Sweet potato IbGSTU7 gene for regulating and controlling drought resistance and skin color of sweet potato and application of sweet potato IbGSTU7 gene

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a sweet potato IbGSTU7 gene for regulating and controlling drought resistance and skin color of sweet potatoes and application of the sweet potato IbGSTU7 gene, the nucleotide sequence of the sweet potato IbGSTU7 gene is shown as SEQ ID NO.1, and the amino acid sequence of the sweet potato IbGSTU7 gene is shown as SEQ ID NO.2. According to the invention, the IbGSTU7 gene is successfully cloned from sweet potatoes for the first time, and the positive regulation effect of the IbGSTU7 gene on the color of sweet potato skins and the positive influence on drought resistance are confirmed. A new genetic resource is provided for sweet potato variety improvement, and research and development of a new variety of high-quality and drought-resistant sweet potatoes can be promoted so as to meet agricultural production and market requirements. By deeply analyzing the potential effect of the IbGSTU7 gene in sweet potato anthocyanin synthesis and drought resistance, a new gene resource and theoretical basis are expected to be provided for sweet potato quality improvement and stress resistance breeding, and the IbGSTU7 gene has important significance in accelerating breeding of sweet potato varieties with high anthocyanin content and stress resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering, and particularly relates to a sweet potato IbGSTU7 gene for regulating the drought resistance and skin color of sweet potatoes and an application thereof. Background Art

[0002] Sweet potato (Ipomoea batatas (L.) Lam) is an important crop for food, feed, and industrial raw materials. Sweet potato is rich in dietary fiber, sugar, protein, vitamins, anthocyanins, mineral elements and other nutrients. Potato peel color is one of the important traits for evaluating the commercial quality of sweet potato, and its color mainly depends on the type of pigment produced. The pigments in sweet potato mainly include carotenoids and anthocyanins. Carotenoids give sweet potato a light yellow to orange appearance, and anthocyanins give sweet potato a red to blue-purple hue. Anthocyanins, as a natural antioxidant, have multiple functions such as regulating the body's immunity, anti-tumor, anti-aging, lowering blood sugar, and enhancing memory. Therefore, the content of anthocyanins in sweet potato has become a key target trait in sweet potato breeding.

[0003] In recent years, there have been many studies on the biosynthetic pathway of plant anthocyanins. Anthocyanins are produced from the precursor substance phenylalanine through a series of enzymatic reactions. They are an important branch of the flavonoid synthesis pathway and are very conserved in higher plants. Anthocyanins in plants are synthesized in the endoplasmic reticulum and then transferred to the vacuole for storage. This process effectively prevents cell damage caused by excessive accumulation of anthocyanins in the cytoplasm. The anthocyanin content in plants is closely related to the stress resistance of plants. Under drought stress, the redox balance in plant cells is disturbed and reactive oxygen accumulates excessively. If anthocyanins are lacking to remove excessive reactive oxygen, it will cause damage to cell components and metabolic dysfunction, which seriously affects the growth and development of plants. Therefore, there is an urgent need to provide a new strategy for sweet potato to cope with drought stress. Summary of the invention

[0004] The purpose of the present invention is to provide a sweet potato IbGSTU7 gene for regulating the drought resistance and potato skin color of sweet potatoes, so as to improve the drought resistance of sweet potatoes and regulate the potato skin color.

[0005] The technical solution adopted by the present invention is:

[0006] The invention provides a sweet potato IbGSTU7 gene for regulating the drought resistance and skin color of sweet potatoes. The nucleotide sequence of the sweet potato IbGSTU7 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0007] The invention provides a recombinant overexpression vector comprising the sweet potato IbGSTU7 gene.

[0008] The present invention provides a method for preparing the recombinant overexpression vector, comprising the following steps:

[0009] A pair of primers is synthesized, and the primer sequences are shown in SEQ ID NO.12 and SEQ ID NO.13; total RNA is extracted from sweet potato, reverse transcribed into cDNA, and the sweet potato IbGSTU7 gene is amplified by PCR using the cDNA as a template using the primers; after the overexpression vector is digested, it is connected with the sweet potato IbGSTU7 gene and transformed to obtain the recombinant overexpression vector.

[0010] Preferably, the overexpression vector comprises any one of pCAMBIA1300, pCAMBIA1301, pCAMBIA1302 and pCAMBIA1305.

[0011] Preferably, the restriction endonucleases used for digestion of the overexpression vector are SpeⅠ and BstEⅡ.

[0012] The present invention provides an application of the sweet potato IbGSTU7 gene or the recombinant overexpression vector, which promotes the accumulation of anthocyanins in the sweet potato by overexpressing the sweet potato IbGSTU7 gene, so as to achieve the purpose of regulating the color of the potato peel.

