Plant insect resistance-related protein UGT198 and its application

Abstract

The present invention discloses a plant insect-resistant protein UGT198 and its application, and specifically discloses the nucleic acid sequence and protein sequence of the plant insect-resistant protein UGT198 gene. It also discloses the application of the plant insect-resistant protein UGT198, specifically that transgenic plants overexpressing the UGT198 gene can improve resistance to gypsy moth larvae and can also delay the development of gypsy moth larvae. The present invention relates to the field of genetic engineering technology, and specifically provides a plant insect-resistant protein UGT198 and its application. The endogenous insect-resistant glycosyltransferase UGT198 gene of poplar is used as an entry point to explore its resistance to gypsy moth larvae. The results show that transgenic plants overexpressing the gene can improve resistance to gypsy moth larvae and can delay the development of the larvae. An endogenous insect-resistant gene is identified through this scheme, which can provide a new gene resource for insect-resistant poplar breeding.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, in particular to a plant insect resistance-related protein UGT198 and an application thereof. Background Art

[0002] Poplar is an important industrial timber and ecological tree species. During large-scale afforestation in my country, there are problems such as single species, excessive area, and unreasonable age structure, which lead to serious pest and disease phenomena and bring great threats to the healthy development of poplar plantations. Therefore, there is an urgent need to cultivate new insect-resistant poplar varieties. Poplar is a model tree species for forest genetic engineering research. Compared with other forest trees, poplar genetic engineering has made rapid progress. The use of genetic engineering methods to cultivate insect-resistant poplars has important scientific significance and application value. At present, the main sources of insect-resistant genes are Bacillus thuringiensis genes (Bt genes) of microorganisms, plant protease inhibitor genes, and genes of animal origin. Due to factors such as the insect-resistant stability and biosafety of these exogenous genes, their application is limited. Therefore, digging out endogenous insect-resistant genes in poplars and stimulating their own defense capabilities is of great significance to the cultivation of new insect-resistant poplar varieties.

[0003] Glycosylation is a crucial molecular modification reaction in organisms. Glycosylation products have numerous biological functions, such as playing a crucial role in the diversity and complexity of plant secondary metabolites, as well as in plant defense and stress resistance. Glycosylation in plants is performed by a large family of enzymes called glycosyltransferases (GTs). GTs represent a large gene family of enzymes. As of August 2021, the carbohydrate-active enzyme database, CAZy, classifies known glycosyltransferases into 114 families. GT1 is the largest of the GT gene families and, because it uses UDP-glucose as a glycosyl donor, is also known as the UGT family. The N-terminal regions of the plant UGT gene family are highly diverse in sequence, while the C-terminal regions contain a highly conserved sequence of 44 amino acid residues, known as the plant secondary product glycosyltransferase (PSPG) box. The sugar acceptor and sugar donor binding domains are located in Rossmann-like domains at the N- and C-termini, respectively. UGT gene family members share 40% or more amino acid sequence similarity, and subfamily members share 60% or more amino acid similarity. With the continuous completion of genome sequencing of various plants, UGT gene families have been identified in many plants, such as Arabidopsis thaliana (107), rice (215), maize (147), upland cotton (274), cassava (121), etc. In addition, Lorenzo et al. identified UGT gene families in 12 species (including poplar), but only conducted phylogenetic reconstruction studies on this multigene family.

