Lithocarpus litseifolius sweet substance related gene and application thereof

Abstract

The invention belongs to the technical field of biology, and relates to a gene related to synthesis of a sweet substance of lithocarpus litseifolius, which is used for resource evaluation and genetic background analysis of the sweet substance of lithocarpus litseifolius germplasm, metabonomics research of sweet substance difference, transcriptomics and biological information research of a sweet substance synthesis related enzyme gene and the like. A plurality of genes related to synthesis of the Lithocarpus litseifolius sweet substances are screened, the most important genes are Cluster-69084.4 (PGT1), and the most important genes further comprise Cluster-58209.2 (4CL), Cluster-68818.0 (CHI), Cluster-56317.2 (CHS) and the like. The related gene provided by the invention can be used for evaluating or breeding germplasm resources of Lithocarpus litseifolius, the Lithocarpus litseifolius can be developed into a novel natural sweetening agent, the source of the sweetening agent is widened, and the gene has very important theoretical significance and practical value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to genes related to sweet substances in Lithocarpus litseifolius and their applications. Background Art

[0002] Lithocarpus litseifolius [Hance] Chun is an evergreen tree of the genus Lithocarpus in the Fagaceae family. It is a characteristic plant resource of new food raw materials that widely distributes in the Hunan Xuefeng Mountain range area and uses young leaves as tea drinks (commonly known as sweet tea and Yao tea). "Xupu Yao tea" has been listed as a national geographical indication protection product. Lithocarpus litseifolius is rich in flavonoids and has the effects of lowering blood sugar, blood pressure, and antioxidant.

[0003] Lithocarpus litseifolius has strong adaptability, a large biomass, and strong plant germination ability. New leaves germinate in spring, summer, and autumn, and mature leaves can be picked throughout the year. After being picked, new branches can grow from the leaf axils. Even if the whole plant is cut down, multiple new branches can germinate from the roots. It is a very characteristic resource. It is mainly distributed in mountain forests at an altitude of 200 - 2,000 m south of the Yangtze River, likes sunlight and is drought-tolerant. The resources in Hunan, Jiangxi and other provinces are particularly rich. According to incomplete statistics, the distribution area of Lithocarpus litseifolius in Hunan Province is about 133,000 hm 2 2, and the area of Lithocarpus litseifolius in Xupu County is about 0.53 hm 2 2. The annual output of fresh young leaves of wild Lithocarpus litseifolius in the whole country is about 8,000 tons, and the annual output of fresh young leaves in the Xuefeng Mountain area of Hunan is 1,600 tons.

[0004] Currently, the industrial development of Lithocarpus litseifolius is still mainly based on tea products, including granular tea, instant tea, tea bags, and tea substitute beverages, etc. The added value is low. Generally, only young stems and leaves can be used, so the output is also low. And there is also the problem that some consumers find it difficult to accept because of the overly sweet taste. Sweeteners are important raw materials in the food industry. Excessive intake of high-calorie carbohydrate substances can cause diseases such as obesity, diabetes, hyperlipidemia, and dental caries, which has become a major social problem. Under the global trend of sugar reduction, new natural sweeteners represented by mogroside have broad market prospects, and the development and utilization of natural sweetener plant resources have received more and more attention. Therefore, based on the characteristics of high sweetness, low calories, and blood sugar lowering of Lithocarpus litseifolius, developing it into a new natural sweetener to completely or partially replace sucrose and produce low-sugar or sugar-free foods especially meets the needs of people with diabetes, obesity, etc. It can not only broaden the source of sweeteners but also make full use of Lithocarpus litseifolius resources.

[0005] The development of new natural sweeteners from Lithocarpus litseifolius mainly has the following problems: First, the sweet substances have not been systematically analyzed; second, the quality differences of different germplasms are large, there is no stable excellent strain, and the genetic basis of excellent quality traits is lacking; third, the biosynthesis mechanism of sweet substances is not clear. SUMMARY OF THE INVENTION

[0006] In view of this, the object of the present invention is to provide genes related to the synthesis of sweet substances in Lithocarpus litseifolius, as well as a series of industrial applications of these genes in Lithocarpus litseifolius germplasm.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] 1. Provide genes related to the synthesis of sweet substances in Lithocarpus litseifolius, and the most important of the related genes include Cluster-69084.4 (PGT1).

[0009] Further, the related genes also include Cluster-58209.2 (4CL), Cluster-68818.0 (CHI), and Cluster-56317.2 (CHS).

[0010] Further, the related genes also include Cluster-24268.12 (CHI), Cluster-69541.2 (C4H), Cluster-48439.0 (4CL), Cluster-59728.2 (F3’H), Cluster-67928.0 (CHS).

