Clone And Function Verication of The OPR Gene Related to Linoleic Acid Synthesis

Background: Fatty acid composition and content affect rapeseed oil quality. Fatty acid synthesis-related genes in rapeseed have been studied globally by researchers. Nevertheless, rapeseed oil is mainly composed of seven different fatty acids, and each fatty acid was regulated by different genes, furthermore different fatty acid contents affect each other, which needs continuous and in-depth research to obtain more clear results. Results: In this paper, broad-scale miRNA expression proles were constructed and 21 differentially expressed miRNAs were detected. GO enrichment analysis showed that most up-regulated proteins were involved in transcription factor activity and catalytic activity. KEGG pathway enrichment analysis indicated that 20 pathways involving 36 target genes were enriched, of which the bna00592 pathway may be involved in fatty acid metabolism. The results were veried using a Quantitative Real-time PCR (RT-PCR) analysis, and it was found that the target gene of bna-miR156b>c>g was the OPR (12-oxo-phytodienoic acid reductase). Four copies of OPR gene were found, and the over-expression vectors (pCAMBIA1300-35s-OPR and pCAMBIA1300-RNAi-OPR) were constructed to verify their functions. In T 1 and T 2 plants, OPR-OE (OPR Over-Expression strain) signicantly increased linoleic acid content (T 1 12.56%, T 2 7.185%) and OPRi (OPRi RNA-interference strain) decreased linoleic acid content (T 1 5.98%, T 2 0.86%). Conclusions: This is the rst study to provide four copies of the OPR gene that regulates LA metabolism, can be used for the molecular mechanism of LA and optimizing fatty acid proles in oilseed for breeding programs. involved in fatty acid metabolism. A total of 20 pathways were enriched by KEGG pathway, involving 36 target genes, of which 15 may be involved in the regulation of fatty acid metabolism; the reliability of the results was veried using the RT-PCR analysis. This result provided a basis for subsequent functional verication. quantity of the puried RNA were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Santa Clara, USA) and RNA 6000 nanokit (Agilent Technologies, Palo Alto, Santa Clara, USA). sRNAs with lengths of 18–30 nt were separated and puried using 15% denaturing polyacrylamide gel electrophoresis. Consequently, sRNAs fractions were ligated to the 5′ lectrophoresters using T4 RNA ligase (Epicentre, America). The adapter-ligated fragments were then reverse transcribed and amplied by performing PCR with a pair of adapter complementary primers. These PCR products were puried and sequenced using IlluminaHiseq XTEN (Illumina, USA). Construction of the sRNA libraries and deep sequencing were carried out by Oebiotech Genomics (Shanghai, China).


Background
Brassica campestris L. (rapeseed) is one of the most important oil crops in the world [1,2]. The quality of rapeseed oil mainly depends on its fatty acid composition, especially the proportion of three main unsaturated fatty acids: oleic acid (C 18 : 1 ), linoleic acid (C 18 : 2 ), and linolenic acid (C 18 : 3 ) [3]. Many studies have shown that rapeseed oil with a high unsaturated fatty acid content will have better health effects and can prevent the occurrence of cardiovascular diseases [4]. Linoleic acid (LA) can prevent or reduce the incidence of cardiovascular diseases and being used for the prevention and treatment of hypertension, hyperlipidemia, angina pectoris, coronary heart disease, atherosclerosis, and senile obesity [5,6]. LA can be used as a precursor of ultra-long chain polyunsaturated fatty acids. α-linolenic acid (ALA) and γ-linolenic acid (GLA) have been hydrogenated from linoleic acid using desaturase [7]. miRNAs are short noncoding regulatory RNAs that regulate gene expression via post-transcriptional repression [8,9]. In plants, miRNAs are involved in various biological processes, including the regulation of plant development [10], architecture formation [11], photosynthesis [9], tolerance to biotic and abiotic stresses [12,13,14,15]. In recent years, several new miRNAs have been found in oilseed rape [16,17,18]. The process of seed development is a period that starts at embryo development and ends when dry seeds are mature; it is a key stage that affects the seed size, oil production, protein content, and antinutritional accumulation of rapeseed. Furthermore, gene expression changes during seed development [19,20,21]. Recently, miRNAs and their target transcripts involved in fatty acid and lipid metabolism have been studied in different development of B.napus seed, which is considered the third largest oil crop worldwide [22,23]. Some miRNAs involved in acetyl-CoA generate and carbon chain desaturase were also observed; nevertheless, the total number of known miRNAs and their functions in B. napus are still unknown [23].