[0004] UGTs (glycosyltransferases) have a wide range of physiological functions. Some glycosyltransferases contribute to plant resistance by catalyzing the synthesis of small molecules such as terpenes, steroids, phenylpropanoids, and flavonoids. For example, the rice glucosyltransferase UGT gene, Os07g32020, contributes to rice insect resistance by regulating the synthesis of naringenin, a biochemical precursor of a major flavonoid compound. The soybean GmUGT gene is a major factor in resistance to leaf-feeding insect pests. CRISPR / Cas9 mutants of GmUGTs enhance resistance to cotton bollworm and Spodoptera litura. Overexpression lines are susceptible to these two pests. Differences in metabolite and gene expression suggest that GmUGTs contribute to enhanced resistance to leaf-feeding insects by altering flavonoid content and the expression patterns of genes involved in flavonoid biosynthesis and defense. In the flavonoid and flavonol biosynthesis pathway, upregulation of UGT gene expression promotes the synthesis of rutin and rutin, both of which are toxic to insects. These studies indicate that UGT gene family members play an important role in regulating plant defense against insect pests. However, their roles in inducing pest resistance remain relatively limited, with only exploratory studies conducted. Furthermore, there are few reports on the involvement of poplar UGT genes in insect defense. Therefore, identifying potential insect-resistance-related UGT genes in poplar could provide genetic resources for enhancing insect resistance in poplars through regulating endogenous genes, which is of great significance for the breeding of insect-resistant transgenic poplars. Summary of the Invention

[0005] In response to the above situation, in order to solve the problem that the insect-resistant genes used in the currently cultivated transgenic insect-resistant poplars are mostly derived from other species, and there are factors such as unsatisfactory insect-resistant effects or uncertain safety, which leads to limited application, the present invention provides a plant insect-resistant related protein UGT198 and its application. In this scheme, the poplar endogenous insect-resistant related glycosyltransferase UGT198 gene is used as the entry point to explore its resistance to the larvae of the American white moth. The results show that transgenic plants overexpressing this gene can improve resistance to the larvae of the American white moth and can delay the development of the larvae.

[0006] The present invention provides the following technical solution: The present invention proposes a plant insect resistance-related protein UGT198, the nucleic acid sequence of which is as follows:

[0007]

[0008] The protein sequence of UGT198 is as follows:

[0009] MEEALVLYPSPPIGHLVSMVELGKLLLTHRPSLSIHILIAASPYVAGKANKYMATVSANVPSIDFHHLPIVTPVSTNITHHEELTLEVLHLSKPHVHEELLNISKRYKIHGLVMDFFCTS GLSVATELDIPSYFFLTSGACFLAFFLYLPTLHQKTSKSFKDMKDHYLDIPGLPPLLATDLPNPFLDRDNQAYQHFLDDFATQFPQASGIMINTFELLESRVVKAISDGLCVPNNRTPPIS CIGPLIVADDKRGGSGKNSPEDVHECLSWLDSQPSQSVVFLCFGSLGLFTKEQLWEIATGLEENSGQRFLWVVRNPPSHNLKVAIKEQGDPDLDSLLPEGFLERTKERGYVVKSWAPQVAI VNHSSVGGFVTHCGWNSTLEAVYAGLPMVAWPLYAEQRLNRVVLVEEMKLALSMNESEDGFVSADEVEKKVRGLMESKEGKMIRERALAMKNEARAALSEGGSSHVAVSKLLESWKHEK.

[0010] At the same time, the present invention also proposes the application of plant insect resistance-related protein UGT198, specifically, transgenic plants overexpressing the UGT198 gene can improve resistance to the larvae of the American white moth.

[0011] Furthermore, the transgenic plants overexpressing the UGT198 gene can also delay the development of nymphalid moth larvae.

[0012] The beneficial effects achieved by the present invention using the above-mentioned structure are as follows: The plant insect-resistant protein UGT198 and its application proposed in the present invention have the following specific advantages: This scheme uses the endogenous insect-resistant glycosyltransferase UGT198 gene of poplar as the starting point to explore its resistance to the larvae of the American white moth. The results show that transgenic plants overexpressing this gene can improve resistance to the larvae of the American white moth and delay the development of the larvae. Through this scheme, an endogenous insect-resistant gene was identified, which can provide a new gene resource for insect-resistant poplar breeding, thereby effectively solving the problem that the insect-resistant genes used in the currently cultivated transgenic insect-resistant poplars are mostly derived from other species, and there are factors such as unsatisfactory insect-resistant effects or uncertain safety, which leads to limited applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0014] Figure 1 This is the real-time fluorescence quantitative PCR analysis of the UGT198 gene;

[0015] Figure 2 This is the diagram of transformation of Escherichia coli and Agrobacterium tumefaciens by recombinant overexpression vector;