[0011] Further, the related genes also include Cluster-45928.0, Cluster-55957.0, Cluster-76905.1, Cluster-24268.0, Cluster-69541.1, Cluster-66263.0, Cluster-66580.1, Cluster-68258.0, Cluster-44706.0, Cluster-59829.1, Cluster-69084.3, Cluster- {56317.1}, Cluster-38495.0, Cluster-55957.1, Cluster-68258.4, Cluster-55165.0, Cluster-69084.1 or Cluster-68258.1.

[0012] Among the related genes described in any of the above, the sweet substances are phloretin, phloridzin, trilobatin, 3-hydroxyphloridzin, or neohesperidin dihydrochalcone.

[0013] Further, the related genes that are significantly positively correlated with each sweet substance are as follows:

[0014] Genes that are extremely significantly positively correlated with phloridzin include: CHS (Cluster-56317.2, Cluster-67928.0, Cluster-24268.12), CHI (Cluster-68818.0), C4H (Cluster-45928.0, Cluster-55957.0), 4CL (Cluster-48439.0, Cluster-58209.2), PAL (Cluster-76905.1), PGT1 (Cluster-69084.4), F3’H (Cluster-59728.2).

[0015] Genes that are extremely significantly positively correlated with 3-hydroxyphloridzin include: CHI (Cluster-24268.0), C4H (Cluster-69541.1, Cluster-69541.2), 4CL (Cluster-66263.0, Cluster-66580.1, Cluster-68258.0), PGT1 (Cluster-44706.0, Cluster-59829.1, Cluster-69084.3).

[0016] Genes that are extremely significantly positively correlated with phloretin include: CHS (Cluster-56317.1, Cluster-56317.2, Cluster-67928.0), CHI (Cluster-24268.12, Cluster-38495.0), C4H (Cluster-45928.0, Cluster-55957.0, Cluster-55957.1), 4CL (Cluster-48439.0, Cluster-58209.2, Cluster-68258.4), PAL (76905.1), PGT1 (Cluster-69084.4), F3’H (Cluster-59728.2).

[0017] Genes that are extremely significantly positively correlated with trifolin include: CHS (Cluster-55165.0), PGT1 (69084.1).

[0018] Genes that are significantly positively correlated with neohesperidin dihydrochalcone include: CHI (Cluster-24268.0), C4H (Cluster-69541.1, Cluster-69541.2), 4CL (Cluster-66263.0, Cluster-66580.1, Cluster-68258.0, Cluster-68258.1), PAL (Cluster-76905.1), PGT1 (Cluster-69084.4), F3’H (Cluster-59728.2).

[0019] The accession numbers of the above genes in NCBI are PRJNA1252376.

[0020] 2. Based on the related genes of any one of the above, the present invention also provides an application of the related genes in screening Litsea cubeba germplasm.

[0021] The present invention also provides qRT-PCR primer information for 18 genes related to the synthesis of sweet substances, and the specific sequences are shown in SEQ ID No.1 - SEQ ID No.36.

[0022] 3. The third aspect of the present invention provides an application of the related genes in promoting the synthesis of sweetness in Litsea cubeba germplasm.

[0023] 4. The present invention also provides an application of the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium of the related genes in cultivating new Litsea cubeba germplasm.

[0024] 5. The present invention also provides a glycosyltransferase gene in Litsea cubeba, and the glycosyltransferase genes are PGT1 (Cluster-59829.0), PGT1 (Cluster-69084.4).

[0025] The beneficial effects of the present invention are as follows: Based on the previous research on the variation rules of the content of sweet substances in Lithocarpus litseifolius germplasm resources in different regions of the country, including different parts, tree ages, leaf ages, harvesting periods, and processing technologies, the present invention further selects high-sweet Lithocarpus litseifolius germplasm resources for genetic characteristic analysis of chloroplast and mitochondrial genomes, and conducts metabolome and transcriptome research on Lithocarpus litseifolius germplasm resources with different sweet substances, and excavates and conducts bioinformatics analysis on the enzyme genes related to the synthesis of sweet substances. Finally, a total of 43 genes related to the synthesis of sweet substances in Lithocarpus litseifolius are screened, among which the most important one is Cluster-69084.4 (PGT1), and the second includes Cluster-58209.2 (4CL), Cluster-68818.0 (CHI), and Cluster-56317.2 (CHS), etc. The screening of a large amount of data ensures the reliability of the genes provided related to the synthesis of sweet substances in Lithocarpus litseifolius. Further using the related genes, the germplasm resources of Lithocarpus litseifolius can be evaluated or selected, and Lithocarpus litseifolius can also be developed into a new type of natural sweetener, broadening the source of sweeteners, providing theoretical support for the development and industrialization of new sweeteners from Lithocarpus litseifolius, and having very important theoretical significance and practical value. Two glycosyltransferase genes related to the synthesis of sweet substances are excavated, namely PGT1 (Cluster-59829.0) and PGT1 (Cluster-69084.4), finding an entry point for future sweetener development. It also provides new insights into the synthesis of sweet substances in Lithocarpus litseifolius. It can break through the current situation that the industrial development of Lithocarpus litseifolius mainly focuses on traditional tea products with low added value. Through the related genes, Lithocarpus litseifolius is upgraded from traditional tea beverages to the field of new natural sweeteners, providing a new direction for the development and utilization of Lithocarpus litseifolius germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to make the objectives, technical solutions, and beneficial effects of the present invention clearer, the present invention provides the following drawings for description:

[0027] Figure 1 PCA analysis diagram of the gene expression values of the samples.

[0028] Figure 2 Analysis of differentially expressed genes related to the synthesis pathway of sweet substances in Lithocarpus litseifolius.

[0029] Figure 3 Correlation analysis of metabolites and synthase genes in the synthesis pathway of sweet substances in Lithocarpus litseifolius.

[0030] Figure 4 qRT-PCR analysis of the relative expression levels of enzyme genes related to the synthesis pathway of sweet substances. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] Next, in combination with the accompanying drawings in the embodiments of the present invention, the technical solutions of the preferred embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. For the experimental methods without specific conditions noted in the embodiments, they are usually carried out under conventional conditions or according to the conditions recommended by the manufacturers.

[0032] Example 1

[0033] Materials: The Litsea leaf-like Lithocarpus from the standardized planting base of Hunan Aokang Biotechnology Co., Ltd. and the surrounding wild areas was used as the test material, and it was identified as Lithocarpus litseifolius [Hance] Chun of the genus Lithocarpus in the Fagaceae family. The standardized planting base of Litsea leaf-like Lithocarpus is located in Heiniutian Village, Yanxi Township, Liangyaping Town, Xupu County, Hunan Province (110.58°E, 27.74°N), with an altitude of about 676 m, an average annual temperature of 16.9 °C, an average annual rainfall of 1539.1 mm, and an average annual frost-free period of 286 days.

[0034] In the early stage of the present invention, the variation rules of the contents of five sweet substances, namely phloridzin, phloretin, 3-hydroxyphloretin, neohesperidin dihydrochalcone, and phloretin, in Litsea leaf-like Lithocarpus with different parts (see Table 1 for results), tree ages, leaf ages, harvesting periods, and processing technologies were systematically studied. The results showed that phloridzin and phloretin are the two sweet substances with the highest proportions.

[0035] Table 1 Variation of the contents of different sweet substances in different parts of Litsea leaf-like Lithocarpus

[0036]

[0037] Note: Different lowercase letters in the same column indicate significant differences in different treatments of the same substance (P < 0.05), and the same applies hereinafter.

[0038] Example 2

[0039] Thirty-eight germplasm resources of Litsea leaf-like Lithocarpus in this study were collected from 12 provinces such as Yunnan, Sichuan, Hunan, and Guizhou, and transplanted in the germplasm resource nursery of Litsea leaf-like Lithocarpus in Hunan Yaotea Engineering Technology Research Center (110.58°E, 27.74°N). The specific germplasm resource information is shown in Table 2.

[0040] In April 2022, in the germplasm resource nursery of *Litsea coreana* var. *lanceolata*, three plants were randomly selected from each germplasm, and young leaves (one bud with three leaves) on the current year's new shoots were collected in the east, south, west, and north directions. Mature leaves were collected in the same way in September of the same year, blanched at 105°C for 5 minutes, dried at 65°C, and pulverized with a universal pulverizer for the determination of the contents of phlorizin, phloretin, 3-hydroxyphloretin, neohesperidin dihydrochalcone, and phloretin.

[0041] Table 2 Germplasm Resource Information

[0042]

[0043] Evaluation of *Litsea coreana* var. *lanceolata* Germplasm Resources Based on the Content of Sweet Substances

[0044] The results of the contents of sweet substances in the young leaves of different *Litsea coreana* var. *lanceolata* germplasm resources are shown in Table 3. The content of phlorizin in the young leaves was between 58.80 mg / g and 144.29 mg / g. The content of phlorizin in SP2 was the lowest (P<0.05), and the content of phlorizin in HX4 was the highest (P<0.05). The content of phloretin was between 1.75 mg / g and 16.87 mg / g, the content of 3-hydroxyphloretin was between 0.32 mg / g and 4.08 mg / g, the content of neohesperidin dihydrochalcone was between 0.18 mg / g and 2.51 mg / g, and the content of phloretin was between 0.24 mg / g and 4.84 mg / g. The contents of the five sweet substances differed greatly.