The development of molecular biology technology has greatly promoted the research on LA breeding in rapeseed. ( ) In terms of mining the function of genes, a study showed signi cant correlation between FAD3 and LA content [24], while another study revealed that the overexpression of SsDGAT1 signi cantly affected LA content (about 16%) [25]. McD6DES generates a double bond at the carboxyl end of LA, which reduces the LA content [26]; BJULFY increases LA content (approximately 5%) [27]. The single nucleotide mutation of FAD3 exon from G to A was screened from the rapeseed "Alboglabra" treated with an Ethyl Methyl Sulfone (EMS) solution, and the mutant materials with high LA content and low alanine content (about 2.0%) were obtained [28]. ( ) In terms of screening the genes, a genome wide association study (GWAS) discovered 53 and 24 SNP related to LA in 2013Cq and 2014Cq rapeseed materials, respectively [29]; when combined with RT-PCR, 95 candidate genes were found to be highly related to LA and other fatty acid metabolism [30]. ( ) In terms of locating the genes, FAD2 gene on chromosome A05 was found to be associated with LA content, and a minor gene regulating LA content was found on chromosome A09 [31]; twenty QTLs related to LA were detected using two spring rapeseed varieties, "Polo" and "Topas", and subsequently distributed in seven linkage groups: A01, A02, A03, A05, C01, C03, and C09 [32]. miRNA technology has been adopted to nd several miRNAs in enzymes used for carbon chain desaturation, while being used in studies on fatty acid and lipid metabolism in rapeseed [22]. Employing this technology can be conducive to the breeding of rapeseed [23]. Therefore, we sequenced rapeseed material using miRNA technology. Then, we excavated four copies of OPR genes. The four copies of gene overexpression and RNAi vectors were transferred into A. thaliana. This is the rst study to report that all copies of OPR gene could directly regulate the synthesis of linoleic acid. These ndings will enhance the understanding of LA metabolism in rapeseed.

Overview of sRNA sequencing results
We have already upload data and ensure the deposited data is made public (NCBI) : https://www.ncbi.nlm.nih.gov/sra/PRJNA760803, accession number PRJNA760803. A total of 22,744,964 (low oleic acid rapeseed materials, A) and 26,060,122 (HOAR materials, B) raw reads were generated from the sequencing machine. After removing the adaptor sequences, ltering out low quality tags, and cleaning up sequences derived from adaptor ligation, 20,912,776 (A) and 23,710,938 (B) clean reads were obtained. Consequently, the bioinformatic analysis of these clean reads were carried out ( Table 1). The size distribution patterns of the original and unique reads were displayed in Figure 1A. Small RNAs (24 nt) were the most abundant in all the samples. In addition, the clean reads exhibited 87.45% (A) and 88.26% (B) homology with the reference genome sequences. The sRNA sequencing results were of high quality and reliable and can be used for further functional analysis.
Differentially expressed miRNAs identi cation and their function analysis The clustering analysis method was used to investigate the similarity between samples by calculating the differential miRNA distance between low oleic acid rapeseed (A) and HOAR (B) ( Figure 1B). As shown, three samples of high or low oleic acid contents were found in a cluster, suggesting that these miRNAs might have similar biological functions.
The target genes were then subjected to GO functional and KEGG Pathway analyses. In many cases, multiple terms were assigned to the same miRNA. Thus, 133 putative target genes were associated with 21 differentially expressed miRNAs and distributed into the following subcategories: 62 "Biological process", 32 "Cellular component", and 39 "Molecular function" ( Figure 1D). Under "Biological process", most of the target genes were related to "transcription", and "regulation of transcription". Within the "Cellular component" category, "nucleus" and "cytosol" were observed as much as "cytoplasm". Among genes in the "Molecular function" category, most potential functions were related to "transcription factor activity", "DNA binding", and "Catalytic activity". The distribution of target genes indicated that rapeseed underwent active metabolization.