[0013] The present invention provides an application of the sweet potato IbGSTU7 gene or the recombinant overexpression vector, and improves the drought resistance of the sweet potato by overexpressing the sweet potato IbGSTU7 gene.

[0014] The present invention also provides an application of the recombinant overexpression vector, using the recombinant overexpression vector to construct an overexpression plant to regulate potato skin color and / or improve the drought resistance of sweet potatoes;

[0015] Among them, the method for constructing the overexpression plant includes the following steps: transferring the recombinant overexpression vector into Agrobacterium competent cells to prepare an infection solution; using the infection solution to infect sweet potato cells, and sequentially performing co-cultivation, delayed culture, selection culture, ABA-induced culture and subculture to obtain an overexpression plant.

[0016] Preferably, the Agrobacterium competent cells include any one of EHA105, A208SE and LBA4404.

[0017] Preferably, when the infection solution is used to infect sweet potato cells, the sweet potato cells used for infection are embryonic suspension cells.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a sweet potato IbGSTU7 gene for regulating the drought resistance and potato skin color of sweet potatoes, wherein the nucleotide sequence of the sweet potato IbGSTU7 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The present invention successfully clones the IbGSTU7 gene from sweet potatoes for the first time, and confirms its positive regulatory effect on potato skin color and its positive influence on drought resistance. The present invention provides a new genetic resource for sweet potato variety improvement, and can promote the research and development of high-quality, drought-resistant sweet potato new varieties to meet agricultural production and market needs.

[0020] The present invention is based on the analysis of potato skin transcriptome sequencing data of red potato skin Sushu No. 8 and its yellow potato skin natural mutant Zhengshu 20, and screens out an unreported GST family gene, IbGSTU7. The protein characteristics of the gene were analyzed by bioinformatics analysis and subcellular localization experiments, and it was found that the protein is acidic, hydrophilic, stable protein, and located in the cytoplasm. The expression characteristics of the IbGSTU7 gene were analyzed by real-time fluorescence quantitative PCR, and it was found that it was expressed in multiple tissues of sweet potato, among which the expression level in potato skin was the highest. Drought, high salt and exogenous abscisic acid can induce the expression of the gene in leaves. Transgenic sweet potato plants overexpressing the IbGSTU7 gene were obtained by Agrobacterium tumefaciens-mediated genetic transformation technology. The identification results showed that the anthocyanin content of the transgenic sweet potato skin was significantly higher than that of the wild-type plant WT, and the expression levels of anthocyanin synthesis pathway related genes IbPAL and IbUFGT were also increased. In addition, compared with WT, the hydrogen peroxide H of the transgenic plants under drought stress was increased. 2 O 2 The content was significantly reduced, the expression of IbMDHAR and IbPOD, genes related to the active oxygen scavenging system, was significantly upregulated, the fresh weight and root length were increased, and the drought resistance was significantly improved. Anthocyanins have the functions of scavenging active oxygen, reducing cell damage, maintaining osmotic balance, etc., and can enhance the resistance of plants to abiotic stresses such as drought. The present invention promotes the breeding speed of new varieties of high-anthocyanin drought-resistant sweet potatoes, and provides new gene resources and theoretical basis for sweet potato quality improvement and stress resistance breeding.

[0021] GSTs, as a key enzyme involved in a variety of biological processes, play an important role in the synthesis and transport of anthocyanins. However, there are relatively limited reports on the research of GST genes in sweet potatoes, especially the GSTU subfamily genes, and the specific functions of the GSTU subfamily genes in the accumulation of anthocyanins in sweet potatoes have not been reported. In addition, the traditional sweet potato breeding cycle is long and the range of variation is limited, while genetic engineering can accurately improve the quality, shorten the cycle, and improve the breeding efficiency. The present invention aims to provide new gene resources and theoretical basis for the quality improvement and stress resistance breeding of sweet potatoes by deeply analyzing the potential role of the IbGSTU7 gene in the anthocyanin synthesis and drought resistance of sweet potatoes, which is of great significance for accelerating the breeding of sweet potato varieties with high anthocyanin content and stress resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the tertiary structure analysis of IbGSTU7 protein.

[0023] Figure 2 Subcellular localization of IbGSTU7 protein.

[0024] Figure 3 The expression of IbGSTU7 gene in different tissues of Sushu No. 8.

[0025] Figure 4 The responses of IbGSTU7 gene to 100 μM ABA, 200 mM NaCl and 20% PEG6000 treatment.

[0026] Figure 5 Related pictures of overexpressing sweet potato plants, among which A is the embryonic callus of Li Zixiang; B is the formation of Hyg-resistant callus; C is the formation of somatic embryos; D is the regeneration of the pseudo-transgenic plant; E is the grown-up pseudo-transgenic plant; F is the complete pseudo-transgenic plant; G is the propagation of transgenic test tube seedlings; H is the greenhouse acclimatization of transgenic seedlings; I is the field planting of transgenic seedlings.