[0045] Table 3 Contents of Sweet Substances in Young Leaves of Different *Litsea coreana* var. *lanceolata* Germplasm

[0046]

[0047]

[0048] Example 3

[0049] Experimental materials: The source of *Litsea coreana* var. *lanceolata* was the same as before. Leaves of two *Litsea coreana* var. *lanceolata* germplasms with large differences in the content of sweet substances (HX4, from Xupu, Hunan; SP2, from Panzhihua, Sichuan) were selected. The young leaf samples of *Litsea coreana* var. *lanceolata* plants from the Xupu, Hunan provenance were named HXN, the open-faced leaf samples were named HXD, and the old leaf samples were named HXW. The young leaf samples of *Litsea coreana* var. *lanceolata* plants from the Panzhihua, Sichuan provenance were named SPN, the open-faced leaf samples were named SPD, and the old leaf samples were named SPW. There were 3 samples of each type, for a total of 18 samples. They were immediately frozen in liquid nitrogen on-site and then taken back to the laboratory and stored in a -80°C refrigerator for subsequent metabolomics determination and transcriptomics research.

[0050] Perform metabolomics research on the above samples to analyze the differential metabolites in the synthesis pathway of the sweet substances in Lithocarpus litseifolius. Results: (1) A total of 1,292 metabolites were identified. Among them, flavonoids accounted for the largest proportion, 24.23% (313 species), followed by phenolic acids at 16.87% (218 species), lipids at 11.15% (144 species), others at 8.98% (116 species), amino acids and their derivatives at 8.28% (107 species), organic acids at 7.04% (91 species), terpenoids at 6.97% (90 species), alkaloids with a relatively small proportion (5.96%, 77 species), lignans and coumarins (5.03%, 65 species), nucleotides and their derivatives at 4.95% (64 species), and tannins with the least proportion (0.54%, 7 species). The flavonoids identified in the 6 samples included: 97 species of Flavones flavonoid components, 124 species of Flavonols flavonols, 24 species of Chalcones chalcones, 30 species of Flavanones flavanones, 7 species of Other Flavonoids other flavonoids, and 12 species of Flavanonols dihydroflavonols. A total of 528 metabolites were annotated. (2) Trilobatin had a higher expression level in the young leaves of the Hunan Xupu germplasm (HX4), phloridzin, phloretin, and neohesperidin dihydrochalcone had higher expression levels in the old leaves of the Sichuan Panzhihua germplasm (SP2), and 3-hydroxyphloridzin had a higher expression level in the old leaves of the Hunan Xupu germplasm (HX4). (3) The results of screening differential metabolites showed that 457, 445, and 435 differential metabolites were detected in different germplasms at three periods, mainly including flavonoids, phenolic acids, etc., among which flavonoid differential metabolites accounted for the largest proportion. The changes in these differential metabolites were mainly involved in specific pathways such as the biosynthesis of flavonoids (ko00941), the biosynthesis of flavones and flavonols (ko00944), etc.

[0051] Example 4

[0052] Further perform transcriptomics research on two germplasms with relatively large differences in the content of sweet substances (HX4, Hunan Xupu; SP2, Sichuan Panzhihua), screen differentially expressed genes, and conduct a combined analysis with metabolomics to study the enzyme genes related to the synthesis of sweet substances in Lithocarpus litseifolius, providing support for clarifying the synthesis mechanism of sweet substances in Lithocarpus litseifolius. The samples are the same as in Example 3.

[0053] Main test reagents: EASYspin Plus Complex Plant RNA Kit (Aidlab Biotechnologies Co., Ltd.); Prime Script TM II 1st Strand c DNA Synthesis Kit; Premix Ex Taq TMII (Tli RNase H Plus) (Takara Biotechnology (Dalian) Co., Ltd.)

[0054] After extracting the sample RNA using the kit, the purity of the RNA was detected using a spectrophotometer, and the RNA concentration was measured and its integrity was detected using a fluorometer and a bioanalyzer. After further detecting, library construction, and library quality control of the samples, the samples were sequenced on the machine. Trinity was used to splice and remove redundancy from the clean reads, and the longest transcript in the cluster was used as Unigene for analysis. The BLAST software was used for Unigene sequence alignment to obtain the annotation information of Unigene. Then, FPKM analysis was performed.