Expression pattern of bna-miR156b>c>g gene was detected by RT-PCR To con rm the results of the miRNA sequence analysis, 21 annotated differentially expressed miRNAs were compared to the B. napus genome using BLAST [33] ( Figure 2B).
Moreover, the expressions of fatty acid metabolism related to differential miRNAs, such as bna-miR396a, bna-miR156b>c>g, and their target genes, were studied in different developmental stages ( Figure 2C). The bna-miR396a has opposite expression pattern with its target gene, at rst, the bna-miR396 had upregulated expression, until the bud stage reached the peak, and the expression decreased with the growth stage; bna-miR156b>c>g had an opposite expression pattern with its target gene, it had down-regulated expression in the whole growth stages, and the expression decreased with the growth stages. Differentially expressed miRNAs and their target genes were related to fatty acid metabolism in bna-mi156b>c>g at different developmental stages ( Figure 1C). In contrast to the expression pattern of bna-miR156b>c>g and its target gene, the expression level was down-regulated throughout the whole growth stage of the plant, and the expression level gradually decreased with the growth process of rapeseed.

Cloning of OPR genes in rapeseed and bioinformatic analysis
Target gene: bna-miR156b>c>g was cloned by miRNA sequencing, and four copies were detected: GSBRNA2T00012422001, GSBRNA2T00135385001, GSBRNA2T00082938001, and GSBRNA2T00094910001, which were named OPR1, OPR2, OPR3, and OPR4, respectively. Both OPR1 and OPR3 were 1119 bp, OPR2 and OPR4 were 1125 bp and 1122 bp, respectively ( Figure 3A). DNAMAN 7.0 software was used to compare the cloned target sequence with the rapeseed sequence published on the Brassica napus Genome Browser website. Different base position ( Figure 3B), homology were more than 99% with the published sequences ( Figure 3C). OPR1, OPR3, OPR4 had no base difference with the published sequence, there were 10 base differences between OPR2 and published sequence and the homology was 99.11%. Preliminary identi cation of OPR2 and OPR3 were located in A genome and OPR4 and OPR1 in C genome was conducted.
The number of four copies of OPR gene amino acids ranged from 372 bp to 374 bp, with a molecular weight of about 41 ku; The encoded amino acids were acidic (< 7), unstable (< 40), exhibited a fat coe cient of about 75, and belonged to fat-soluble proteins. Predicted subcellular localization of proteins were encoded by different copies of OPR genes and we found that these four proteins were located in the cytoplasm. We found that the four copies were all extracellular proteins without a transmembrane structure. Predicted protein secondary structures were summarized in Table 4.
The predicted tertiary structure model of the protein shows that the tertiary structures of OPR1, OPR2, OPR3, and OPR4 were all adapted to the 12-O-plant dienoate reductase model (integrated with the crystal structure of At1g76680 protein in A. thaliana), but their conformations were slightly different ( Figure 3D). It has been reported that the protein structure A. thaliana of At1g76680 is similar to that of yeast ScOYE1 [34].
Vector construction and transformation of A. thaliana.
The recombinant vector was transformed into A. thaliana and the obtained A. thaliana was detected. The T 1 transgenic A. thaliana seeds were screened using hygromycin ( Figure 5A) and the results of hygromycin primer identi cation ( Figure 5B) showed that each copy of A. thaliana OPR had been successfully transformed, and the target plasmid T-DNA had been inserted into the genome of A. thaliana.

Analysis of fatty acid composition and fatty acid content
The transformation methods in A. thaliana, reference Clough and Bent (1998) [35]. fatty acid composition was detected by gas chromatography [36]. we obtained fatty acid composition results of the T 1 and T 2 generations. Fatty acid composition of A. thaliana T-DNA insertion lines in table 5, and transformation materials fatty acid composition in the contrast T1/T2. The contents of oleic acid and stearic acid in OPR1i were signi cantly increased and the LA content decreased signi cantly; OPR1-OE will lead to a signi cant increase in palmitic acid and LA content; OPR2i signi cantly increased stearic acid content and decreased LA content; and OPR2-OE increased LA content.