[0027] Figure 6 Quantitative PCR analysis of sweet potato plants overexpressing IbGSTU7

[0028] Figure 7 For the phenotypic analysis of transgenic sweet potato tubers, A: parallel samples of tubers from 3 WT plants; B: parallel samples of tubers from 3 OE-3 plants; C: parallel samples of tubers from 3 OE-6 plants; D: parallel samples of tubers from 3 OE-7 plants; E is the cross section of the corresponding tuber in A; F is the cross section of the corresponding tuber in B; G is the cross section of the corresponding tuber in C; H is the cross section of the corresponding tuber in D.

[0029] Figure 8 To identify the pigment content in the skin and flesh of transgenic sweet potatoes, A: carotenoid content; B: total anthocyanin content in the skin; C: total anthocyanin content in the flesh.

[0030] Fig. 9 This is the detection of the expression levels of genes related to anthocyanin synthesis in transgenic sweet potato plants. A to C are the relative expression levels of IbCHI, IbPAL and IbUFGT, respectively.

[0031] Fig.10 Phenotypic analysis of transgenic sweet potato plants under drought stress, A: 3 parallel samples of WT; B: 3 parallel samples of OE-3; C: 3 parallel samples of OE-6; D: 3 parallel samples of OE-7.

[0032] Fig.11 To identify the morphological and physiological indicators of transgenic sweet potato plants under drought stress, A to C are fresh weight, root length and H 2 O 2 content.

[0033] Fig.12 This is the identification of the expression levels of genes related to reactive oxygen scavenging in transgenic sweet potato plants under drought stress. A and B are the relative expression levels of IbMDHAR and IbPOD, respectively. DETAILED DESCRIPTION

[0034] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0035] The inventive concept of the present invention is as follows:

[0036] The anthocyanin content in plants is closely related to their stress resistance. Under drought stress, the redox balance in plant cells is disrupted and reactive oxygen accumulates excessively. If anthocyanins are lacking to remove the excessive reactive oxygen, it will cause cell component damage and metabolic dysfunction, seriously affecting the growth and development of plants. Therefore, there is an urgent need to provide a new strategy for sweet potato to cope with drought stress.

[0037] Based on this, the present invention provides a sweet potato IbGSTU7 gene for regulating sweet potato drought resistance and potato skin color, the nucleotide sequence of the sweet potato IbGSTU7 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0038] In order to make those skilled in the art better understand that the technical solution of the present invention can be implemented, the present invention is further described below in conjunction with specific embodiments. In the description of the present invention, if not otherwise specified, the reagents used are all commercially available, and the methods used are all conventional techniques in the art. The abbreviation table of the present invention is shown in Table 1.

[0039] Table 1 Abbreviations

[0040] Abbreviation Full name AS Acetosyringone CS Ceftriaxone Sodium Hyg Hygromycin B

[0041] Example 1

[0042] A sweet potato IbGSTU7 gene, specifically as follows:

[0043] 1. Cloning of IbGSTU7 gene.

[0044] Total RNA was extracted from the sweet potato variety Sushu No. 8 and then reverse transcribed into cDNA. The sequence of primer IbGSTU7-F is shown in SEQ ID NO.3, and the sequence of primer IbGSTU7-R is shown in SEQ ID NO.4. LA Taq enzyme of TaKaRa Company was used to amplify the ORF sequence of IbGSTU7 gene. The cloning PCR reaction system of IbGSTU7 gene is shown in Table 2.

[0045] Table 2 PCR reaction system for cloning of IbGSTU7 gene

[0046] Reagents Amount used, μL <![CDATA[10×LA PCR BufferⅡ,Mg 2+ Plus]]> 5 dNTPs 8 IbGSTU7-F 1 IbGSTU7-R 1 LA Taq 0.5 Template DNA 2 <![CDATA[ddH 2 The]]> 32.5

[0047] PCR amplification program: 94℃5min; 35 cycles: 94℃30s, 55℃30s, 72℃90s; 72℃7min, stored at 4℃. After PCR, add 2μL of 10× Loading buffer and mix well. Take the PCR product for 1% agarose gel electrophoresis detection, and use the Quanshijin Company kit to purify the target fragment.

[0048] Using TaKaRa's pMD TM 19-T Vector Cloning Kit was used to connect the target fragment to the pMD19-T vector. The reaction system was as follows: Solution I 5 μL; target fragment recovery product 4 μL; pMD19-T Vector 1 μL. Ligation was carried out at 16℃ for 30 minutes and storage was carried out at -20℃.