[0055] The summary of the sequencing data is shown in Table 4. The number of Raw Reads for the 18 samples was between 39,840,530 and 52,125,442, and the number of Clean Reads was between 38,218,308 and 51,209,020. The Error Rate was 0.03% for all samples, the GC content was between 43.64% and 44.74%, and the values of Q20 and Q30 were greater than 97.00% and 92.00% respectively. The sequencing results met the requirements of the experiment and further analysis could be carried out.

[0056] Table 4 Sequencing Data

[0057]

[0058] After transcript splicing and assembly, a total of 276,574 Transcript sequences were obtained. The average length of the Transcript sequences was 998 bp, the lengths of N50 and N90 were 1,756 and 388 bp respectively, and the total number of bases was 276,014,540. 152,214 Unigene sequences were obtained. The average length of the Unigene sequences was 1,304 bp, the lengths of N50 and N90 were 1,999 and 563 bp respectively, and the total number of bases was 198,543,509. In the above analysis, through Trinity splicing and Corset clustering, the transcript and Unigene sequences in Lithocarpus litseifolius were successfully obtained, providing a basis for subsequent gene expression and functional annotation. The accession number of the transcript uploaded to NCBI is PRJNA1252376.

[0059] Gene function annotation: The annotation situations in each database are shown in Table 5: A total of 152,214 Unigenes were annotated. The number of annotated Unigenes was between 55,363 and 105,625. The annotated Unigenes were 76,515, 68,016, 103,829, 55,363, 72,066, and 63,864 respectively, accounting for 50.27%, 44.68%, 68.21%, 36.37%, 47.35%, and 41.96% respectively.

[0060] Table 5 Annotation statistical table

[0061]

[0062] Gene expression quantification: The overall distribution of sample gene expression levels is shown in Table 6. The alignment rates of 18 samples were all above 80.00%, the highest being 85.52% and the lowest being 83.35%. This indicates that most clean reads can be successfully mapped to the reference sequence, suggesting that the sequencing data quality is high and suitable for subsequent analysis.

[0063] Table 6 Alignment statistical table

[0064]

[0065] After statistical analysis of the correlation between samples, the square of the Pearson correlation coefficient (R 2 ) was 1 for all, indicating very good repeatability between samples and good correlation between different germplasms at the same growth stage. This result is consistent with the PCA analysis. PCA analysis was performed on the gene expression values of 18 samples in this study, and the results are as Figure 1 shown. The differences between sample groups of different Litsea elongata germplasms at different times are obvious, while the differences within groups are small.

[0066] Screening of differentially expressed genes: The number of up-regulated genes and down-regulated genes in each group was as follows: For SPN_vs_HXN, the number of up-regulated genes was 12,079 and the number of down-regulated genes was 13,477. For SPD_vs_HXD, the number of up-regulated genes was 12,783 and the number of down-regulated genes was 11,334. For SPW_vs_HXW, the number of up-regulated genes was 13,571 and the number of down-regulated genes was 10,926.

[0067] KEGG enrichment analysis of differentially expressed genes: In the SPN vs HXN group, 21,135 genes were distributed into 145 enriched pathways. Among them, the P-values of 16 pathways were < 0.05, and those of 6 pathways were < 0.01, namely Biosynthesis of various plant secondary metabolites, Isoflavonoid biosynthesis, Monoterpenoid biosynthesis, Ubiquinone and other terpenoid - quinone biosynthesis, etc.

[0068] In the SPD vs HXD group, 19,612 genes were distributed into 143 enriched pathways. Among them, the P-values of 20 pathways were < 0.05, and those of 10 pathways were < 0.01, namely Biosynthesis of various plant secondary metabolites, Aminoacyl - tRNA biosynthesis, Ribosome biogenesis in eukaryotes, Stilbenoid, diarylheptanoid and gingerol biosynthesis, Other glycan degradation, etc.

[0069] In the SPW vs HXW group, 18,600 genes were distributed into 143 enriched pathways. Among them, the P-values of 22 pathways were < 0.05, and those of 11 pathways were < 0.01, namely Phenylpropanoid biosynthesis, Ribosome biogenesis in eukaryotes, Flavonoid biosynthesis, Isoflavonoid biosynthesis, etc.

[0070] GO analysis of differentially expressed genes: In the comparison of the number of annotated genes in cellular components and biological processes for SPD vs HXD, SPW vs HXW, and SPN vs HXN, in terms of cellular components, the number of annotated genes for cellular anatomical entity was the largest, with 12,840, 12,361, and 13,247 respectively. Among them, the number of upregulated genes was relatively large, reflecting the significant differences in these cellular components under different conditions. The number of annotated genes for protein-containing complex was the second in each comparison, ranging from 1,222 to 1,979. In terms of biological processes, the number of annotated genes for cellular process was the largest, with 9,926, 9,642, and 10,257 respectively. Metabolic process also ranked relatively high in each group, with the number of annotated genes ranging from 6,583 to 8,895.