[0016] Figure 3 This is the sequencing alignment of poplar UGT198;

[0017] Figure 4 is the PCR detection diagram of the transgenic line;

[0018] Figure 5 This is a comparison chart of the relative expression levels of the UGT198 gene in the transgenic lines;

[0019] Figure 6 This is a schematic diagram of the selective feeding of hystrix moth larvae on poplar leaves that overexpress the UGT198 gene;

[0020] Figure 7 This is a schematic diagram of the forced feeding of hystrix moth larvae on poplar leaves overexpressing the UGT198 gene;

[0021] Figure 8 Schematic diagram of the effect of feeding transgenic plants on the growth and development of hystrix moth larvae. DETAILED DESCRIPTION

[0022] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0023] Example:

[0024] This example performs expression analysis, detection, and related insect resistance tests on the plant insect resistance-related protein UGT198 gene provided in the invention content of this solution.

[0025] 1. Experimental Setup

[0026] (1) Expression analysis of UGT198 gene

[0027] For salt and drought stress, the wild-type "741 Poplar" tissue culture seedlings were transplanted into the greenhouse. When they grew to 12-15 cm, 200 mmol / L NaCl and 0.6% PEG6000 aqueous solution were used to irrigate the poplar seedlings, and samples were taken after 24 hours. Samples of different tissues (roots, stems and leaves) of the poplar were taken for tissue-specific expression analysis. For pest treatment, 10 American white moth larvae were used to treat poplar leaves in the same period, and samples were taken at different time points (0, 2, 4, 8 and 12 hours) for real-time fluorescence quantification. Three biological replicates were set for each batch of samples. After sampling, they were quickly frozen with liquid nitrogen and placed at -80°C for use. Total RNA was extracted and reverse transcribed using the extraction kit of Tiangen Company, and the extraction was performed according to the method in the instruction manual. Primer5.0 software was used to design fluorescence quantitative primers to identify the expression pattern of the UGT198 gene in different tissues, drought and salt stress, and different feeding times.

[0028] (2) Cloning of UGT198 gene and construction of overexpression vector

[0029] Based on the gene sequence of Populus trichocarpa PtUGT198, specific primers F / R were designed using Primer 5.0 software to amplify the target gene from Populus euphorbiae 107. The amplified product was ligated into the overexpression vector pNC-Cam2304-35S and transformed into competent Escherichia coli DH5α. Positive clones were sent to the company for sequencing. Single clones with correct sequencing results were used to extract the plasmid by shaking and transformed into competent Agrobacterium GV3101 cells.

[0030] (3) Poplar genetic transformation

[0031] Agrobacterium containing the UGT198 gene was propagated and shaken to an OD600 of approximately 0.6-0.8 for poplar transformation. Sterile tissue culture seedlings of the 741 poplar variety, 3-6 weeks old and robust, were selected. The third to fifth leaves, roughly uniform in size, shape, and color, were removed from the top and placed on a Petri dish lined with filter paper. Three to four evenly incised wounds were made across the veins of each leaf, and the leaves were spread flat on pre-culture medium for 1-3 days. Genetic transformation was performed using the leaf disc method within a clean bench. Pre-cultured leaves were inoculated with the resuspension and shaken at 28°C and 150 rpm for 10-15 minutes to allow the Agrobacterium to fully adhere to each leaf. The leaves were removed and spread flat on sterile filter paper. The bacterial suspension on the leaf surface was blotted dry, and then the leaves were spread flat on co-culture medium and co-cultured in the dark at 25°C for 2-4 days. The co-cultivated explants were transferred to a selective differentiation medium and cultured under a 16h / 8h photoperiod at 28°C until resistant buds were induced. When the induced buds reached approximately 1 cm in length, they were transferred to a selective rooting medium. After approximately two weeks, adventitious roots were induced, forming complete plantlets, preliminarily confirming the overexpression strain.