[0049] The ligation product was transformed into E. coli competent cells Trans 5α, and after plating and culturing, the positive clones detected by PCR were picked and sent to Sangon Biotech Co., Ltd. for sequencing. An equal volume of 50% glycerol was added to the E. coli liquid containing the target gene with the correct sequencing results, and the cells were frozen at -80°C. After sequencing, the nucleotide sequence of the IbGSTU7 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0050] The E. coli monoclonal bacterial solution containing the target gene with the correct sequencing results was selected for activation, and the plasmid was extracted using the plasmid extraction kit of Quanshijin Company. The extracted target plasmid was stored in a -20°C refrigerator.

[0051] 2. Analysis of protein characteristics of IbGSTU7 gene.

[0052] 2.1. Bioinformatics analysis of IbGSTU7 protein.

[0053] TMHMM analysis showed that the IbGSTU7 protein had no transmembrane structure. ProtParam analysis showed that the molecular weight of the IbGSTU7 protein was 25.10 kDa, the theoretical isoelectric point was 5.61, the instability coefficient was 38.92, and the average hydrophobicity index was -0.148. The protein was acidic, hydrophilic, and stable. SOPMA analysis showed that the IbGSTU7 protein was composed of α-helix, extended chain, β-turn, and random coil, accounting for 53.42%, 13.7%, 4.52%, and 28.31% respectively. Figure 1 The predicted three-dimensional structure of IbGSTU7 protein.

[0054] 2.2. Subcellular localization of IbGSTU7 protein.

[0055] Primers were designed based on the ORF sequence of the cloned IbGSTU7 gene with the stop codon removed, and the upstream primer was modified with restriction endonuclease SacⅠ to obtain the upstream primer IbGSTU7-p1300-F, the sequence of which is shown in SEQ ID NO.5; the downstream primer was modified with restriction endonuclease BamHI to obtain the downstream primer IbGSTU7-p1300-R, the sequence of which is shown in SEQ ID NO.6. The recombinant pMD19-T plasmid containing the IbGSTU7 gene was used as a substrate to amplify the IbGSTU7 gene sequence and obtain the target gene ORF fragment.

[0056] The pCAMBIA1300-GFP empty vector was double-digested with restriction endonucleases SacⅠ and BamHI.

[0057] The target gene ORF fragment was connected to the pCAMBIA1300-GFP vector after restriction digestion using T4 DNA Ligase from TaKaRa Company. The reaction system is shown in Table 3.

[0058] Table 3 Reaction system for connecting ORF fragments to pCAMBIA1300-GFP vector

[0059] Element Dosage, μL Target gene ORF fragment 6 pCAMBIA1300-GFP vector after restriction digestion 2 T4 DNA Ligase buffer 2 T4 DNA Ligase 1 <![CDATA[ddH 2 The]]> 9 Total 20

[0060] After overnight ligation at 16°C, the cells were transformed into competent E. coli cells Trans 5α. After plating and culturing, positive clones detected by PCR were picked and sent to Sangon Biotech Co., Ltd. for sequencing verification.

[0061] The plasmid was extracted from the Escherichia coli culture liquid with correct sequencing, and then the recombinant vector was double-digested and verified using SacⅠ and BamH I. The monoclonal culture liquid verified to be correct by enzyme digestion was added with an equal volume of glycerol and stored in a -80°C refrigerator.

[0062] The constructed pCAMBIA1300-IbGSTU7-GFP recombinant vector was introduced into Agrobacterium competent EHA105. The pCAMBIA1300-IbGSTU7-GFP recombinant vector and the pCAMBIA1300-GFP empty vector were transiently expressed in tobacco leaves by Agrobacterium infection method using 8-leaf Nicotiana benthamiana as the material. After culturing in the dark for 8 hours, the leaves were placed under the light conditions of 16 hours of light and 8 hours of darkness, and cultured at 28°C for 72 hours. The epidermis of pCAMBIA1300-IbGSTU7-GFP was peeled off to make a slide, which was placed under a laser confocal microscope to observe the fluorescence signal; the leaves of pCAMBIA1300-GFP were not peeled off, and the leaves directly used were placed under a laser confocal microscope to observe the fluorescence signal.

[0063] The results showed that the control group transferred with the empty vector pCAMBIA1300-GFP had fluorescence signals in the nucleus, cell membrane and cytoplasm, while the fluorescence signal in the fusion expression vector showed a discontinuous distribution around the cell membrane. Combined with the prediction of the online website Cell-PLoc 2.0 that the IbGSTU7 protein is localized in the cytoplasm, and the results of TMHMM analysis that the IbGSTU7 protein has no transmembrane structure, it shows that the fluorescence signal is consistent with the characteristics of cytoplasmic localization. Therefore, the IbGSTU7 protein is localized in the cytoplasm. Figure 2 .