[0071] The transcription factor annotation is shown in Table 7. The transcription factor families with relatively large numbers of genes are bHLH (194), C2H2 (163), C3H (149), bZIP (147), AP2 / ERF-ERF (135), SNF2 (134), WRKY (119), SET (114), and FAR1 (104).

[0072] The numbers of differentially expressed transcription factors between different groups were: SPN vs HXN (863), SPD vs HXD (911), and SPW vs HXW (848). The specific up- and down-regulated transcription factor situations are shown in Table 8.

[0073] Table 7 Prediction of transcription factor families in Lithocarpus litseifolius

[0074]

[0075]

[0076] Table 8 Numbers of differentially expressed transcription factors among different treatments

[0077]

[0078] Forty-three genes related to the synthesis of five sweet substances (phloretin, phloridzin, trilobatin, 3-hydroxyphloridzin, and neohesperidin) in Lithocarpus litseifolius were screened from the significantly differentially expressed genes, and a pathway map containing the heatmap of the expression levels of each gene in the biosynthesis of five sweet substances in Lithocarpus litseifolius was constructed, as Figure 2 shown.

[0079] PAL, C4H, 4CL, and CHS are involved in the early enzymatic reactions of the synthesis of sweet substances in Lithocarpus litseifolius. CHS (Cluster-56317.1, Cluster-56317.2, Cluster-67928.0), 4CL (Cluster-66263.1, Cluster-66580.0, Cluster-68258.1, Cluster-48439.0) were significantly up-regulated in SPW. PAL (Cluster-76905.1), C4H (Cluster-55957.0, Cluster-45928.0), CHS (Cluster-56317.1, Cluster-67928.0), 4CL (Cluster-74078.6, Cluster-68258.4, Cluster-54095.0) were significantly up-regulated in SPD. CHS (Cluster-55165.0), 4CL (Cluster-54568.0, Cluster-74078.6, Cluster-66283.1) were significantly up-regulated in SPN. PAL (Cluster-76905.1), C4H (Cluster-69541.1, Cluster-69541.2, Cluster-45928.0, Cluster-55957.1), CHS (Cluster-56317.2, Cluster-67928.0), 4CL (Cluster-66283.0, Cluster-66580.1, Cluster-58209.2, Cluster-68256.1) were significantly up-regulated in HXW. PAL (Cluster-65438.0), 4CL (Cluster-54095.0, Cluster-68258.0) were significantly up-regulated in HXD. CHS (Cluster-55165.0, Cluster-65027.0), 4CL (Cluster-73418.0, Cluster-68258.0) were significantly up-regulated in HXN.

[0080] PGT1, F3’H, and C12RT1 are involved in the terminal enzymatic reaction of the synthesis of sweet substances in Lithocarpus litseifolius. PGT1 (Cluster-69084.4) was significantly upregulated in SPW. PGT1 (Cluster-59829.0, Cluster-59829.3) was significantly upregulated in SPD. PGT1 (Cluster-69084.1, Cluster-59829.3), PGT1 (Cluster-41508.0, Cluster-69084.4, Cluster-59829.1, Cluster-44706.0, Cluster-69084.3) were significantly upregulated in HXW. PGT1 (Cluster-44706.0, Cluster-69084.3) was significantly upregulated in HXD. PGT1 (Cluster-59829.1, Cluster-69084.1) was significantly upregulated in HXN. F3’H (Cluster-59728.2) was significantly upregulated in SPW and HXW. C12RT1 (Cluster-54209.4) was significantly upregulated in HXN.

[0081] Correlation analysis of metabolites and synthase genes in the sweet substance synthesis pathway of Lithocarpus litseifolius is as follows Figure 3As shown, phloridzin was extremely significantly positively correlated with CHS (Cluster-56317.2, Cluster-67928.0, Cluster-24268.12), CHI (Cluster-68818.0), C4H (Cluster-45928.0, Cluster-55957.0), 4CL (Cluster-48439.0, Cluster-58209.2), PAL (Cluster-76905.1), PGT1 (Cluster-69084.4), and F3’H (Cluster-59728.2). 3-Hydroxyphloridzin was extremely significantly positively correlated with CHI (Cluster-24268.0), C4H (Cluster-69541.1, Cluster-69541.2), 4CL (Cluster-66263.0, Cluster-66580.1, Cluster-68258.0), and PGT1 (Cluster-44706.0, Cluster-59829.1, Cluster-69084.3). Phloretin was extremely significantly positively correlated with CHS (Cluster-56317.1, Cluster-56317.2, Cluster-67928.0), CHI (Cluster-24268.12, Cluster-38495.0), C4H (Cluster-45928.0, Cluster-55957.0, Cluster-55957.1), 4CL (Cluster-48439.0, Cluster-58209.2, Cluster-68258.4), PAL (76905.1), PGT1 (Cluster-69084.4), and F3’H (Cluster-59728.2). Naringin was extremely significantly positively correlated with CHS (Cluster-55165.0) and PGT1 (69084.1). Neohesperidin dihydrochalcone was extremely significantly positively correlated with CHI (Cluster-24268.0), C4H (Cluster-69541.1, Cluster-69541.2), 4CL (Cluster-66263.0, Cluster-66580.1, Cluster-68258.0, Cluster-68258.1), PAL (Cluster-76905.1), PGT1 (Cluster-69084.4), and F3’H (Cluster-59728.2).

