A method for identifying gene duplication and functional differentiation in Rosaceae genera.

Abstract

This invention belongs to the fields of bioinformatics and molecular biology, specifically relating to a method for identifying gene duplication and functional differentiation in Rosaceae genera. By analyzing MAPK family proteins in rose and Arabidopsis thaliana, the invention identifies HT1, ANP3, Raf42, and Raf24 proteins that are duplicated and functionally differentiated in rose. Further application of bioinformatics and molecular biology techniques to Rosaceae plants reveals that only ANP3 exhibits duplication and functional differentiation in all Rosaceae plants. This invention provides a molecular-level reference for exploring the function of the MAPK family and the functional differentiation among ANP3-like genes in Rosaceae, laying the foundation for future research and identification of other duplicated and functionally differentiated genes in Rosaceae.

Description

Technical Field

[0001] This invention belongs to the fields of bioinformatics and molecular biology, specifically relating to a method for identifying gene replication and functional differentiation in Rosaceae genera. Background Technology

[0002] Common plants in the Rosaceae family include wild roses, rose bushes, strawberries, pears, apples, and raspberries. These plants not only provide a food source for humans, but some also have medicinal uses. Therefore, plants in the Rosaceae family are widely cultivated worldwide due to their significant economic value.

[0003] Orthologous genes within the same family and genus often undergo duplication through random replication, tandem replication, and whole-genome duplication. The functional differentiation resulting from this duplication is a driving force behind functional diversity among different plants within the same family and has significant evolutionary importance. With the advancement of the genome era, an increasing number of Rosaceae plants have undergone whole-genome sequencing. This has greatly facilitated the study of key functional genes in Rosaceae plants. However, a method for identifying gene duplication and functional differentiation in Rosaceae is currently lacking, hindering future molecular-based optimization of Rosaceae plant quality at the genetic level. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for identifying gene duplication and functional differentiation in Rosaceae genera. By analyzing MAPK family proteins in roses and Arabidopsis thaliana, the method identifies HT1, ANP3, Raf42, and Raf24 proteins that are duplicated and functionally differentiated in roses. Further, using bioinformatics and molecular biology techniques, this method is extended to Rosaceae plants, revealing that only ANP3 exhibits duplication and functional differentiation in all Rosaceae plants. This provides a reference for exploring the function of the MAPK family at the molecular level and the functional differentiation among ANP3-like genes in Rosaceae, laying the foundation for future research and identification of other duplicated and functionally differentiated genes in Rosaceae.

[0005] This invention is achieved by providing a method for identifying gene replication and functional differentiation in Rosaceae genera, comprising the following steps:

[0006] 1) Select a representative plant variety a from the Rosaceae family. Download the whole genome sequence of plant variety a and the conserved domain model of the MAPK protein family from the genome database of plant variety a. Use the conserved domain model of the MAPK protein family to screen the protein database of plant variety a. Using the Arabidopsis MAPK protein family as a reference, search for the proteins of the screened plant variety a one by one under certain criteria. Keep the proteins that meet the criteria and screen the proteins that meet the criteria one by one to find proteins that contain the conserved domain of the MAPK protein family.

[0007] 2) Obtain the chromosomal locations of MAPK genes from the genome database of plant variety a, visualize them, and estimate tandem duplication events according to certain standards;

[0008] 3) Obtain a certain number of AtMAPK, AtMAPKK, and AtMAPKKK genes, and construct an ML phylogenetic tree together with the MAPK protein of plant variety a. At the same time, download the RPKM values ​​of multiple tissue parts from the expression database of plant variety a, and visualize the nwk file generated by the ML phylogenetic tree and the RPKM files of multiple tissue parts.

[0009] 4) Analyze the orthologous proteins in Arabidopsis thaliana and plant variety a. Based on the ML phylogenetic tree, identify the proteins that replicate in plant variety a relative to Arabidopsis thaliana proteins. Perform sequence alignment on the screened proteins and record the amino acid base variations in conserved regions. At the same time, screen whether the genes that replicate in plant variety a show differences in expression in different tissues and organs. Genes that show differential expression and double in number are retained for the next step of analysis.

[0010] 5) The proteins that have replicated and differentiated in plant variety a, as selected in step 4), are further searched for in other varieties of Rosoidae and Maloideae according to certain criteria. The selected homologous proteins are then used to construct an ML phylogenetic tree together with the MAPK protein of plant variety a. The results are then visualized. The phylogenetic trees of other varieties of Rosoidae and Maloideae are modified according to the phylogenetic trees in NCBI and then visualized.