The LA content in OPR3i decreased signi cantly; OPR3-OE signi cantly increased LA content; OPR4i signi cantly decreased LA content; and OPR4-OE increased LA content signi cantly.
Each copy of OPR-OE increased LA content, with an average increase of 12.56% in T 1 generation and 7.185% in T 2 generation. Subsequently, LA content in OPRi gene was signi cantly decreased, with an average decrease of 5.98% in T 1 generation and 0.86% in T 2 generation.
As shown in Table 5, oleic, linolenic, arachidonic, and erucic acids with the same variation trend as that of the fatty acid composition were selected for variance analysis. The results (Table 6) showed that the linolenic acid content in OPR1i signi cantly increased, while OPR2i signi cantly increased the linolenic acid content. Both OPR3-OE and OPR4-OE affected the content of arachidonic acid, which decreased signi cantly. In addition, OPR4i had no signi cant effect on the arachidonic acid content. Discussion miRNA expression and enrichment analysis miRNA is a type of sRNA that can regulate gene expression level and has an important regulatory effect on transcription. miRNAs have been found and reported in many plant species. In recent years, the number of known miRNAs has increased continually, such as A. thaliana [37] and Oryza sativa [38, 39]. B. napus L. has a relatively high genome size and complexity [33], and the number and function of miRNAs in B. napus L. have not been adequately studied; which suggests that many miRNAs have not yet been discovered, especially in seeds.
In this study, MiRNA libraries were constructed from self-pollinated seeds that were collected 20-35 d after pollination of rapeseed with high oleic acid content in the near-isogenic lines. Clean reads exhibited 87.45% (A) and 88.26% (B) homology with the referenced genome sequences ( Table 1). The small RNAs (24 nt) were most abundant in all the samples ( Figure 1A). In addition, the clustering analysis results showed that three samples of high or low oleic acid contents were found in a cluster, revealing that these miRNAs might have similar biological functions ( Figure 1B). Moreover, the above data indicated that the miRNA sequencing results in this study were of high quality and reliability, and can thus be used for further functional analysis. A total of 21 differentially expressed miRNAs were detected ( Table 2, Supplemental Table 2), including 9 (42.86%) up-regulated and 12 (57.14%) down-regulated genes ( Figure 1C). The present study provided a holistic view of HOAR immature seed miRNAs; the differential miRNAs and related putative target genes and their expressions obtained in this study can be utilized to study the molecular mechanism of fatty acids. To ensure the accuracy of the results, combination of RT-PCR technologies to further verify. Genes related to Cd stress, such as BNPCS1, BNGSTU12, and BNGSTU5, have previously been discovered using GO and KEGG pathways [40]. A total of 13 differentially expressed miRNAs were con rmed by RT-PCR, and a hypothetical model of cadmium response mechanism in Brassica napus was proposed on this basis [41].
In our study, Bna-miR156b>c>g may be involved in fatty acid metabolism. A total of 20 pathways were enriched by KEGG pathway, involving 36 target genes, of which 15 may be involved in the regulation of fatty acid metabolism; the reliability of the results was veri ed using the RT-PCR analysis. This result provided a basis for subsequent functional veri cation.
We found that OPR gene has four copies in rapeseed, with OPR1 and OPR4 located in the C genome and OPR2 and OPR3 located in the A genome. The OPR gene has been identi ed in several species and there are often multiple copies. Three OPRs were found in A. thaliana and tomato [42], 6 in peas [43], and 8 in corn [44]. Meanwhile, rice comprised 13 OPRs [45] and wheat had 48 OPRs [46]. Multi-copy genes are ubiquitous in plants and play an important role in maintaining plant genetic stability; however, they have hindered molecular breeding research. The loss of a few copies of gene function often does not cause phenotypic changes, and the probability of simultaneous mutation of multiple copy sites is too low to create a gene family or multiple copies of genes change simultaneously [47]. Rapeseed is an allotetraploid with many multi-copy genes. Conventional molecular breeding research methods are di cult to obtain phenotypic multi-copy gene mutants. Handa found that the main DNA sequence of the protein coding region was highly conserved between rapeseed and A. thaliana [48]. Transformation with A. thaliana as a receptor is helpful to study the function of multi-copy genes in rapeseed. In this study, we found four copies of OPR gene in rapeseed. Based on these multi-copy genes, we preliminarily transformed a single copy in A. thaliana. In the future, we will consider transferring multiple copies simultaneously to study the functionality of OPR genes in depth.