[0082] In the statistics of genes encoding enzymes related to the synthesis of sweet substances in *Lithocarpus litseifolius* Turquoise module, CHI (25.0%, 3) and C4H (25.0%, 3) accounted for the largest proportion, followed by CHS (16.7%, 2) and 4CL (16.7%, 2), PGT1 (8.3%, 1) and F3H’ (8.3%, 1). In the statistical analysis of transcription factors in *Lithocarpus litseifolius* Turquoise module, a total of 9 major categories were identified, namely bHLH (12.5%, 26), MYB (9.6%, 20), MYB-related (8.2%, 17), ERF (7.2%, 15), C2H2 (7.2%, 15), HD-ZIP (7.2%, 15), GRAS (5.8%, 12), TCP (4.8%, 10), others (37.5%, 78).

[0083] A significant correlation network analysis was performed on the *Lithocarpus litseifolius* Turquoise-transcription factor-key enzyme genes. Under the conditions that r is greater than 0.95 and p is less than 0.05, the core genes of the Turquoise module were screened. Nine core genes were screened, namely Cluster-69084.4 (PGT1), Cluster-56317.2 (CHS), Cluster-68818.0 (CHI), Cluster-24268.12 (CHI), Cluster-69541.2 (C4H), Cluster-48439.0 (4CL), Cluster-58209.2 (4CL), Cluster-59728.2 (F3’H), Cluster-67928.0 (CHS). Among them, the strongest correlations were Cluster-69084.4 (PGT1), followed by Cluster-58209.2 (4CL), Cluster-68818.0 (CHI) and Cluster-56317.2 (CHS).

[0084] Example 5 Bioinformatics Analysis of the Gene Encoding the Terminal Synthetase of Sweet Substances in *Lithocarpus litseifolius*

[0085] The Arabidopsis thaliana UGT family protein sequence data used was sourced from the website (http: / / www.p450.kvl.dk / UGT shtml), and 19 Arabidopsis thaliana UGTs sequences were obtained separately from the NCBI database (http: / / www.maize sequence.org / in dex.html). The apple PGT1 family protein sequence data was sourced from the website (https: / / www.rosaceae.org / speciesmalus / malus xdomestica / genome GDDH13v1.1), including MDP0000219282 (MdPGT1, Accession no. EU246349; UGT88F1, ACZ44840; MdP2'GT, KT444675), MDP0000163017 (UGT71K1, ACZ44835), MDP0000215525 (UGTA15, AAZ80472), and MDP0000617956 (MdPh-4'-OGT, AY786997). The PGT1, C12RT1, and F3’H family protein sequences of Lithocarpus litseifolius were sourced from the transcriptome sequencing data of Lithocarpus litseifolius in our research group. qPCR detection was performed on 18 genes related to sweet substance synthesis screened from the transcriptome. The specific amplification primers are shown in Table 9. The preparation of the real-time fluorescence quantitative reaction system (total volume 20 μL) is shown in Table 10.

[0086] Table 9 Information of primers for qRT-PCR verification

[0087]

[0088]

[0089] Table 10 Real-time fluorescence quantitative reaction system

[0090]

[0091] As shown in Table 11, a total of 8 members of the PGT gene family were identified in Lithocarpus litseifolius, and the gene ID numbers were Cluster-45425.0, Cluster-59829.0, Cluster-59829.1, Cluster-59829.2, Cluster-69084.2, Cluster-69084.1, Cluster-69084.3, and Cluster-69084.4. One member of the C12RT1 gene family was identified, with the gene ID number Cluster-54209.2. One member of the F3’H gene family was identified, with the gene ID number Cluster-59728.2.