[0011] 6) RNA was extracted from multiple sites of plant variety a and reverse transcribed. The expression changes of replication genes in the MAPK family of plant variety a were detected by real-time quantitative PCR. Transcriptome data of one variety from the Rosinoideae and Maloideae subfamilies were downloaded and the expression changes of replication genes were analyzed. The 1.5kb promoter sequences of replication genes of the MAPK family were extracted from one variety from the Rosinoideae and Maloideae subfamilies. The cis-acting elements on the promoters were analyzed and the differences between the genes were analyzed.

[0012] Preferably, in step 1), the protein database of plant variety a is filtered using the hmmsearch program with a hidden Markov model of the conserved domains of the MAPK protein family and a query file with an E value ≤ 10⁻¹⁰.

[0013] Using the Arabidopsis MAPK protein family in the TAIR database as a reference, and with the criteria of E value ≤ 10⁻⁵ and homology > 50%, the proteins of the screened plant variety a were searched one by one using the BLASTp program.

[0014] The searchable proteins were then screened one by one using Pfam, Smart, and NCBI CD Search programs to identify proteins containing conserved domains of the MAPK protein family.

[0015] Further preferred, in step 2), the chromosomal location of the obtained MAPKs genes includes the start site and the end site; tandem repeat events are estimated according to the following criteria: first, the distance between the positions of the tandem repeat genes on the chromosome does not exceed 100Kb and the sequence similarity exceeds 80%; second, there is no insertion of other genes between the two repeat genes.

[0016] Further optimization, in step 3), 20 AtMAPK genes, 10 AtMAPKK genes and 80 AtMAPKKK genes were obtained and used with the MAPK protein of plant variety a to construct an ML phylogenetic tree using the IQ-Tree V1.6 JTT+F+I+G4 model, with 1000 bootstraps set; the number of tissue parts was 11, namely closed flowers, fruits, axillary buds, stamens, leaves infected with gray mold, young leaves and stems, leaves under water stress, floral meristems and early floral organs, and dormant axillary buds.

[0017] Further optimization revealed that the proteins screened in step 4) that replicate and functionally differentiate in plant variety a are HT1, Raf24, Raf42, and ANP3.

[0018] Further optimization, in step 5), the BLASTp program is used to search for each of the raspberry and strawberry in the Rosine subfamily and the peach, pear and apple in the Maloideae subfamily, with parameters set to E value ≤ 10-5 and homology > 60% standard; the screened homologous proteins are used together with the MAPK protein of plant variety a to construct an ML phylogenetic tree using the IQ-Tree V1.6 JTT+F+I+G4 model, and 1000 bootstraps are set.

[0019] Further optimization involves selecting the stem tips, young stems, leaves, closed flowers, and roots of plant variety a to extract RNA for reverse transcription.

[0020] Further optimization revealed that plant variety a was a rose.

[0021] Compared with the prior art, the advantages of the present invention are as follows:

[0022] 1. This invention is the first to conduct MAPK family gene mining and analysis based on the whole genome sequence of rose, which is relatively systematic and comprehensive;

[0023] 2. This invention can quickly identify replicated and functionally differentiated genes in the Rosaceae family by using the MAPK family genes that replicate and functionally differentiate in roses.

[0024] 3. This invention discovered that the ANP3 gene underwent replication events throughout the Rosaceae family, and that different ANP3 genes exhibited functional differences. The rose ANP3-1 gene was associated with abiotic stress, the strawberry ANP3-1 gene was associated with both stress and hormones, but the ANP3-2 gene was not. Attached Figure Description

[0025] Figure 1 The chromosomal locations and statistical results of the MAPK, MAPKK, and MAPKKK family genes of this invention are shown below.

[0026] Figure 2 Phylogenetic and spatiotemporal expression analysis of MAPKs and RcWD40s in rose and Arabidopsis thaliana;

[0027] Figure 3 Phylogenetic, spatiotemporal expression and sequence alignment analysis of Raf24, Raf42, ANP3 and HT1 in rose and Arabidopsis thaliana;

[0028] Figure 4 Phylogenetic analysis of Raf24, Raf42, ANP3 and HT1 in Arabidopsis and Rosaceae;

[0029] Figure 5 Expression and promoter analysis of Raf24, Raf42, ANP3 and HT1 in rose, strawberry and apple. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0031] 1. Discovery and identification of MAPK, MAPKK, and MAPKKK.