[0064] 3. Analysis of expression characteristics of IbGSTU7 gene.

[0065] 3.1. Expression analysis of IbGSTU7 gene in different tissues of sweet potato.

[0066] Total RNA was extracted from the samples of the fourth inverted stem, the fifth inverted leaf, the root, the peel, and the flesh of the wild-type Sushu No. 8 plant planted for 10 weeks, and reverse transcribed into cDNA. The expression level of the IbGSTU7 gene was determined by fluorescence quantitative PCR. The upstream primer QIbGSTU7-F was designed according to the ORF sequence of the IbGSTU7 gene, and the sequence is shown in SEQ ID NO.7; the downstream primer QIbGSTU7-R, and the sequence is shown in SEQ ID NO.8. The internal reference gene is the sweet potato actin gene IbActin, gene accession number AY905538, the upstream internal reference primer sequence is shown in SEQ ID NO.9, and the downstream internal reference primer sequence is shown in SEQ ID NO.10. 2 –ΔΔCt The relative expression level of IbGSTU7 gene was analyzed by the method. Three biological replicates were set for each experiment. Figure 3 As shown, the IbGSTU7 gene was expressed in all tissues of Sushu No. 8. The expression level in the stem was recorded as 1. The expression level of the IbGSTU7 gene in the potato peel was the highest, which was 62 times that of the stem. Figure 3 The S, L, PR, SSR and FSR refer to stems, leaves, roots, peels and flesh respectively.

[0067] 3.2. Expression analysis of IbGSTU7 gene under different stresses.

[0068] The experiment was conducted using the test tube seedlings of the sweet potato variety Sushu No. 8, which had grown for about 4 weeks. After the test tube seedlings were opened and placed in an artificial climate box for 3 days to acclimate, the culture medium on the roots was washed and placed in water for 1 day. The test tube seedlings of Sushu No. 8 were placed in 100μM ABA, 200mM NaCl and 20% PEG6000 for stress treatment, and the leaves were sampled at 0h, 1h, 3h, 6h, 12h, 24h and 48h. Three replicates were taken at each time point, and each replicate included samples from 3 different plants. The total RNA of the samples was extracted, reverse transcribed into cDNA, and then the expression spectrum of the IbGSTU7 gene under different treatments was analyzed by fluorescent quantitative PCR. Figure 4 As shown in the figure, the expression level of IbGSTU7 at 0 hours of stress treatment was set as the control value, recorded as 1. The IbGSTU7 gene was significantly induced under the three stresses. After 100 μM ABA treatment for 1 hour, the expression level of IbGSTU7 increased to 31 times that of the control, decreased to 11 times after 3 hours, and reached a peak after 6 hours, which was 33 times that of the control; after 200 mM NaCl treatment for 6 hours, the expression level of IbGSTU7 increased to 70 times that of the control, decreased after 12 hours, and reached a peak at 24 hours, which was 138 times that of the control; when treated with 20% PEG6000, the expression level of IbGSTU7 generally showed a trend of first increasing and then decreasing, reaching the highest value after 3 hours of treatment, which was 57 times that of the control.

[0069] Example 2

[0070] The application of IbGSTU7 gene is as follows:

[0071] 1. Obtaining sweet potato plants overexpressing the IbGSTU7 gene.

[0072] Primers were designed based on the ORF sequence of the cloned IbGSTU7 gene, and the upstream primer was modified with restriction endonuclease SpeⅠ to obtain the upstream primer IbGSTU7-p1302-F, the sequence of which is shown in SEQ ID NO.11. The downstream primer was modified with restriction endonuclease BstEⅡ to obtain the downstream primer IbGSTU7-p1302-R, the sequence of which is shown in SEQ ID NO.12. The recombinant pMD19-T plasmid containing the IbGSTU7 gene was used as a template for PCR amplification of the target gene. Total RNA can also be extracted from sweet potatoes, reverse transcribed into cDNA, and the IbGSTU7 gene can be amplified by PCR using cDNA as a template.

[0073] The overexpression vector pCAMBIA1302 was digested with SpeⅠ and BstEⅡ, and the digested pCAMBIA1302 vector was connected with the PCR purified and recovered product amplified by the corresponding enzymes to obtain the pCAMBIA1302-IbGSTU7 recombinant overexpression vector.

[0074] The recombinant overexpression vector pCAMBIA1302-IbGSTU7 was transferred into Agrobacterium tumefaciens competent cells EHA105 to prepare infection solution, and the sweet potato variety Lizixiang, which has light red skin, white flesh and poor stress resistance, was used as the receptor for transformation.