Regulation function of OPR genes to fatty acid
In this study, we found that OPR genes may affect the metabolism of linoleic acid and each copy was transferred separately into A. thaliana. The LA content of OPR-OE transgenic plants was signi cantly increased (T 1 12.56%, T 2 7.185%), while the LA content of OPRi transgenic plants was signi cantly decreased (T 1 5.98%, T 2 0.86%). These results have rarely been reported before. However, it has been described that the ClOPR genes, particularly ClOPR2 and ClOPR4, signi cantly upregulated by exogenous jasmonic acid, salicylic acid, and ethylene treatments in watermelon [49]. Virus-induced gene silencing (VIGS) analysis suggested that knockdown of GhOPR9 could increase the susceptibility of cotton to V. dahliae infection [50]. OPR gene was cloned from Oryza sativa; the overexpression of OPR genes was found to enhance the stress resistance of tobacco to heavy metal Cd 2+ [51]. Previous studies concluded that OPR genes were widely involved in abiotic stress processes [52]. In addition, OPR genes involved in fatty acid β oxidation, cilinolenic acid reduction, and the octadecanoic acid metabolic pathway [53]. However, there were few reports on OPR genes regulating OA, LA, or saturated fatty acid synthesis. In our study, we found that OPR genes directly affect the synthesis of linoleic acid and indirectly affect the content of other fatty acids ( Figure 6), which is consistent with the theoretical pathway wherein OPR genes regulate JA synthesis using alpha-linolenic acid (18:3) [53,54]. Thus showing a greater correlation between OPR genes and linoleic acid content and not α-linolenic acid (ALA).
A. thaliana and rapeseed are both cruciferous plants and current studies have shown that their gene functions are basically the same, It is of practical signi cance to study in model plants [48]. We found the optimizing quality fatty acids in A. thaliana can be changed by regulating OPR genes. Therefore, OPR may be involved in regulating linoleic acid synthesis and improving fatty acid composition in rapeseed. This is the rst study which discovered that the OPR gene can regulate LA metabolism. Therefore, this study is a good reference for studies researching the molecular mechanism of linoleic acid synthesis and molecular breeding in rapeseed.
In this study, 20 pathways were enriched using the KEGG pathway through high-throughput sequencing, of which 15 may be involved in the regulation of fatty acid metabolism. The reliability of the results was veri ed by performing the RT-PCR analysis, which provided a basis for subsequent functional veri cation. We excavated a target gene OPR, bna-miR156b>c>g, from rapeseed that may be related to fatty acid synthesis and identi ed the function of OPR genes through transformation of A. thaliana. Importantly, OPR gene was transferred separately into A. thaliana. The LA content of OPR-OE transgenic plants signi cantly increased (T 1 12.56%, T 2 7.185%), and the LA content of OPRi transgenic plants signi cantly decreased (T 1 5.98%, T 2 0.86%). In addition, by performing a bioinformatics analysis, we found four copies of the OPR gene in the cytoplasm that were located on chromosomes A and C. In this study, by detecting the fatty acid content of different generations of transgenic A. thaliana, the four copies of OPR gene that can directly affect LA content and indirectly affect other high quality fatty acids were discovered for the rst time. These results can be used in breeding programs aimed at optimizing fatty acid pro les in rapeseed [55].

Plant materials and growth conditions
The near-isogenic rapeseed lines with high (81.4 %) and low (56.2 %) oleic acid contents were provided by Rapeseed Molecular Breeding of Hunan Agricultural University, the strain was originally cultivated by National Oil Improvement Center of Hunan Agricultural University. The materials exhibited stable traits and were planted in the experimental eld of Hunan Agricultural University, China (Changsha, China) with standard agronomic methods [56]. The seeds 20 d after pollination were quickly frozen in liquid nitrogen and stored at -80 °C for subsequent studies [57]. The sample treatment method adopted in this study is the same with our previous study [58].
