Application of ZmCPK28 gene or encoding protein thereof in regulation and control of crop carbohydrate anabolism

Abstract

The invention discloses an application of a ZmCPK28 gene or an encoded protein thereof in regulation and control of crop carbohydrate anabolism, and belongs to the technical field of gene engineering. By observing the phenotypes of an overexpression material and a mutant material of the ZmCPK28 gene under different nitrogen concentration conditions, the phenotype and the overground part biomass of the wild type B73-329 are superior to those of the mutant material and have no obvious difference from those of the overexpression material. Meanwhile, the ZmCPK28 gene has the function of regulating and controlling carbohydrate synthesis of corn seedlings, for example, overexpression of the ZmCPK28 gene can improve the content of sucrose and trehalose and reduce the content of glucose and fructose, and a theoretical basis is provided for clarification of the effect of ZmCPK28 in corn growth and development and sugar anabolism.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a gene ZmCPK28 , or an encoded protein thereof, and application thereof in regulating carbohydrate synthesis metabolism of crops. BACKGROUND

[0002] Calcium ion (Ca 2+ ) is a ubiquitous second messenger in plants, and is widely involved in regulating plant development and response to biological and non-biological stresses. In the Ca 2+ -mediated signal transduction process, the transmission of signals depends not only on the change of Ca 2+ concentration, but also on the synergistic action of multiple functional proteins, including calmodulin (CaM), calmodulin-like protein (CML), CBL-like B subunit protein (CBL), and calcium-dependent protein kinase (CDPK / CPK). Among them, CPK has dual functions of Ca 2+ sensor and effector, which can not only perceive Ca 2+ signals, but also directly convert signals into downstream phosphorylation reactions, so as to realize signal amplification and transduction. CPK is considered to be a multifunctional participant in regulating plant non-biological stress management, and CPKs exist in almost all parts of plants, indicating that these kinases have high potential in regulating various signal transduction pathways and have a significant impact on plant growth and development. As a calcium signal sensor, CPK is involved in responses to non-biological stresses such as drought, salinity, low temperature, and high temperature. For example, cotton GhCPK33 enhances salt tolerance by regulating reactive oxygen species (ROS) homeostasis. In addition, CPK is involved in processes such as pollen development, root growth, and fruit ripening in crops, for example, Medicago MtCPK1 regulates nodule formation, and banana MaCPK regulates fruit ripening. In Arabidopsis, AtCPK10 is involved in the regulation of abscisic acid and Ca 2+ of stomata under drought conditions, AtCPK23 plays an important role in plant response to drought and salt stress, and overexpression of AtCPK6 can enhance the salt and drought tolerance of plants.

[0003] At present, some CPK proteins in plants have been reported in aspects such as nutrition, adversity, and growth and development, but most of the researches are concentrated on Arabidopsis, and there are few reports about the synthesis of carbohydrates in maize under low nitrogen stress, and the mechanism is not very clear. Therefore, it is a technical problem to be solved in the field to mine key genes for regulating the synthesis of carbohydrates in plants, and to provide new gene resources for the creation of high-yield and high-quality maize. SUMMARY

[0004] The technical problem to be solved by the application is to provide a gene ZmCPK28The application of genes or their encoded proteins in regulating crop carbohydrate synthesis metabolism is to solve the technical problem of unclear regulatory genes for carbohydrate synthesis in crops.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is to provide a ZmCPK28 Application of genes or their encoded proteins in regulating crop carbohydrate synthesis metabolism, ZmCPK28 The nucleotide sequence of the gene is shown in SEQ ID NO: 1. ZmCPK28 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2.

[0006] On the basis of the above technical solution, the present invention can also be improved as follows: further, ZmCPK28 The gene positively regulates the synthesis of sucrose and trehalose in crops, and negatively regulates the synthesis of glucose and fructose in crops.

[0007] Furthermore, by overexpression ZmCPK28 Genes that increase the sucrose and trehalose content of crops and reduce the glucose and fructose content; by knocking out or silencing ZmCPK28 Gene that reduces the sucrose and trehalose content of crops and increases the glucose and fructose content.

[0008] Furthermore, the crop is corn.

[0009] The present invention also discloses a method comprising ZmCPK28 Gene plasmid.