[0075] The specific method is as follows: Use the Agrobacterium tumefaciens infection solution containing the recombinant overexpression vector pCAMBIA1302-IbGSTU7 to infect the pretreated chestnut-fragrant embryonic suspension cells. After co-culture, a one-week delayed culture is carried out, followed by selective culture using hygromycin. The embryonic cell clusters without resistance gradually turn brown and die, while the embryonic cell clusters with resistance grow well, with a bright yellow appearance and a hard texture. After ABA-induced culture, the embryonic cell clusters gradually change from yellow to green, and then dedifferentiate to form somatic embryos. The somatic embryos gradually differentiate into roots and buds, and after transfer and subculture, they eventually grow into complete pseudo-transgenic plants. The results are shown in Figure 5 The culture medium formula and culture conditions are shown in Table 4.

[0076] Table 4 Medium formula and culture conditions

[0077]

[0078] In Table 4, “ / ” indicates that there is no such item.

[0079] The genomic DNA of the proposed transgenic plants was extracted using the simple CTAB method, and the upstream primer 1302-35S-F, the sequence of which is shown in SEQ ID NO.13; the downstream primer GSTU7-324-R, the sequence of which is shown in SEQ ID NO.14, was used to perform PCR positive identification of the proposed transgenic plants. The identification results showed that a total of 10 positive IbGSTU7 overexpression sweet potato lines were obtained, which were named OE-1, OE-2, OE-3, OE-4, OE-5, OE-6, OE-7, OE-8, OE-9 and OE-10. The expression level of the IbGSTU7 gene was determined by fluorescent quantitative PCR, as shown in Figure 6 , OE-3, OE-6 and OE-7 strains with higher expression levels were selected for test tube seedling propagation, greenhouse acclimatization and field planting, and subsequent experiments were carried out after the tubers were harvested.

[0080] 2. The IbGSTU7 gene is used to promote the accumulation of anthocyanins in sweet potatoes as follows:

[0081] The potato peels of WT and transgenic lines OE-3, OE-6, and OE-7 were sampled using a 1-cm diameter round hole sampler. During the sampling process, the potato flesh was removed as much as possible to ensure that each replicate contained eight round holes with a total weight of 0.2 g, and each line included three biological replicates. The samples were submitted to Sanshu Biotechnology Co., Ltd. for the determination of carotenoid and total anthocyanin content. Figure 7 As shown in Figure 2, compared with WT, the potato peels of the IbGSTU7 overexpression lines all showed a darker red color. The results of the determination of the total anthocyanins and carotenoids in potato peels are shown in Figure 2. Figure 8 The carotenoid content of the three transgenic lines was not significantly different from that of WT, but the total anthocyanin content was significantly higher than that of WT. The anthocyanin content of OE-3, OE-6 and OE-7 was 3.29, 2.86 and 3.03 times that of WT, respectively. Therefore, the overexpression of IbGSTU7 gene promoted the accumulation of anthocyanins in potato peel.

[0082] The potato tubers of WT and transgenic lines OE-3, OE-6 and OE-7 were peeled, sliced ​​horizontally and 0.1 g of potato flesh was sampled. Each line included three biological replicates and the samples were sent to Sanshu Biotechnology Co., Ltd. for the determination of total anthocyanin content. Compared with WT, the color of the potato flesh of the IbGSTU7 overexpression line was slightly darker, and the total anthocyanin content showed an upward trend, but it did not reach a significant difference level. Figure 8 Overexpression of IbGSTU7 gene had no significant effect on anthocyanin accumulation in potato flesh.

[0083] Fluorescence quantitative PCR was used to analyze the expression levels of phenylalanine ammonia lyase gene IbPAL, chalcone isomerase gene IbCHI and flavonoid-3-O-glucosyltransferase gene IbUFGT, genes related to anthocyanin synthesis pathway, in WT and transgenic lines OE-3, OE-6 and OE-7. The upstream and downstream primer sequences of fluorescence quantitative PCR of IbPAL gene are shown in SEQ ID NO.15 and SEQID NO.16, the upstream and downstream primer sequences of fluorescence quantitative PCR of IbCHI gene are shown in SEQ ID NO.17 and SEQ ID NO.18, and the upstream and downstream primer sequences of fluorescence quantitative PCR of IbUFGT gene are shown in SEQ ID NO.19 and SEQ ID NO.20. The results of fluorescence quantitative PCR showed that in the three transgenic lines, the expression level of IbCHI was slightly higher than that of WT, and the expression levels of IbPAL and IbUFGT were significantly higher than those of WT, as shown in Figure 2. Fig. 9 Overexpression of the IbGSTU7 gene promoted the expression of genes related to the anthocyanin biosynthesis pathway.

[0084] The above results indicate that the IbGSTU7 gene positively regulates the accumulation of anthocyanins in sweet potato.