[0032] (4) Obtaining and transplanting transgenic seedlings

[0033] When adventitious roots reach approximately 1 cm in length, select transgenic seedlings in good growth condition and harden them in a well-ventilated area with the sealing film removed for 3 days. Remove the seedlings from the tissue culture flasks, rinse the culture medium around the roots with clean water, and maintain the integrity of the root system as much as possible. Plant them in pots with a 1:1:1 ratio of garden soil: nutrient soil: vermiculite and cultivate them in a greenhouse. Initially, cover the seedlings with plastic wrap to maintain a high humidity level to prevent wilting and death due to water loss. Once new leaves appear, gradually remove the film and transfer the seedlings to the greenhouse environment.

[0034] (5) Positive plant identification and RT-PCR detection

[0035] When each strain grew to 10-15 cm, genomic DNA was extracted from the overexpressing and wild-type "741 Poplar" plants. PCR was then performed on the transgenic lines using the DNA of each strain as a template, a plasmid containing the target gene as a positive control (CK+), and wild-type "741 Poplar" as a negative control (CK-) to confirm whether the transgenic lines contained the UGT198 target gene. To analyze the relative expression of UGT198 in the different overexpressing poplar strains, total RNA was extracted from wild-type "741 Poplar" and each transgenic strain, reverse-transcribed into cDNA, and fluorescent quantitative PCR analysis was performed using wild-type "741 Poplar" as a control to determine the relative expression level of the UGT198 gene.

[0036] (6) Indoor insect feeding test of transgenic strains

[0037] A. Selective feeding test

[0038] Young leaves from wild-type and different transgenic lines were placed in the same incubator along with 30 nymphalid larvae. The petioles were then placed in moist floral mud for a preferential feeding experiment. During the 12-hour feeding period, leaf area loss was recorded for each line, and larval activity was observed.

[0039] B. Mandatory insect feeding test

[0040] The third and fourth fresh young leaves of wild-type and transgenic poplar plants were placed in an incubator to feed nymphal larvae of the American cunning moth (Cyprinus cuneiformis). Fifteen larvae were used per treatment, with at least three replicates. Poplar leaves were scanned before and after feeding to observe leaf loss. Leaf feeding was observed every two hours for 12 hours, and leaf loss was recorded at the 12-hour mark.

[0041] The strains with better feeding effects and relatively higher expression levels were comprehensively selected to conduct long-term forced feeding experiments, and the changes in larvae weight and length were recorded by weighing and measuring every day.

[0042] The experiments were carried out in an incubator at a temperature of 27±2°C, a relative humidity of 65±5%, and a light / dark cycle of 16h / 8h. Each experiment was repeated at least three times.

[0043] 2. Measurement results

[0044] (1) Expression analysis of UGT198 gene

[0045] Figure 1 This is a diagram of real-time fluorescence quantitative PCR analysis of the UGT198 gene.

[0046] Figure 1 (a) Relative expression of UGT198 gene in different tissues; Figure 1 (b) Relative expression level of UGT198 gene after salt and drought stress treatment; Figure 1 (c) The relative expression levels of UGT198 gene in gypsy moth larvae before feeding, after feeding for 2h, 4h, 8h and 12h.

[0047] Real-time quantitative PCR analysis of untreated poplar samples revealed that the UGT198 gene was expressed in leaves, stems, and roots, with the expression level in stems significantly higher than in roots and leaves. The relative expression level in stems was 7.39 times that in leaves, which was a significant difference. The relative expression level in roots was 2.22 times that in leaves, which was not a significant difference ( Figure 1 a). In the stress treatment, it was found that after 24 hours of PEG6000 and NaCl treatment, the relative expression levels of UGT198 gene were significantly different from those of the control, with the expression levels being 4.57 times and 3.17 times that of CK, respectively ( Figure 1 b). The UGT198 gene showed an up-regulation trend after the poplar leaves were eaten by the nymphs of the American moth at different times. Its relative expression level was significantly different from that of the leaves that were not eaten, but the difference at each time point was not significant ( Figure 1 c), indicating that the UGT198 gene may play an important role in regulating insect resistance in poplar.