[0010] The present invention also discloses a method comprising ZmCPK28 Overexpression vector of a gene or plasmid.

[0011] The present invention also discloses a method comprising ZmCPK28 Genes, plasmids or overexpression vectors, and preparations for regulating carbohydrate anabolism in crops.

[0012] The beneficial effects of the present invention are: 1. The present invention is based on observation ZmCPK28 The phenotypes of the gene overexpression materials and mutant materials under different nitrogen concentration conditions showed that the phenotype and aboveground biomass of the wild type B73-329 were better than those of the mutant materials, and had no significant difference with the overexpression materials.

[0013] 2. The content of photosynthetic products of overexpression materials and mutant materials under different nitrogen concentration conditions was determined. The experiment showed that ZmCPK28 Genes that regulate carbohydrate synthesis in maize seedlings, such as overexpression ZmCPK28 Genes can increase the content of sucrose and trehalose and reduce the content of glucose and fructose, which can help to clarify ZmCPK28The results provide a theoretical basis for the role of the gene in the growth and development of corn and sugar synthesis metabolism. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Phenotype identification results of corn mutant material zmcpk28 -1, zmcpk28 -2 and wild type B73-329; Figure 2 Phenotype identification results of corn mutant material zmcpk28 -1 and zmcpk28 -2; Figure 3 Phenotype identification results of corn overexpression material ZmCPK28 OE -1 and ZmCPK28 OE -2; Figure 4 Phenotype identification results of corn overexpression material ZmCPK28 OE -1 and ZmCPK28 OE -2, mutant material zmcpk28 -1 and zmcpk28 -2 and wild type material B73-329; Figure 5 Changes in biomass of corn overexpression material ZmCPK28 OE -1 and ZmCPK28 OE -2, mutant material zmcpk28 -1 and zmcpk28 -2 and wild type material B73-329; Figure 6 Changes in glucose content of corn overexpression material ZmCPK28 OE -1 and ZmCPK28 OE -2, mutant material zmcpk28 -1 and zmcpk28 -2 and wild type material B73-329; Figure 7 Changes in fructose content of corn overexpression material ZmCPK28 OE -1 and ZmCPK28 OE -2, mutant material zmcpk28 -1 and zmcpk28 -2 and wild type material B73-329; Figure 8 Changes in trehalose content of corn overexpression material ZmCPK28 OE -1 and ZmCPK28 OE -2, mutant material zmcpk28 -1 and zmcpk28 -2 and wild type material B73-329. DETAILED DESCRIPTION

[0015] The following describes the specific embodiments of the present application in order to facilitate the understanding of the present application by those skilled in the art. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments not mentioned by the manufacturers are all conventional products that can be purchased on the market. However, it should be clear that the present application is not limited to the scope of the specific embodiments. For those skilled in the art, it is obvious that all the changes within the spirit and scope of the present application defined and determined by the appended claims are obvious and all the inventions utilizing the concept of the present application are within the scope of protection.

[0016] ZmCPK28 The nucleotide sequence of the gene is as follows:

[0017] Zea mays ZmCPK28 The genomic DNA of the gene consists of 4199 bases, and the gene is derived from the inbred line ND101, and the number in the corn genome database is Zm00001d006479.

[0018] ZmCPK28 The amino acid sequence of the protein encoded by the gene is as follows: MGQCCSRAPAPDSGRGGTNGYGYSNQAKPAQTPSSYNPPQQPAEVRYTPPATNPPVVPPVPVPPKPTADTILGKQYEDVRSVYSLGKELGRGQFGVTYLCTEVASGKQYACKSISKRKLTSKADREDIRREIQIMQHLSGQPNIVEFRGAYEDKSNVHVVMELCAGGELFDRIIAKGHYTERAAATICRAVVNVVNICHFMGVMHRDLKPENFLLASKEENAMLKATDFGLSVFIEEGKMYRDIVGSAYYVAPEVLKRSYGKEIDVWSAGVILYILLSGVPPFWAETEKGIFDAILHEEIDFESQPWPSISESAKDLVRKMLTRDPKKRLTSAQVLQHSWLREGGGASDKPIDSAVLSRMKQFRAMNKLKKMALKVIASNLNEEEIKGLKQMFMNMDTDNSGTITYEELKAGLAKLGSKLSEAEVKQLMEAADVDGNGSIDYVEFITATMHRHKLERDEYLFKAFQYFDKDNSGFITRDELESALIEHEMGDTSTIKEIISEVDTDNDGRINYEEFCAMMRGGMQQPMRLK (SEQ ID NO: 2).

