under high vacuum at 5kV. The diameters of 200 starch granules were measured for each strain, and the granules were divided into three groups based on their diameters: Group a, starch granules with a diameter greater than 10µm; Group b, with a diameter of 5µm-10µm; and Group c, with a diameter less than 5µm. The results are shown in the figure below. Figure 4 shown.

[0106] Depend on Figure 4 It is known that ZmSUS1 The starch granules of the genetically modified corn were larger than those of the control ( Figure 4 ad). The diameters of most starch granules in transgenic materials are concentrated in the interval of group a, while the diameters of most starch granules in wild-type materials are concentrated in the interval of group b ( Figure 4 e). The average diameter of wild-type starch granules is 8.87µm. ZmSUS1 The average starch granule diameters of the transgenic maize lines L1, L2, and L3 were 9.83µm, 10.40µm, and 9.80µm, respectively. Compared to the wild-type, the average starch granule diameter of the transgenic grains was significantly increased.

[0107] Example 5: Transfer ZmSUS1 Improved drought resistance of genetically modified corn

[0108] 1. The conversion obtained in Example 2 ZmSUS1 The genetically modified maize L1, L2 and L3 lines and the wild-type maize line (control group, CK) were subjected to drought treatment and then rewatered. Figure 5 shown.

[0109] The specific treatment method is as follows: the transgenic and control corn are subjected to water-deprivation and drought treatment before flowering. The soil moisture content of each pot of corn is measured before treatment to ensure that the moisture content is consistent. After all the plants wilt, they are rehydrated and their recovery is observed.

[0110] Depend on Figure 5 It can be seen that after the transgenic and control corn were deprived of water and drought treatment before flowering, the leaves of the control group plants drooped on the seventh day, while the transgenic lines L1, L2, and L3 remained upright and in good growth condition; after all the lines wilted, they were rehydrated. After about 2 hours of rehydration, the transgenic lines L1, L2, and L3 began to stretch, while the control group had not yet recovered.

[0111] 2. The conversion obtained in Example 2 ZmSUS1 Genetic maize L1, L2 and L3 lines and wild-type maize lines (control group) were subjected to simulated drought treatment. Figure 6 shown.

[0112] The specific treatment method is as follows: transgenic and control corn were subjected to simulated drought treatment using 16% concentration of PEG at the two-leaf and one-heart stage.

[0113] Depend on Figure 6 It can be seen that the leaves of the control group began to wilt after 3 days of treatment, while ZmSUS1 The transgenic corn seedlings grew well; after seven days of treatment, all four groups wilted, but the leaves and roots of the transgenic lines were longer than those of the control group. This indicates that the transgenic corn obtained using the method of the present invention exhibits significant drought resistance, confirming that the method of the present invention is a green, efficient, and sustainable solution for stabilizing or minimizing yield losses in corn under drought and water shortage stress, and has broad application prospects.

Claims

1. A method for increasing amylose content and drought tolerance in maize by overexpressing sucrose synthase gene, characterized in that, The method is to overexpress sucrose synthase gene in corn ZmSUS1 , obtain high amylose content and high drought resistance of transgenic corn; the nucleotide sequence of the sucrose synthase gene ZmSUS1 is shown as SEQ ID NO.

1.

2. The method for constructing a transgenic maize having a high amylose content and a high drought resistance according to claim 1, characterized by, The method comprises the following steps: (1) Cloning of sucrose synthase gene from cDNA of corn kernel ZmSUS1 The gene ZmSUS1 is then ligated into plant expression vector pU130 to obtain plasmid vector pU130-BAR-MCS; The sucrose synthase gene ZmSUS1 The nucleotide sequence of the sucrose synthase gene is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO.

2. (2) Transforming the plasmid vector pU130-BAR-MCS into Agrobacterium strains, and then using the recombined Agrobacterium strains obtained through screening to infect corn young embryos, and after recovery culture, screening culture and differentiation culture, transgenic corn is obtained.

