Use of gene encoding gibberellin 3beta-hydroxylase of <i>Glycine max</i>, GmGA3ox1

Abstract

Provided is a use of gene encoding gibberellin 3B-hydroxylase of G. max, GmGA3ox1. The use of gibberellin 3-hydroxylase gene of G. max, GmGA30x1, set forth in SEQ ID NO:1, is in genetic engineering of seed weight of Arabidopsis thaliana and Glycine max. In A. thaliana, overexpression of GmGA3ox1 can complement the low seed weight phenotype of an atga3ox1 mutant. In G. max, overexpression of the excellent haplotype of the gene can significantly improve the seed weight of G. max.

Claims

1. A method for genetic engineering of seed weight or length of Glycine max, comprising using a sequence of Glycine max; wherein the method for genetic engineering comprises: using DNA of leaves of Jurong Flat Green Bean as a template, amplifying the DNA using primers set forth in SEQ ID NO: 19 and SEQ ID NO: 20 to obtain a sequence of Glycine max with a length of 4187 bp, ligating the sequence of Glycine max into a pCAMBIA3301 vector without a CaMV 35S promoter to obtain a pCAMBIA3301-GmGA3ox1 plant overexpression vector, wherein a representative sample has been deposited under CGMCC deposit number 34555, transferring the pCAMBIA3301-GmGA3ox1 plant overexpression vector into Agrobacterium tumefaciens strain EHA105, and using the resulting EHA105 transformant to transform Glycine max cultivar Williams 82.

2. A method for improving the yield of Glycine max, comprising the following steps: overexpressing a protein of Glycine max or a gene encoding the protein of Glycine max, in Glycine max; wherein the overexpressing step comprises: using DNA of leaves of Jurong Flat Green Bean as a template, amplifying the DNA using primers set forth in SEQ ID NO: 19 and SEQ ID NO: 20 to obtain a sequence of Glycine max with a length of 4187 bp, ligating the sequence of Glycine max into a pCAMBIA3301 vector without a CaMV 35S promoter to obtain a pCAMBIA3301-GmGA3ox1 plant overexpression vector, wherein a representative sample has been deposited under CGMCC deposit number 34555, transferring the pCAMBIA3301-GmGA3ox1 plant overexpression vector into Agrobacterium tumefaciens strain EHA105, and using the resulting EHA105 transformant to transform Glycine max cultivar Williams 82.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 depicts Agarose gel electrophogram of cloning GmGA3ox1 by PCR. Target fragment size is 1137 bp, Marker: DL2000Plus.

(2) FIG. 2 depicts tissue expression pattern of GmGA3ox1, N=3.

(3) FIG. 3 depicts the expression level of GmGA3ox1 is positively correlated with G. max seed weight.

(4) FIGS. 4A-C depict GUS staining of GmGA3ox1 transgenic A. thaliana, N=3; (4A) GUS staining of GmGA3ox1.sub.pro (1957): GUS transgenic A. thaliana; (4B) GUS staining of GmGA3ox1.sub.pro (1005): GUS transgenic A. thaliana; (4C) GmGA3ox1.sub.pro (487): GUS transgenic A. thaliana.

(5) FIG. 5 depicts subcellular localization analysis of GmGA3ox1 protein. GFP: GFP fluorescence; Light: bright field; Merge: fusion protein; 35S: GFP: unloading control; 35S: GmGA3ox1-GFP: GFP-tagged GmGA3ox1 protein; scale: 20 m.

(6) FIGS. 6A-D depict overexpression of GmGA3ox1 in atga3ox1 mutant improves the seed weight of A. thaliana. (6A) The rosette leaf phenotypes of 4-week-old A. thaliana wild type (Col-0), atga3ox1 mutant and three 35S: GmGA3ox1/atga3ox1 transgenic lines; (6B) Mature plant phenotypes of 7-week-old Col-0, atga3ox1 mutants and three 35S: GmGA3ox1/atga3ox1 transgenic lines. (6C) Seed phenotypes of Col-0, atga3ox1 mutant and three 35S: GmGA3ox1/atga3ox1 transgenic lines; scale=1 mm; (6D) Comparison of 1000-seed weight between atga3ox1 mutant and three 35S: GmGA3ox1/atga3ox1 transgenic lines, N=9; the error bar represents SEM. Statistical analysis is performed using a two-tailed t test, **: P<0.01; ***: P<0.001.