[0019] The wild type corn used below is B73-329 (WT), which is described in the following document: [1] Wang F, Cui P, Huang Y, et al. Molecular mechanisms of phosphorus uptake and redistribution in maize [C] / / 2018 National Plant Biology Conference Proceedings. China Agricultural University, Center for Crop Functional Genomics and Molecular Breeding, State Key Laboratory of Plant Physiology and Biochemistry, 2018: 218.

[0020] [2] The application number is CN202011525282.9, and the patent name is “Application of CBL9 and its encoding gene in regulating plant salt-alkali tolerance”.

[0021] Available to the public from China Agricultural University.

[0022] Example 1 Obtaining overexpression materials and mutant materials 1. ZmCPK28 Obtaining and identifying gene-edited transgenic corn plants (1) Corn CRISPR-Cas9 mutant materials zmcpk28 -1: Using conventional CRISPR-Cas9 technology, wild-type corn B73-329 CPK28 -1 gene was knocked out and obtained after transformation and identification.

[0023] (2) Corn CRISPR-Cas9 mutant materials zmcpk28 -2: Using conventional CRISPR-Cas9 technology, wild-type corn B73-329 CPK28 -2 gene was knocked out and obtained after transformation and identification.

[0024] Identification results such as Figure 1 As shown in the figure, compared with the wild type, the mutant material zmcpk28 -1 CPK28 -1 The gene sequence is missing two bases, mutant material zmcpk28 -2 CPK28 -2 The gene sequence is missing one base.

[0025] 2. Corn ZmCPK28 Preparation of gene overexpression plants Maize ZmCPK28 The CDS sequence of the gene is 1596 bp long and encodes a protein containing 531 amino acids. Gene-specific primers were designed based on the CDS sequence. The primer sequences are as follows: ZmCPK28 -1-F: 5'-GTGAATTCCAATATAAACGTT-3' (SEQ ID NO: 3); ZmCPK28 -1-R: 5'-GGACTTGCAGGCGTACTGCTTA-3' (SEQ ID NO: 4); ZmCPK28 -2-F: 5'-GTGAATTCCAATATAAACGTT-3' (SEQ ID NO: 5); ZmCPK28-2-R: 5'-GGACTTGCAGGCGTACTGCT-3' (SEQ ID NO: 6); The gene was amplified by PCR technology ZmCPK28 The 20.0 μL amplification system included: CL Buffer 10.0 μL, -1-F primer (10 μmol / μL) 1.0 μL, -1-R primer (10 μmol / μL) 1.0 μL, template DNA 1.0 μL, CL enzyme 0.5 μL, and ddH2O 6.5 μL. ZmCPK28 -1-F primer (10 μmol / μL) 1.0 μL, ZmCPK28 -1-R primer (10 μmol / μL) 1.0 μL, template DNA 1.0 μL, CL enzyme 0.5 μL, and ddH2O 6.5 μL.

[0026] The 20.0 μL amplification system included: CL Buffer 10.0 μL, -2-F primer (10 μmol / μL) 1.0 μL, ZmCPK28 -2-F primer (10 μmol / μL) 1.0 μL, ZmCPK28 -2-R primer (10 μmol / μL) 1.0 μL, template DNA 1.0 μL, CL enzyme 0.5 μL, and ddH2O 6.5 μL.

[0027] The PCR program was as follows: 94°C pre-denaturation for 1 min; 98°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 90 s (TaKaRa LA Taq 1 Kb / 1 min), 34 cycles; 72°C post-extension for 10 min; and 4°C storage.

[0028] Subsequently, 30 μL of the PCR product was used for 1% agarose gel electrophoresis, the target band was cut, and the gel was recovered using a Zhuangmeng Biological Gel Recovery Kit.