[0032] Using HMMER software, 96 MAPK proteins were identified in the rose genome based on PF00069. These proteins were then classified into three types—RcMAPKKKs(70), RcMAPKKs(10), and RcMAPKs(16)—based on sequence alignment with Arabidopsis thaliana. They were then named RcMAPKKK1–RcMAPKKK70, RcMAPKK1–RcMAPKK10, and RcMAPK1–RcMAPK16 (…) based on their positions on the rose chromosome. Figure 1 A).

[0033] 2. Chromosomal localization and tandem duplication analysis of MAPK, MAPKK, and MAPKKK.

[0034] To further investigate the genetic differences and gene replication patterns of RcMAPKs, the physical location of RcMAPKs on rose chromosomes was analyzed. Figure 1 B). RcMAPKs are widely but unevenly distributed across the seven rose chromosomes. Chr2 contains 25 RcMAPK genes (the most abundant), approximately 0.28 per Mb of genome sequence. Chr1, on the other hand, has only 5 genes, the lowest abundance, approximately 0.07 per Mb of genome sequence. Further observation revealed uneven distribution of RcMAPK genes across different chromosomes. RcMAPK genes are more likely to appear on the upper arms (Chr3 and Chr7), lower arms (Chr1, Chr4, and Chr6), or both arms (Chr2 and Chr5). To further refine the distribution of the three types of RcMAPK genes, RcMAPKKK and RcMAPK genes were observed to be distributed on all seven chromosomes, but the RcMAPKK gene only appeared on Chr2, Chr5, and Chr7. Meanwhile, 21 members were identified on Chr2, Chr3, Chr4, Chr5, and Chr7, exhibiting characteristics of tandem replication events. The results indicate that, in addition to random replication, tandem replication plays a role in the amplification of the rose RcMAPKs gene.

[0035] 3. Phylogenetic tree and expression analysis of MAPK, MAPKK, and MAPKKK.

[0036] To further understand gene duplication events in the RcMAPKs family, a phylogenetic tree of Rose and Arabidopsis MAPKs was constructed. Based on AtMAPKs, all Rose MAPKs proteins were assigned to specific clades. All these proteins were divided into three major clades (MAPKKK, MAPKK, and MAPK), and based on previous phylogenetic analysis, the MAPKKK family was further refined into three subgroups (MEKK, Raf, and ZIK).

[0037] The expression of all MAPK genes in roses is summarized as follows: In different tissues, most genes in the MEKK clades were expressed at lower levels than in other clades or subpopulations. Ten RcMAPKKK genes showed the highest expression in active axillary buds, leaves under water stress, and early flower buds, indicating that these genes function in these specific tissue sites.

[0038] 4. Analysis of replication and functional differentiation of HT1, Raf24, Raf42 and ANP3 in roses.

[0039] Based on the phylogenetic tree results ( Figure 2 and Figure 3 The study found that only one copy of HT1, Raf42, ANP3, and raf24 was found in Arabidopsis thaliana, while all of them were replicated in roses. Furthermore, the expression of these two genes differed in different parts of the plant, indicating that these genes underwent functional differentiation. Figure 4 A). However, although the other two copies of the AT1G04700 and AT3G58640 genes were duplicated in roses, there was no difference in the expression of the duplicated genes. This suggests that these genes may not have functional diversity. Figure 4 B). Then, rename RcMAPKKK40 to RcHT1-1, RcMAPKKK64 to RcHT1-2, RcMAPKKK6 to RcRaf42-1, RcMAPKKK5 to RcRaf42-2, RcMAPKKK32 to RcANP3-1, RcMAPKKK23 to RcANP3-2, RcMAPKKK43 to RcRaf24-1, and RcMAPKKK44 to RcRaf24-2.

[0040] 5. Analysis of ANP3 replication and functional differentiation in Rosaceae

[0041] To further investigate the potential for functional differentiation of RcHT1s, RcRaf42s, RcANP3s, and RcRaf24s after duplication in the rose genome, we compared their protein sequences with corresponding homologous proteins in Arabidopsis thaliana. Figure 3 C). Detailed comparison results showed that the kinase domains of RcHT1-1, RcRaf24-1, RcANP3-1, and RcRaf24-1 differed from those of RcHT1-2, RcRaf24-2, RcANP3-3, and RcRaf24-4 at positions 84, 23, 26, and 24, respectively. These positions are potential sites of protein-protein interaction, which may lead to differences in the function of these replicated genes.