3. The construction method of claim 2, wherein, In step (1), the construction method of the plasmid vector pU130-BAR-MCS is specifically as follows: The cDNA of corn kernel was used as template to amplify sucrose synthase by PCR ZmSUS1 sequences; Then sucrose synthase ZmSUS1 The sequence was connected to the 35S promoter. bar The gene was used as a selection marker in the plant expression vector pU130 to obtain the plasmid vector pU130-BAR-MCS.

4. The construction method of claim 2, wherein, In step (2), the transformation of Agrobacterium is performed as follows: EH105 Agrobacterium is selected as the transformation strain, 3 μl of the plasmid containing the pU130-BAR-MCS expression vector is added into 50 μl of the Agrobacterium competent cells, ice bath for 30 minutes, liquid nitrogen freezing for 5 minutes, 37 °C water bath for 5 minutes; then 800-1000 μl of YEP liquid medium is added, and the culture is performed at 25-28 °C, 180-250 rpm for 3 hours; the bacterial liquid is taken out and coated on the solid YEP medium containing rifampicin and kanamycin, and placed at 25-28 °C in the dark for 3-4 days; the colonies are taken for colony PCR verification, and sequencing is performed; the Agrobacterium with correct sequencing is preserved and used for infection.

5. The construction method of claim 2, wherein, In step (2), the infection is performed as follows: a. Selecting corn inbred line KN5585 as the receptor inbred line, taking the ear 10-12 days after pollination, removing the bracts, and in a sterile workbench, first treating with 70% alcohol for 5-6 minutes, then washing with sterile water for 4-5 times, peeling off the 1.5-2 mm young embryos, and placing the scutes upwards on the young embryo induction medium, and culturing at 28 °C in the dark to induce callus; after 2-3 weeks, the induced callus is transferred to the subculture medium for subculture, and subcultured every 2 weeks, and used for Agrobacterium infection; b. Picking the Agrobacterium single colony containing the pU130-BAR-MCS expression vector, adding 5-6 mL of YEP (Kan) medium, and culturing overnight; centrifuging at 5000-6000 rpm for 5-10 minutes at room temperature, and collecting the bacterial bodies; suspending the bacterial bodies in the infection liquid containing acetosyringone, and making OD600=0.6-0.8, mixing, and waiting for use; activating the prepared infection liquid at 25-28 °C on a shaker at 180 rpm for 1-2 hours, and used for infection; collecting the embryogenic callus of corn, placing in a sterile triangular flask, and then pouring the infection liquid, and shaking the large callus pieces to make the callus fully contact with the Agrobacterium, and infecting for 15-20 minutes; taking out the callus, and inoculating on the co-culture medium for 3 days of culture at 19-22 °C in the dark, and completing the infection.

6. The construction method of claim 2, wherein, In step (2), the recovery culture is performed as follows: The surface of the callus is washed with sterile water containing antibiotics for 3-5 times, the liquid is discarded when the added water is no longer turbid, the callus is transferred into a flat dish with filter paper, the water on the surface of the callus is blown dry in a super-clean bench, and the callus is transferred into recovery medium and cultured in dark at 28°C for 7-10 days to complete recovery culture; The screening culture is performed according to the following method: After the recovery culture, the transformed callus is transferred into screening medium added with glufosinate, screened for two weeks, then new screening medium is replaced, and the callus is cultured in dark at 25-28°C for 15-20 days, then the callus is scattered and transferred into new screening medium for continuous screening, a total of 2 rounds of screening, and the callus is cultured for 30-40 days to complete the screening culture.

7. The construction method of claim 2, wherein, In step (2), the differentiation culture is performed according to the following method: The resistant callus is transferred into differentiation medium, cultured in dark at 25-28°C for 7-10 days, then transferred into a light culture box and cultured at 25-28°C until the regenerated sprout grows to 3-5 cm, then transferred into rooting medium, and after a large number of strong roots grow, the callus is hardened for 2-3 days, washed with root medium, and transplanted into sterilized nutrient soil, then transplanted into a field after hardened in a room for 7 days to obtain transgenic corn with high amylose content and high drought resistance.

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