(7) FIGS. 7A-G. The excellent haplotype with GmGA3ox1 overexpression improves the seed weight of G. max. (7A) Seed phenotypes of Williams 82 (W82) and two GmGA3ox1-OE transgenic lines; (7B) Schematic diagram of seed length, width, height of G. max; (7C) The relative expression level of GimGA3ox1 in W82 and two GmGA3ox1-OE transgenic lines, N=3; (7D) 100-seed weight of W82 and two GmGA3ox1-OE transgenic lines, N=10; (7E) Seed length of W82 and two GmGA3ox1-OE transgenic lines, N=10; (7F) Seed width of W82 and two GmGA3ox1-OE transgenic lines, N=10; (7G) Seed height of W82 and two GmGA3ox1-OE transgenic lines, N=10; the error bar represents SEM. Statistical analysis is performed using a two-tailed t test, **: P<0.01; ***: P<0.001; ns: not significant.

(8) FIGS. 8A-F depict PCR detection of transgenic A. thaliana and G. max; (8A) Detection of GmGA3ox1.sub.pro (1957): GUS transgenic A. thaliana, target fragment size of 1957 bp, M: Marker DL2000, P: positive plasmid, ck: A. thaliana wild type, 1-6: different GmGA3ox1.sub.pro (1957): GUS transgenic plants, H.sub.2O: blank control; (8B) Detection of GmGA3ox1.sub.pro (1005): GUS transgenic A. thaliana, target fragment size of 1005 bp, M: Marker DL2000, P: positive plasmid, ck: A. thaliana wild type, 1-6: different GmGA3ox1.sub.pro (1005): GUS transgenic plants, H.sub.2O: blank control; (8C) Detection of GmGA3ox1.sub.pro (487): GUS transgenic A. thaliana, target fragment size of 487 bp, M: Marker DL2000, P: positive plasmid, ck: A. thaliana wild type, 1-6: different GmGA3ox1.sub.pro (487): GUS transgenic plants, H.sub.2O: blank control; (8D) PCR detection of A. thaliana atga3ox1 mutant by setting a specific amplification time, and in the set time, a fragment with fragment size of 2000 bp cannot be amplified in atga3ox1 mutant, but can be amplified in the wild-type plant Col-0, M: Marker DL2000, Col-0: A. thaliana wild-type Col-0, atga3ox1: A. thaliana atga3ox1 mutant, H.sub.2O: blank control; (8E) PCR detection of GmGA3ox1 transgenic A. thaliana, target fragment size of 1137 bp, M: Marker DL2000, Col-0: A. thaliana wild type Col-0, atga3ox1: A. thaliana atga3ox1 mutant, H.sub.2O: blank control, 1-6: different 35S: GmGA3ox1/atga3ox1 transgenic plants; (8F) PCR detection of GmGA3ox1 transgenic G. max, target fragment size of 800 bp, M: Trans5K DNA Marker, P: positive plasmid, ck: wild type G. max W82, H.sub.2O: blank control, 1-6: different GmGA3ox1 transgenic plants.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(9) The present disclosure is further described below in combination with drawings and Examples.

(10) The methods used in the following Examples are conventional methods unless otherwise specified.

Example 1

(11) 1) Cloning of Gibberellin 3-Hydroxylase Gene of G. max, GmGA3ox1

(12) The leaves of G. max variety, Nannong 1138-2, were taken and ground with a mortar, then added to a 1.5 mL EP tube containing lysis buffer. After full oscillation, the obtained mixture were transferred to a 1.5 mL EP tube to extract total RNA (Total RNA Kit, Tiangen, Beijing, China). The quality of total RNA was identified by Formaldehyde denatured gel electrophoresis, and the content of RNA was determined by spectrophotometer. The first strand of cDNA was obtained by using the total RNA as a template for reverse transcription according to the instructions of the reverse transcription kit (TaKaRa Primer Script TM RT reagent kit, Japan) provided by TaKaRa company in Japan, and amplified by PCR. The PCR procedure was as follows: pre-denaturation at 95 C. for 3 min, 35 cycles of denaturation at 95 C. for 15 sec, annealing at 60 C. for 15 sec, extension at 72 C. for 90 sec; at last holding at 72 C. for 5 min, followed by keeping in constant temperature of 12 C., then the cDNA of Nannong 1138-2 was obtained.