The seeds of wild-type (WT) A. thaliana (ecotype: Columbia) were bought from Think Gene Biological Technology Co., LTD Shanghai, China. Plants were grown under greenhouse conditions: 24 °C with a photoperiod of 18.5 h-light/5.5 h-dark, with a light intensity of 6500 lx.
sRNA library construction and high throughput sequencing Total RNA was extracted from frozen seeds using the Trizol reagent (Sigma Aldrich, St. Louis, MO, USA), according to the manufacturer's instructions. The quality and quantity of the puri ed RNA were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Santa Clara, USA) and RNA 6000 nanokit (Agilent Technologies, Palo Alto, Santa Clara, USA). sRNAs with lengths of 18-30 nt were separated and puri ed using 15% denaturing polyacrylamide gel electrophoresis. Consequently, sRNAs fractions were ligated to the 5′ lectrophoresters using T4 RNA ligase (Epicentre, America). The adapter-ligated fragments were then reverse transcribed and ampli ed by performing PCR with a pair of adapter complementary primers. These PCR products were puri ed and sequenced using IlluminaHiseq XTEN (Illumina, USA). Construction of the sRNA libraries and deep sequencing were carried out by Oebiotech Genomics (Shanghai, China).
Bioinformatic analysis of the sRNAs sequencing data Clean reads were generated after eliminating the low-quality reads, poly As, reads smaller than 18 nt, and gener adaptor contaminants and subsequently inserting nulls. Using bowtie software [59], the clean reads were aligned against the NCBI Gen Bank [60], B. napus oilseed genome [33], and Rfam databases (version 10.0). Reads annotated into the noncoding RNA categories, including rRNA, tRNA, snRNA, and snoRNA were ltered. The remaining sRNA sequences were aligned against the mi RBase21.0 [61]. The nearly matched sequences (less than two mismatches) were considered to be the known miRNAs. The unannotated sRNAs were further analyzed to predict the novel miRNAs using the Mirdeep2 software. The secondary structures of premiRNAs were also predicted using the RNAfold software.
To reveal the continuous changes in expression of miRNAs during the biosynthesis process, the variation in expression was analyzed in immature seed libraries of high oleic acid rapeseeds (HOAR). The frequency of miRNAs was normalized as transcripts per million (TPM) for further analysis. In addition, miRNAs were assessed using the negative binomial distribution test, with P < 0.05 and absolute value of log2 (treatment/control) > 1.5 being considered as differentially expressed. Moreover, the similarity between samples was investigated by the clustering method. The Blast 2 GO software with default parameters was applied to determine the functional annotation and categorization of the target genes  Table 2.

Cloning of OPR genes in rapeseed and bioinformatic analysis
Four copies of OPR genes were found using miRNA sequencing homologous cloning. Total RNA was extracted from the rapeseed leaf samples using the CTAB method adopted by Niu et al. (2018) [66], with minor modi cations. cDNA was produced by implementing reverse transcription, which was followed by PCR ampli cation (Table 7). According to each copy sequence of OPR, the physical and chemical properties of corresponding proteins were analyzed by using online websites such as the ExPASY-Protparam Tool and modifying the methods described by Li

Vector construction and transformation of A. thaliana
Speci c RNAi primer (Table 8) cDNA was used as a template. The base before the 5'-end enzyme digestion site was used for the construction of recombinant plasmids (the same below), according to the method adopted by Li et al. (2021) with minor modi cations [69]. The cloned OPR sequence was digested with BamH I and Sal I (Table 7) and recombinant with pCAMBIA1300-35s vector (Kangyan Corporation, China) to construct pCAMBIA1300-35s-OPR vector.
Similarly, the pCAMBIA1300-RNAi-OPR vector (Kangyan Corporation, China) was reconstructed using a double digestion process in vitro (Table 8)     Note: the underline indicates the restriction site. the same as Table 8.