[0042] The study also investigated whether HT1, Raf42s, ANP3, and Raf24 also replicated and functionally differentiated in other Rosaceae plants. Analysis revealed that species in the Rosinoideae subfamily (raspberry, rose, strawberry) and peach have 2 or 3 copies of the HT1 gene, while other species (pear and apple) have only 1 copy. Figure 4 A and 5E). Since there have been no recent WGD events in the Rosaceae family, this duplication of HT1-type genes likely occurred before the division of Rosaceae plants. Duplication of ANO3-type genes has been confirmed in all Rosaceae species. Figure 4 C and 4E). This result demonstrates that the duplication of ANP3 class genes may occur earlier than in all Rosaceae plants. Furthermore, the number of ANP3 class genes is greater in the Maloideae subfamily than in the Rosae subfamily (C and 4E). Figure 4 C and 4E). This indicates that ANP3 replicated before the differentiation of Rosaceae, but after the separation of Rosaceae and Maloideae, ANP3 replicated further in Maloideae. The number of Raf42s and Raf24s varied irregularly (from 1 to 3). Figure 4 The results (B, 4D, and 4E) indicate that these genes may originate from random or tandem replication. Based on these results, the next focus will be on whether the expression of ANP3-class genes, which replicate in all Rosaceae families, is diversified.

[0043] First, the expression patterns of ANP3 in these two rose varieties were detected using real-time quantitative PCR. The expression levels of RcANP3-1 in shoot apex meristems, young shoots, young leaves, closed flowers, and roots were significantly higher than those of RcANP3-2. Figure 5 The expression level of RcANP3-1 in RO was approximately 6 times that of RcANP3-1. ANP3s expression in strawberry (a representative species of the Rosoidae subfamily) was also analyzed. Figure 5 F) and apple (a representative species of the subfamily Maloideae) Figure 5 Expression of ANP3-1 in strawberry and apple tissues was higher than that of other genes, indicating that ANP3-1 is a major gene in these species. The expression results also demonstrate that ANP3 expression varies within the Rosaceae family. To further elucidate the reasons for this ANP3 expression differentiation, potential cis-elements in the 1.5 kb upstream of the ANP3 transcriptional starting site (TSS) were analyzed. Figure 5RcANP3-1 possesses more cis-elements associated with abiotic and biotic stresses, such as 12 MYB binding sites, while RcANP3-2 is induced by hormones. Both rose ANP3 types contain cis-elements related to growth and development. FvANP3-1 contains more stress-related cis-elements (7 MYB binding sites) and hormones, while FvANP3-2 does not. The four apple ANP3 types each possess different transcription factor binding sites. These results demonstrate the diverse regulatory potential of post-replication ANP3 genes.

[0044] Previous studies have shown that ANP3 controls the development and immunity of flowers and roots in Arabidopsis and tobacco. Plant immunity is frequently activated by various biotic stresses (such as pathogens), and this is one of the strategies for self-protection. All ANP3-class genes in the Rosaceae family possess conserved functions, but the upstream regulatory factors differ. Figure 5 K). ANP3-1 in roses and strawberries and ANP3-3 in apples seem to focus more on immunity (K). Figure 5 K).

[0045] Simultaneously, the expression patterns and potential cis elements of HT1s, Raf24s, and Raf42s generated by random replication, tandem replication, or WGD in Rosaceae were compared. Figure 5 AB Figure 5 DE、 Figure 5 G-5I and Figure 5 Interestingly, most of these genes also differ in expression patterns and transcription factor binding sites.

[0046] In summary, this application presents a systematic whole-genome analysis of the rose MAPKs family, including the identification and analysis of MAPKs family genes, chromosomal localization patterns, and tandem repeat analysis. An ANP3-type gene that replicates and functionally differentiates within the Rosaceae family was identified from the MAPKs family. These analyses will contribute to elucidating the functions and molecular mechanisms of the MAPK family and ANP3-type genes, providing new insights for research on various abiotic stresses affecting roses. They also provide a standard analytical procedure for analyzing other replicating and functionally differentiated genes in the Rosaceae family.