[0085] 3. IbGSTU7 gene is used to improve drought resistance of sweet potato, as follows:

[0086] WT and transgenic lines OE-3, OE-6 and OE-7 were subcultured on MS medium containing 20% ​​PEG6000, respectively. The culture conditions were 27°C, light intensity 3000 Lux, and daily illumination for 13 h. After 4 weeks of treatment, the growth status of the transgenic lines was significantly better than that of WT, as shown in Figure 2. Fig.10 The fresh weight and root length of the plants were measured and it was found that the fresh weight and root length of the transgenic lines were significantly higher than those of the WT under drought stress conditions. Fig.11 Plants overexpressing the IbGSTU7 gene showed superior growth performance when faced with drought stress. 0.1 g of leaves from WT and transgenic lines were taken for H 2 O 2 The results showed that the H 2 O 2 The content of H in the overexpression lines under drought stress was significantly lower than that in WT. 2 O 2 The levels were significantly reduced and the tolerance to drought stress was higher.

[0087] Fluorescence quantitative PCR was used to identify the expression levels of the reactive oxygen scavenging related genes monodehydroascorbate reductase gene IbMDHAR and peroxidase gene IbPOD in WT and transgenic lines OE-3, OE-6 and OE-7. The upstream and downstream primer sequences of the IbMDHAR gene fluorescence quantitative PCR are shown in SEQ ID NO.21 and SEQ ID NO.22, and the upstream and downstream primer sequences of the IbPOD gene fluorescence quantitative PCR are shown in SEQ ID NO.23 and SEQ ID NO.24. The results of fluorescence quantitative PCR showed that the expression levels of IbMDHAR and IbPOD genes in the three transgenic lines were significantly higher than those in WT, as shown in Figure 2. Fig.12 IbGSTU7 may improve the plant's tolerance to drought by increasing the expression of genes related to reactive oxygen species scavenging.

[0088] The above results showed that overexpression of IbGSTU7 gene enhanced the drought resistance of sweet potato.

[0089] Figure 3 , Figure 4 , Figure 6 , Figure 7 , Figure 8 , Fig.11 and Fig.12 In the table, “*” indicates statistically significant difference, “**” indicates P < 0.01, and “*” indicates P < 0.05.

[0090] The nucleotide sequence of the IbGSTU7 gene is shown in SEQ ID NO.1; the amino acid sequence of the IbGSTU7 gene is shown in SEQ ID NO.2.

[0091] SEQ ID NO.1:

[0092] 。

[0093] SEQ ID NO.2:

[0094] MAEVKVLGSWGSPFSRRVEVALTLKGVEYEFLEQDLFNKAPLLKSNPVHKKVPVLLHHGNPIPESLLILEYIDDTFPGTPILPKHPYERAMARFWANFIDDKIVPASEK AVFTKGLEHEKGKEEVCELLEILDNEFKKKKFLGGETIGLADIAGMYVALWLGMREEIMGVELGVTKEKFPHLCRWNEDLLNCEVIKENLPSRDELVAYLRPYYTKVFM。

[0095] The upstream primer IbGSTU7-F for ORF amplification is shown in SEQ ID NO.3, and the downstream primer IbGSTU7-R for ORF amplification is shown in SEQ ID NO.4.

[0096] SEQ ID NO. 3: ATGGCAGAAGTGAAGGTTTT.

[0097] SEQ ID NO. 4: TTACATGAATACCTTGGTGTAGTAG.

[0098] The upstream primer IbGSTU7-p1300-F and the downstream primer IbGSTU7-p1300-R are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0099] SEQ ID NO. 5: CGAGCTCATGGCAGAAGTGAAGGTTTTAG.

[0100] SEQ ID NO. 6: CGGATCCCATGAATACCTTGGTGTAGTAGGG.

[0101] The upstream primer QIbGSTU7-F and the downstream primer QIbGSTU7-R of IbGSTU7 gene fluorescence quantitative PCR are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0102] SEQ ID NO. 7: AGTGAAGGTTTTAGGGTCGTGG.

[0103] SEQ ID NO. 8: GACAGGGTTGGATTTGAGAAGC.

[0104] The upstream primer IbActin-F and the downstream primer IbActin-R of IbActin gene fluorescence quantitative PCR are shown in SEQ ID NO.9 and SEQ ID NO.10.

[0105] SEQ ID NO. 9: AGCAGCATGAAGATTAAGGTTGTAGCAC.

[0106] SEQ ID NO. 10: TGGAAAATTAGAAGCACTTCCTGTGAAC.

[0107] The upstream primer IbGSTU7-p1302-F and the downstream primer IbGSTU7-p1302-R are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0108] SEQ ID NO. 11: GACTAGTATGGCAGAAGTGAAGGTTTT.

[0109] SEQ ID NO. 12: GGGTGACCTTACATGAATACCTTGGTGTAGTAG.