[0029] The T4 DNA Ligase used for ligation was purchased from the NEB company. The 10.0 μL ligation system included: T4 DNA ligase 1 μL, pCXUN-Vector1 DNA 0.4 μL (the pCXUN-Vector1 vector was provided by the Maize Functional Platform of China Agricultural University), insert DNA 8.0 μL, and T4 DNA Ligase 0.15 μL. After adding the sample, it was mixed, separated, and connected at 115°C overnight.

[0030] After the ligation was completed, Escherichia coli DH5α was transformed to obtain pUbi::Zm-UBI1-1. ZmCPK28 The recombinant plasmid was subjected to sequence verification, and after the sequencing was correct, it was submitted to the Maize Transgenic Platform of China Agricultural University for overexpression maize material transformation. The plant expression vector was transformed into maize plants using the Agrobacterium-mediated method, and a ZmCPK28 gene overexpression maize plant was obtained.

[0031] 3、 ZmCPK28 Identification of gene overexpression homozygous lines After the transformation was completed, a ZmCPK28Gene overexpression materials, two strains in total, respectively ZmCPK28 OE -1 and ZmCPK28 OE -2. The materials were germinated, and when they grew to three-leaf-one-heart stage, samples were taken, genomic DNA was extracted for PCR identification (corn genome was extracted by CTAB method, and the reagents were purchased from Beijing Solabio Science and Technology Co., Ltd. Reagent name: 2x CTAB extraction buffer; item number: LS00066, and the specific experimental method was referred to the instruction), and the gene expression in the transgenic plants was verified. ZmCPK28

[0032] Example 2: Phenotype identification The research objects were corn inbred line B73-329 (wild type), mutant materials zmcpk28 -1 and zmcpk28 -2, and overexpression materials ZmCPK28 OE -1 and ZmCPK28 OE -2.

[0033] Seeds of the same period, uniform size and full grains were selected, sterilized with 75% ethanol solution for 15 min, and washed with distilled water for 5 times. The seeds were evenly placed in sterilized glass culture dishes covered with filter paper, and were quantitatively sprayed with distilled water, and were placed in an incubator for germination. After the seeds were white, the seeds with basically consistent germination state were selected and transferred to soil culture pots with pre-absorbed water. The soil culture pots were prepared by crushing and sieving the nutrient soil, and then the soil was equally filled into 12 cm x 12 cm square pots, and the soil was compacted to be about 1 cm from the pot edge. These pots were placed in trays with a volume of 90 cm x 60 cm x 25 cm, respectively filled with clean water. After each pot of soil fully absorbed the same amount of clean water, the remaining solution was poured out. The seeds of wild type strain B73-329, mutant materials zmcpk28 -1 and zmcpk28 -2, and overexpression materials ZmCPK28 OE -1 and ZmCPK28 OE -2 were respectively sowed in the prepared flowerpots (mixed soil of vermiculite: sand = 3: 1), 2 corn seeds were sowed in each pot, and the endosperm side was kept upward. After germination in the artificial climate chamber, the seedlings were hardened to three-leaf-one-heart stage, and sufficient water was ensured during the period. Then the seedlings with consistent growth were grouped for high and low nitrogen treatment. The high nitrogen group (HN) contained 4 mM NO3 - in the high nitrogen nutrient solution, and the low nitrogen group (LN) contained 0.05 mM NO3 - in the low nitrogen nutrient solution. The specific composition is shown in Tables 1-3.

[0034] Table 1: Composition of mother liquor used

[0035] Table 2: Composition of high nitrogen group (HN) nutrient solution ​

[0036] Table 3 Nutrient solution composition of low nitrogen group (LN)

[0037] After the nutrient soil is filled with the nutrient solution, the excess nutrient solution is poured out, and the nutrient solution of the high and low nitrogen groups is applied 2.5 L each time and replaced every two days. The illumination culture is carried out in an environment with a humidity of 70%, and the illumination condition is set to illumination for 16 h (28°C) + darkness for 8 h (26°C). When the corn seedlings of the low nitrogen group (LN) are cultivated to show the nitrogen deficiency phenotype, photographs are taken immediately, and then the relevant physiological indexes are determined.