[0092] Table 11 Physicochemical properties of the terminal synthase gene of the sweet substance in Lithocarpus litseifolius

[0093]

[0094]

[0095] Further analysis was carried out on the phylogenetic evolution of PGT1, C12RT1, and F3’H genes in Lithocarpus litseifolius, the conserved domains of PGT1, C12RT1, and F3’H proteins in Lithocarpus litseifolius, and the prediction of the secondary and tertiary structures of PGT1, C12RT1, and F3’H proteins in Lithocarpus litseifolius. And qRT-PCR verification of the genes related to the synthesis of sweet substances in Lithocarpus litseifolius: 18 DEGs were screened for qRT-PCR verification, with HXN, HXD, and HXW as controls respectively. The research results showed that there was a high consistency between the gene expression patterns detected by qRT-PCR and the sequencing results ( Figure 4 , qRT-PCR analysis of the relative expression levels of the genes related to the synthesis pathway of sweet substances), indicating that the experimental results of transcriptome sequencing were reliable. Combining the joint pathway analysis of transcriptome and metabolome, two glycosyltransferase genes, PGT1 (Cluster-59829.0) and PGT1 (Cluster-69084.4), were screened. The fluorescence protein signals encoded by these two genes were mainly distributed on the cell membrane, with a small amount of expression in the nucleus and cytoplasm.

[0096] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. Genes related to the synthesis of sweet substances in Lithocarpus litseifolius, characterized in that, The related genes include Cluster-69084.4 (PGT1).

2. The related gene according to claim 1, wherein The related genes also include Cluster-58209.2 (4CL), Cluster-68818.0 (CHI), and Cluster-56317.2 (CHS).

3. The related gene according to claim 1, wherein The related genes also include Cluster-24268.12 (CHI), Cluster-69541.2 (C4H), Cluster-48439.0 (4CL), Cluster-59728.2 (F3’H), Cluster-67928.0 (CHS).

4. The related gene according to claim 1, characterized in that, The related genes also include Cluster-45928.0, Cluster-55957.0, Cluster-76905.1, Cluster-24268.0, Cluster-69541.1, Cluster-66263.0, Cluster-66580.1, Cluster-68258.0, Cluster-44706.0, Cluster-59829.1, Cluster-69084.3, Cluster-56317.1, Cluster-38495.0, Cluster-55957.1, Cluster-68258.4, Cluster-55165.0, Cluster-69084.1, or Cluster-68258.

1.

5. The related gene according to any one of claims 1 to 4, characterized in that, The sweet substances are phloretin, phloridzin, trifolin, 3-hydroxyphloridzin, or neohesperidin dihydrochalcone.

6. The related gene according to claim 5, wherein The following related genes are significantly and positively correlated with each of the sweet substances respectively: Genes significantly and positively correlated with phloridzin include: Cluster-56317.2, Cluster-67928.0, Cluster-24268.12, Cluster-68818.0, Cluster-45928.0, Cluster-55957.0, Cluster-48439.0, Cluster-58209.2, Cluster-76905.1, Cluster-69084.4, Cluster-59728.2; Genes significantly and positively correlated with 3-hydroxyphloridzin include: Cluster-24268.0, Cluster-69541.1, Cluster-69541.2, Cluster-66263.0, Cluster-66580.1, Cluster-68258.0, Cluster-44706.0, Cluster-59829.1, Cluster-69084.3; Genes that are extremely significantly positively correlated with phloretin include: Cluster-56317.1, Cluster-56317.2, Cluster-67928.0, Cluster-24268.12, Cluster-38495.0, Cluster-45928.0, Cluster-55957.0, Cluster-55957.1, Cluster-48439.0, Cluster-58209.2, Cluster-68258.4, Cluster-76905.1, Cluster-69084.4, Cluster-59728.2; Genes that are extremely significantly positively correlated with trilobatin include: Cluster-55165.0, Cluster-69084.1; Genes that are extremely significantly positively correlated with neohesperidin dihydrochalcone include: Cluster-24268.0, Cluster-69541.1, Cluster-69541.2, Cluster-66263.0, Cluster-66580.1, Cluster-68258.0, Cluster-68258.1, Cluster-76905.1, Cluster-69084.4, Cluster-59728.

2.

7. Use of the related genes according to any one of claims 1-6 in screening Litsea cubeba germplasm.

8. Use of the related genes according to any one of claims 1-6 in promoting the synthesis of sweetness in Litsea cubeba germplasm.

9. Use of a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing the related genes according to any one of claims 1-6 in cultivating new Litsea cubeba germplasm.

10. The glycosyltransferase gene in Lithocarpus litseifolius, characterized in that, The glycosyltransferase genes are PGT1 (Cluster-59829.0), PGT1 (Cluster-69084.4).

Images