(13) The gene corresponding to GmGA3ox1 (Glyma.07g033800, GeneID: 100795921) was found from NCBI database and Phytozome v12 G. max database. Specific primers were designed according to the nucleotide sequence provided by the database. The primer sequences were set forth in SEQ ID NO: 3 (atgcettete tetccgaagc ct) and SEQ ID NO: 4 (ctaaatatct agattgaag). Genes in coding sequence (CDS) of G. max variety Nannong 1138-2 was amplified. After PCR cloning, the obtained PCR products were purified, ligated and transformed. The positive clones were picked and sequenced. After sequencing, the CDS sequence of GmGA3ox1 gene of G. max with a complete coding region of 1137 bp was obtained, in which the coding sequence is set forth in SEQ ID NO: 1, with a size of 1137 bp (FIG. 1) (nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO:2)).

(14) TABLE-US-00002 Theaminoacidsequencesetforth inSEQIDNO:2is: MPSLSEAFRGHPVYLHHKHSDFNSLQELPDSYSWTQ PHDHHLPNYPSNNKTKIFVPVIDLNHPNAPNLIGH ACKTWGVFQVVNHDIPMSLFSDIQRASLALFSLPL HQKLKAARSPDGVSGYGRARISSFFPKLMWSECFT ILDSPLDLFLKLWPQDYAKYCDIVVEYEAAMKKLA AKLMCLMLASLGITKEDTKWAGPKGEFNGACAALH LNSYPSCPDPDRAMGLAAHTDSTLLTILHQNNVNG LQVLKEGEGWVAVPPLHGGLVINVGDLLHILSNGL YPSVLHRVRVNRTQQRFSVAYLYGPPANVQISPHV KLVGPTRPALYRPVTWNEYLGTKANLFNKALSAVR LSASINGLFDINEDQNNDFQVGFNLDI.
2) Tissue Expression Analysis of GmGA3ox1

(15) In order to identify the expression level of GmGA3ox1 in different tissues, the roots, stems, leaves, flowers, pods and seeds of G. max variety Nannong 1138-2 at different developmental stages were collected: roots, stems and leaves were in stage V4; mature flowers were in stage R2; and seeds and pods were collected 15 days after flowering. The samples were frozen in liquid nitrogen and stored at 80 C. The extraction of total RNA was the same as step 1). The total RNA obtained from the above tissues was used as a template to reverse into cDNA. The fluorescent quantitative primer sequences of GmGA3ox1 were set forth in SEQ ID NO: 5 (agtccacatg tcaagttggtgg) and SEQ ID NO: 6 (cggttaggct ttctgcgtcta). The expression level of GmGA3ox1 gene in different tissues was detected with reference gene Tubulin in G. max as the internal reference. The primer sequences set forth in SEQ ID NO: 7 (cctcgttcga attcgcttttg) and SEQ ID NO: 8 (caactgtctt gtcacttggc at) were used for real-time fluorescence quantitative PCR (Real-time RT-PCR).

(16) GmGA3ox1 was mainly expressed in leaves and stems of G. max, and the expression level of which was low in roots, stems, flowers, pods and seeds (FIG. 2).

(17) 3) Correlation Analysis Between Expression Level of GmGA3ox1 and Seed Weight

(18) 39 G. max materials with different seed weights were selected and planted in greenhouse. The top three leaves of each G. max material were collected at stage V4, and the samples were frozen in liquid nitrogen and stored at 80 C. The extraction of total RNA was the same as step 1). The total RNA obtained from the above tissues was used as a template to reverse into cDNA. The expression level of GmGA3ox1 gene in each G. max material was detected by real-time fluorescence quantitative PCR (Real-time RT-PCR) with Tubulin as the reference gene. The primer sequences were set forth in SEQ ID NO:7 and SEQ ID NO: 8. The correlation between GmGA3ox1 expression level and seed weight was calculated by SPSS 20.0 software.