[0048] (2) UGT198 gene cloning and vector construction

[0049] Figure 2 This is a diagram of the transformation of Escherichia coli and Agrobacterium tumefaciens by recombinant overexpression vector.

[0050] Figure 2 (a) Detection of E. coli transformed with the cloning vector, lane M is a 2000 bp DNA marker, and lanes 1-17 are amplified UGT198 genes; Figure 2 (b) is the overexpression vector transformed into Agrobacterium detection; 1-10 are the amplified UGT198 genes.

[0051] Figure 3 This is a sequence alignment of poplar UGT198; PtrUGT198 is the sequence of Populus trichocarpa; UGT198 is the sequence cloned from Populus europaeus 107.

[0052] The UGT198 gene sequence was cloned from Populus occidentalis 107. Its complete ORF region is 1440 bp, encoding 479 amino acids, with a theoretical molecular weight of 53.14 kDa and an isoelectric point of 5.92. It encodes an unstable hydrophobic protein. The UGT198 gene was constructed into an overexpression vector, and the recombinant overexpression vector pNC-Cam2304-35S-UGT198 plasmid was transformed into Escherichia coli. The electrophoresis diagram of the bacterial solution was ( Figure 2 a) showed that most of the target bands were clear and accurately positioned. The E. coli solution with the correct bands was sent to the company for sequencing. DNAMAN was used to perform Blast comparison on the sequencing results. After sequencing comparison, 16 base mutations were found between the two ( Figure 3 ), indicating that the gene sequences of different plants may vary, and the presence of these variant sites may cause differences in gene expression efficiency. Save the bacterial solution with the correct sequence, extract the plasmid, transform the plasmid into Agrobacterium, and pick a single Agrobacterium colony for target gene PCR verification. The results showed that the target band position was accurate, proving that the recombinant plasmid pNC-Cam2304-35S-UGT198 was successfully transformed into Agrobacterium ( Figure 2 b).

[0053] (3) PCR detection of transgenic poplars

[0054] Figure 4 This is the PCR detection diagram of the transgenic line.

[0055] Figure 4 (a) is the gel electrophoresis of 35S-UGT198, M is a 2000bp DNA marker, CK+ is a positive control (bacterial solution), and CK- is a negative control (wild-type poplar WT). 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 21, and 25 are 13 transgenic poplar lines, Figure 4 (b) is the gel electrophoresis of the Kan gene, CK+ is the positive control (bacterial solution), and CK- is the negative control (wild-type poplar WT). 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 21, and 25 are 13 transgenic poplar lines.

[0056] The genomic DNA of the transgenic poplar plants was extracted and PCR amplified for detection of 35S-UGT198 and the resistance gene Kan. The electrophoresis results were as follows: Figure 4 , 13 overexpression poplar lines all amplified the 35S-UGT198 gene band of about 1440 bp ( Figure 4 a) and the resistance gene Kan band of about 500 bp ( Figure 4 b), indicating that the target gene UGT198 was successfully inserted into the poplar genome.

[0057] (4) Analysis of relative expression of UGT198 gene in transgenic lines

[0058] Figure 5 The figure is a comparison chart of the relative expression levels of the UGT198 gene in the transgenic lines. Figure 5 The same letters indicate no significant difference, and different letters indicate significant difference. The significance level is 0.05.

[0059] Nine robust lines were selected, and three biological replicates were taken from each line to extract total RNA from poplar trees, which was reverse transcribed into cDNA. The relative expression levels of the UGT198 gene in the transgenic lines were detected by qRT-PCR. Figure 5 As shown in the figure, the expression levels of UGT198 gene in the nine transgenic lines were higher than that in wild-type poplar, showing significant differences. The relative expression levels were 8.82 to 45.67 times that of the wild type.

[0060] (5) Selective insect feeding test

[0061] Figure 6 Schematic diagram of the selective feeding of gypsy moth larvae on poplar leaves overexpressing the UGT198 gene.

[0062] Figure 6 (a) The selective feeding of first-instar larvae of the gypsy moth on leaves of the control and transgenic plants for 4 h; Figure 6 (b) The first-instar larvae of the American white moth selectively fed on the leaves of the transgenic and control plants for 12 hours.