[0110] The upstream primer 1302-35S-F and the downstream primer GSTU7-324-R are shown in SEQ ID NO.13 and SEQ ID NO.14.

[0111] SEQ ID NO. 13: GAAGATAGTGGAAAAGGAAGGTG.

[0112] SEQ ID NO. 14: CCTTTGGTAAATACTGCTTTCTCC.

[0113] The upstream primer IbPAL-F and the downstream primer IbPAL-R of IbPAL gene fluorescence quantitative PCR are shown in SEQ ID NO.15 and SEQ ID NO.16.

[0114] SEQ ID NO. 15: CCCTGCAGTGCTAACTACCC.

[0115] SEQ ID NO. 16: GAATAGCCGGGTTCCCACTC.

[0116] The upstream primer IbCHI-F and the downstream primer IbCHI-R of the IbCHI gene fluorescence quantitative PCR are shown in SEQ ID NO.17 and SEQ ID NO.18.

[0117] SEQ ID NO. 17: CCGATCATAGAAGCCGCCAT.

[0118] SEQ ID NO. 18: CTCCGGCATTGAACCCTCTT.

[0119] The upstream primer IbUFGT-F and the downstream primer IbUFGT-R of IbUFGT gene fluorescence quantitative PCR are shown in SEQ ID NO.19 and SEQ ID NO.20.

[0120] SEQ ID NO. 19: CAAACGGAAACGGGTTGGAC.

[0121] SEQ ID NO. 20: CGGTGGGTTCTAGCTTCTGG.

[0122] The IbMDHAR gene fluorescence quantitative PCR upstream primer IbMDHAR-F and PCR downstream primer IbMDHAR-R are shown in SEQ ID NO.21 and SEQ ID NO.22.

[0123] SEQ ID NO. 21: CTACTCCCGTGCCTTTGATT.

[0124] SEQ ID NO. 22: CTCCAAGAATGCACCAACAA.

[0125] The upstream primer IbPOD-F and the downstream primer IbPOD-R of IbPOD gene fluorescence quantitative PCR are shown in SEQ ID NO.23 and SEQ ID NO.24.

[0126] SEQ ID NO. 23: TTCACGACTGCTTCGTTGA.

[0127] SEQ ID NO. 24: TTCTCAACCGCGGTCTTAA.

[0128] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0129] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A sweet potato IbGSTU7 gene for regulating sweet potato drought resistance and potato skin color, characterized in that: The nucleotide sequence of the sweet potato IbGSTU7 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. A recombinant overexpression vector comprising the sweet potato IbGSTU7 gene according to claim 1.

3. The method for preparing a recombinant overexpression vector according to claim 2, characterized in that: The following steps are involved: Synthesize a pair of primers, the primer sequences are shown in SEQ ID NO.12 and SEQ ID NO.13; Total RNA was extracted from sweet potato, reverse transcribed into cDNA, and the sweet potato IbGSTU7 gene was amplified by PCR using the primers with cDNA as template; After the overexpression vector is digested by enzymes, it is connected with the sweet potato IbGSTU7 gene and transformed to obtain the recombinant overexpression vector.

4. The preparation method according to claim 3, characterized in that: The overexpression vector includes any one of pCAMBIA1300, pCAMBIA1301, pCAMBIA1302 and pCAMBIA1305.

5. The preparation method according to claim 3, characterized in that: The restriction endonucleases used for digesting the overexpression vector are SpeⅠ and BstEⅡ.

6. Use of the sweet potato IbGSTU7 gene according to claim 1 or the recombinant overexpression vector according to claim 2, characterized in that: By overexpressing the sweet potato IbGSTU7 gene, the accumulation of anthocyanins in sweet potatoes can be promoted to achieve the purpose of regulating the color of potato peel.

7. Use of the sweet potato IbGSTU7 gene according to claim 1 or the recombinant overexpression vector according to claim 2, characterized in that: Improving drought resistance in sweet potato by overexpressing the sweet potato IbGSTU7 gene.

8. The use of the recombinant overexpression vector according to claim 2, characterized in that: Using the recombinant overexpression vector to construct an overexpression plant to regulate potato skin color and / or improve drought resistance of sweet potatoes; Wherein, the method for constructing the overexpression plant comprises the following steps: Transforming the recombinant overexpression vector into Agrobacterium competent cells to prepare infection solution; The infection solution was used to infect sweet potato cells, and co-culture, delayed culture, selection culture, ABA-induced culture and subculture were performed in sequence to obtain overexpression plants.

9. The use according to claim 8, characterized in that The Agrobacterium competent cells include any one of EHA105, A208SE and LBA4404.

10. The use according to claim 8, characterized in that When the infection solution is used to infect sweet potato cells, the sweet potato cells used for infection are embryonic suspension cells.

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