Claims

1. A method for identifying gene replication and functional differentiation in Rosaceae genera, characterized in that, Includes the following steps: 1) Select a representative plant variety a from the Rosaceae family. Download the whole genome sequence of plant variety a and the conserved domain model of the MAPK protein family from the genome database of plant variety a. Use the conserved domain model of the MAPK protein family to screen the protein database of plant variety a. Using the Arabidopsis MAPK protein family as a reference, search for the proteins of the screened plant variety a one by one under certain criteria. Keep the proteins that meet the criteria and screen the proteins that meet the criteria one by one to find proteins that contain the conserved domain of the MAPK protein family. 2) Obtain the chromosomal locations of MAPK genes from the genome database of plant variety a, visualize them, and estimate tandem repeat events according to certain standards; 3) Obtain a certain number of AtMAPK, AtMAPKK, and AtMAPKKK genes, and construct an ML phylogenetic tree together with the MAPK protein of plant variety a. At the same time, download the RPKM values ​​of multiple tissue parts from the expression database of plant variety a, and visualize the nwk file generated by the ML phylogenetic tree and the RPKM files of multiple tissue parts. 4) Analyze the orthologous proteins in Arabidopsis thaliana and plant variety a. Based on the ML phylogenetic tree, identify the proteins that replicate in plant variety a relative to Arabidopsis thaliana proteins. Perform sequence alignment on the screened proteins and record the amino acid variations in conserved regions. At the same time, screen whether the genes that replicate in plant variety a show differences in expression in different tissues and organs. Genes that show differential expression and double in number are retained for the next step of analysis. 5) The proteins that have replicated and differentiated in plant variety a, as selected in step 4), are further searched for in other varieties of Rosoidae and Maloideae according to certain criteria. The selected homologous proteins are then used to construct an ML phylogenetic tree together with the MAPK protein of plant variety a. The results are then visualized. The phylogenetic trees of other varieties of Rosoidae and Maloideae are modified according to the phylogenetic trees in NCBI and then visualized. 6) RNA was extracted from multiple sites of plant variety a and reverse transcribed. The expression changes of replication genes in the MAPK family of plant variety a were detected by real-time quantitative PCR. Transcriptome data of one variety from the Rosinoideae and Maloideae subfamilies were downloaded and the expression changes of replication genes were analyzed. The 1.5 kb promoter sequences of replication genes of the MAPK family were extracted from plant variety a, Rosinoideae and Maloideae subfamilies, and the cis-acting elements on the promoters were analyzed to analyze the differences between the genes. In step 1), the hmmsearch program is used to analyze the protein database of plant variety a using a hidden Markov model of conserved domains of the MAPK protein family, with an E value ≤ 10. -10 Filter the query files; Using the Arabidopsis MAPK protein family from the TAIR database as a reference, an E value ≤10 was used. -5 The standard of homology >50% was used to search for proteins of the selected plant variety a one by one using the BLASTp program; The proteins that could be searched were screened one by one using Pfam, Smart, and NCBI CD-Search programs to identify proteins containing conserved domains of the MAPK protein family. In step 2), the chromosomal locations of the obtained MAPK genes include start and end sites; tandem repeat events are estimated according to the following criteria: first, the distance between the tandem repeat genes on the chromosome does not exceed 100 kb, and the sequence similarity exceeds 80%; Secondly, there is no insertion of other genes between the two duplicate genes; In step 5), the BLASTp program is used to search for each of the following species in the Rosine subfamily (raspberry, strawberry) and the Maloideae subfamily (peach, pear, apple), with the parameter set to E value ≤ 10. -5 The homology criterion was >60%; the selected homologous proteins were used together with the MAPK protein of plant variety a to construct an ML phylogenetic tree using the IQ-Tree V1.6 and JTT+F+I+G4 models, and 1000 bootstraps were set. The plant variety a is a rose.

2. The method for identifying gene replication and functional differentiation in Rosaceae and genus according to claim 1, characterized in that, In step 3), 20 AtMAPK genes, 10 AtMAPKK genes, and 80 AtMAPKKK genes were obtained and used with the MAPK protein of plant variety a to construct an ML phylogenetic tree using the IQ-Tree V1.6 and JTT+F+I+G4 models, with 1000 bootstraps set. The number of tissue parts was 11, namely closed flowers, fruits, axillary buds, stamens, leaves infected with gray mold, young leaves, stems, leaves under water stress, floral meristems, early floral organs, and dormant axillary buds.

3. The method for identifying gene replication and functional differentiation in Rosaceae and genus according to claim 1, characterized in that, The proteins that replicate and functionally differentiate in plant variety a in step 4) are HT1, Raf24, Raf42 and ANP3.

4. The method for identifying gene replication and functional differentiation in Rosaceae and genus according to claim 1, characterized in that, In step 6), RNA is extracted from the shoot tips, young stems, leaves, closed flowers, and roots of plant variety a and then reverse transcribed.

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