[0063] The wild-type and transgenic lines with good growth were selected for feeding experiments with gypsy moth larvae to test the insect resistance of the overexpression lines. Leaves of the wild-type and overexpression lines were placed in the same incubator to observe the feeding preferences of the gypsy moth on poplar leaves under the conditions available. At 4 hours of treatment, the larvae were mainly concentrated on the leaves of the wild-type line, which had already suffered damage, while larvae were also distributed on the leaves of the transgenic line, but no damage was observed; at 12 hours, the wild-type line suffered serious damage, while there were no larvae distributed on the leaves of the transgenic line ( Figure 6 ).

[0064] (6) Mandatory insect feeding test

[0065] Figure 7 Schematic diagram of the forced feeding of gypsy moth larvae on leaves of poplars overexpressing the UGT198 gene.

[0066] Figure 7 (a) The first-instar larvae of the gypsy moth forced to feed on leaves of the control and transgenic plants for 12 h; Figure 7 (b) Leaf loss rate of the transgenic and wild-type strains after forced feeding by first-instar larvae of the gypsy moth.

[0067] Figure 8 Schematic diagram of the effect of feeding transgenic plants on the growth and development of hystrix moth larvae.

[0068] Figure 8 (a) The first-instar larvae of the gypsy moth forced to feed on leaves of the control and transgenic plants for 9 days; Figure 8 b, 8c are the development of larvae on days 5 and 9 of forced feeding of 1st instar larvae of the cuneiform moth in the control and transgenic plants; Figure 8 d and 8e show the effects on body weight and length of first-instar nymphalids of the American white moth feeding on transgenic plants and control leaves within 1-9 days.

[0069] A. Effects of feeding by nymphalid moth larvae on leaf loss in transgenic plants

[0070] The leaves of each strain were placed in separate incubators to feed nymphal larvae. Four hours before treatment, the leaves of the wild-type strain had already been damaged, while the leaves of the transgenic strains were not eaten. Between 4 and 8 hours, the leaves of the wild-type strain were damaged more severely, and the larvae were more active. The leaves of the transgenic strains were also fed, with larvae distributed on the mesh, but the larval activity was relatively weak. After 12 hours, the overall leaf feeding situation was counted, and it was found that the leaves of the wild-type strain had the most serious damage. Among the transgenic strains, the leaf damage of strain 3 was the most serious, followed by strains 10, 11, and 25, which had relatively less leaf damage ( Figure 7 ).

[0071] B. Effects of transgenic plants on the growth and development of nymphal moth larvae

[0072] The leaves of each strain were placed in separate incubators to feed 1st-instar larvae of the cuneiform moth, and the effects of feeding for different days on the growth and development of the larvae were observed. Figure 8 Figure a shows leaves and larvae of the gypsy moth feeding for nine days. As can be seen from the figure, after nine days of feeding, the transgenic plants showed less loss of leaves 3, 10, 11, and 25 compared to the control, indicating improved insect resistance to a certain extent. Furthermore, the larvae on the transgenic plants developed more slowly and were smaller. Figure 8 bc are larvae of the 5th and 9th day after feeding by cuneae. After feeding, the larvae of the transgenic lines were relatively thin and smaller than the control. Figure 8 de is the changes in body length and weight of hyphalidae larvae during feeding for 1-9 days, Figure 8 d and Figure 8The changing trends of body length and weight were relatively consistent, with the transgenic lines being smaller than the control, indicating that the growth of the larvae of the gypsy moth was inhibited after feeding on the transgenic plants, delaying the growth and development of the larvae.

[0073] The results of these two groups of insect feeding experiments show that when given a choice, the larvae of the gypsy moth tend to feed on leaves of wild-type poplars; when not given a choice, they will also feed on leaves of overexpression strains, but some larvae stay on the mesh. After feeding on leaves, the larvae's activity decreases but they are not fatal. In addition, feeding on leaves of transgenic plants will delay the larvae's growth and development.

[0074] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, material, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, material, or apparatus.

[0075] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

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