[0038] The results are shown in Figures 2-4 , wherein HN represents the high nitrogen treatment group, LN represents the low nitrogen treatment group, and L1-L4 represent the first leaf to the fourth leaf. The results show that the aboveground biomass of the wild type B73-329 (WT) is better than that of the mutant material zmcpk28 -1, zmcpk28 -2. Compared with the wild type material B73-329, the first leaf and the second leaf of the mutant material both show the phenotype of yellowing in advance, which indicates that the zmcpk28 mutant material is sensitive to low nitrogen stress. However, the wild type and the overexpression material ZmCPK28 OE -1, ZmCPK28 OE -2, the aboveground biomass has no obvious difference, but compared with the wild type material B73-329, the first leaf and the second leaf of the overexpression material show the phenotype of delaying yellowing, which indicates that the ZmCPK28 overexpression material is more resistant to low nitrogen stress.

[0039] Example 3 Changes in sucrose content in corn seedlings 1. The experimental steps for extracting soluble sugar in corn seedlings are as follows: 1) 0.25 g of powdered corn seedling leaves is weighed in a 2.0 mL centrifuge tube; 2) 1.5 mL of 80% v / v ethanol aqueous solution is added to each centrifuge tube, and the water bath is heated at 60°C for 10 min; 3) Centrifugation is performed at 6000 rpm for 5 min, and the supernatant is taken to a 10 mL centrifuge tube; 4) The steps 2) and 3) are repeated twice; 5) Centrifugation is performed at 12000 rpm for 5 min, and the supernatant is taken, i.e. the soluble sugar is obtained.

[0040] 2. The kit used for sucrose content determination is Megazyme SUCROSE, D-FRUCTOSE and D-GLUCOSE, and the experiment is carried out according to the instruction manual. The data results are shown in Figure 5 .

[0041] FromFigure 5 The analysis found that the wild type B73-329 sucrose content was 78.02 g / 100 g; the overexpression material ZmCPK28 OE -1, ZmCPK28 OE -2 sucrose content was 134.31 g / 100 g and 106.78 g / 100 g, respectively, significantly higher than the wild type; the mutant material zmcpk28 -1, zmcpk28 -2 sucrose content was 30.16 g / 100 g and 32.79 g / 100 g, respectively, significantly lower than the wild type.

[0042] And after 10 days of high nitrogen treatment, the wild type B73-329 sucrose content in the control group changed to 126.06 g / 100 g, the overexpression material ZmCPK28 OE -1, ZmCPK28 OE -2 sucrose content was 617.17 g / 100 g and 575.62 g / 100 g, respectively, still significantly higher than the wild type; the mutant material zmcpk28 -1, zmcpk28 -2 sucrose content was 93.97 g / 100 g and 101.68 g / 100 g, respectively, lower than the wild type.

[0043] The above results show that ZmCPK28 The gene positively regulates the synthesis of sucrose in corn seedlings, and will increase the sucrose content of the plant after high nitrogen treatment.

[0044] Example 4 Change of glucose and fructose content in corn seedlings The kit used for glucose and fructose content determination is Megazyme SUCROSE, D-FRUCTOSE and D-GLUCOSE, and the experiment is carried out according to the instructions, and the data results are shown in Figure 6 .

[0045] From Figure 6 The analysis found that after 15 days of low nitrogen treatment, the wild type B73-329 glucose content was 29.69 g / 100 g; the overexpression material ZmCPK28 OE -1, ZmCPK28 OE -2 glucose content was 22.99 g / 100 g and 19.15 g / 100 g, respectively, lower than the wild type; the mutant material zmcpk28 -1, zmcpk28 -2 glucose content was 42.52 g / 100 g and 49.61 g / 100 g, respectively, higher than the wild type. The above results show that ZmCPK28 The gene negatively regulates the synthesis of glucose in corn seedlings.

[0046] And after 15 days of high nitrogen treatment, the wild type B73-329 glucose content in the control group changed to 44.44 g / 100 g, the overexpression material ZmCPK28 OE-1, ZmCPK28 OE -2 glucose contents were 33.52 g / 100 g and 44.63 g / 100 g, respectively, lower than the wild type; mutant materials zmcpk28 -1, zmcpk28 -2 glucose contents were 45.40 g / 100 g and 41.57 g / 100 g, respectively, higher than the wild type.

[0047] The above results show that ZmCPK28 The gene negatively regulates the synthesis of glucose in corn seedlings, and will increase the glucose content of the plant after high nitrogen treatment.

[0048] As Figure 7 shown, after 15 days of low nitrogen treatment, the fructose content of wild type B73-329 was 40.32 g / 100 g; overexpression materials ZmCPK28 OE -1, ZmCPK28 OE -2 fructose contents were 34.47 g / 100 g and 27.46 g / 100 g, respectively, significantly lower than the wild type; mutant materials zmcpk28 -1, zmcpk28 -2 fructose contents were 49.86 g / 100 g and 50.06 g / 100 g, respectively, significantly higher than the wild type.

[0049] Similarly, after 15 days of high nitrogen treatment, the fructose content of wild type B73-329 in the control group became 70.12 g / 100 g, and the overexpression materials ZmCPK28 OE -1, ZmCPK28 OE -2 fructose contents were 38.37 g / 100 g and 40.51 g / 100 g, respectively, still significantly lower than the wild type; mutant materials zmcpk28 -1, zmcpk28 -2 fructose contents were 80.25 g / 100 g and 90.83 g / 100 g, respectively, higher than the wild type.

[0050] The above results show that ZmCPK28 The gene negatively regulates the synthesis of fructose in corn seedlings, and will increase the fructose content of the plant after high nitrogen treatment.

[0051] Example 5 Changes in the content of trehalose in corn seedlings The kit used for trehalose content determination is Megazyme TREHALOSE KIT, and the experiment is carried out according to the instructions, and the data results are shown in Figure 8 .

[0052] As Figure 8 can be seen, the trehalose content of wild type B73-329 in the low nitrogen treatment group was 84.25 g / 100 g; overexpression materials ZmCPK28 OE -1, ZmCPK28 OE-2 trehalose contents were 101.71 g / 100 g and 95.64 g / 100 g, respectively, which were significantly higher than that of the wild type; mutant materials zmcpk28 -1, zmcpk28 -2 trehalose contents were 64.52 g / 100 g and 64.65 g / 100 g, respectively, which were lower than that of the wild type. The above results showed that ZmCPK28 The gene positively regulated the synthesis of trehalose in corn seedlings.

[0053] After 15 days of high nitrogen treatment, the trehalose content of the wild type B73-329 in the control group became 95.64 g / 100 g, and the trehalose content of the overexpression material ZmCPK28 OE -1, ZmCPK28 OE -2 trehalose contents were 112.34 g / 100 g and 114.62 g / 100 g, respectively, which were higher than that of the wild type; mutant materials zmcpk28 -1, zmcpk28 -2 trehalose contents were 90.10 g / 100 g and 92.55 g / 100 g, respectively, which were lower than that of the wild type.

[0054] The above results showed that ZmCPK28 The gene positively regulated the synthesis of trehalose in corn seedlings, and would increase the trehalose content of the plant after high nitrogen treatment.

Claims

1. A ZmCPK28 The application of a gene or its encoded protein in regulating carbohydrate anabolism in crops is characterized in that: described ZmCPK28 The nucleotide sequence of the gene is shown in SEQ ID NO:

1. ZmCPK28 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that described ZmCPK28 The gene positively regulates the synthesis of sucrose and trehalose in crops, and negatively regulates the synthesis of glucose and fructose in crops.

3. The use according to claim 2, characterized in that Through overexpression ZmCPK28 Genes that increase the sucrose and trehalose content of crops and reduce the glucose and fructose content; by knocking out or silencing ZmCPK28 Gene that reduces the sucrose and trehalose content of crops and increases the glucose and fructose content.

4. The use according to claim 1 or 2, characterized in that The crop is corn.

5. A plasmid, characterized in that Containing the claim 1 ZmCPK28 Gene.

6. An overexpression vector, characterized in that Containing the claim 1 ZmCPK28 The gene or the plasmid according to claim 5.

7. A preparation for regulating carbohydrate anabolism in crops, characterized in that: Containing the claim 1 ZmCPK28 gene, the plasmid according to claim 5 or the overexpression vector according to claim 6.

Images