(19) The correlation coefficient r between GmGA3ox1 expression level and seed weight was 0.531, and the P value was 0.0005. The results showed that the expression level of GmGA3ox1 gene was positively correlated with seed weight (FIG. 3).

Example 2

(20) 1) Cloning of Promoter Sequence of Gibberellin 3-Hydroxylase Gene of G. max, GmGA3ox1

(21) Leaf DNA of G. max Nannong 1138-2 was used as template for PCR amplification. The primer sequences were set forth in SEQ ID NO: 9 (cagctatgac catgattact cacatcgata aacgaggttt g), SEQ ID NO: 10 (agtgccaagc ttggctgcag attgtgttga atgcttcgg), SEQ ID NO: 11 (cagctatgac catgattact cacgatccca acattcgtg), SEQ ID NO: 12 (agtgccaagc ttggctgcag attgtgttga atgcttcgg), SEQ ID NO: 13 (cagctatgac catgattacc acctagtgag agagaaagaa gg) and SEQ NO: 14 (agtgccaagc ttggctgcag attgtgttga atgcttcgg). The PCR procedure was as follows: pre-denaturation at 95 C. for 3 min; 35 cycles of denaturation at 95 C. for 15 sec, annealing at 60 C. for 15 sec, extension at 72 C. for 90 sec; and at last holding at 72 C. for 5 min, followed by keeping in constant temperature of 4 C. After sequencing, GmGA3ox1 gene promoter sequences with length of 1957 bp, 1005 bp and 487 bp were obtained.

(22) 2) Construction of GUS Staining Vector

(23) The sequences of promoters with different lengths of the three GmGA3ox1 genes were inserted into pCAMBIA1381Z vector, and then the obtained constructed vector was transferred into Agrobacterium tumefaciens strain EHA105 by freeze-thaw method.

(24) 3) GUS Staining of GmGA3ox1 Transgenic A. thaliana

(25) The A. thaliana was transformed by the floral-dip method. The flower buds of A. thaliana were immersed in bacteria solution of the A. tumefaciens strain EHA105 containing GmGA3ox1.sub.pro (1957): GUS, GmGA3ox1.sub.pro (1005): GUS and GmGA3ox1.sub.pro (487): GUS, respectively, and infected for 30 sec to 1 min. The plants were wrapped with preservative film or fresh-keeping bag to keep the humidity and cultured in dark for 24 h. After that, it could be transformed again after a new batch of inflorescence grew out, depending on the growth status of the plants. A. thaliana was put under normal conditions to continue growing and seeds of which were harvested. The roots, stems, leaves, flowers, pods and seeds of three homozygous T3 generation GmGA3ox1.sub.pro (1957): GUS, GmGA3ox1.sub.pro (1005): GUS and GmGA3ox1.sub.pro (487): GUS transgenic A. thaliana lines were selected for GUS staining. The kit used for staining was from Beijing Solarbio Science and Technology Co., Ltd., with Item No. of G3060. In order to detect whether the transgenic A. thaliana is positive. The extracted DNA fragments were detected by PCR using specific primers set forth in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. The PCR detection gel electropherogram was shown in FIG. 8.

(26) GUS staining results showed that the three types of GmGA3ox1 gene promoters all had high activity, and the tissues with the highest activity of these three types of promoters were located in leaves, followed by stems, flowers, seeds, pods and roots (FIGS. 4A-C).

Example 3

(27) 1) Cloning of Gibberellin 3-Hydroxylase Gene of G. max, GmGA3ox1

(28) The first strand of cDNA was synthesized by using the total RNA of G. max variety Nannong 1138-2 leaves as a template for reverse transcription, and then amplified by PCR. The primer sequences were set forth in SEQ ID NO: 15 (cctctagaat gccttcte tecgaagcct) and SEQ ID NO: 16 (ccgctcgaga atatctagat tgaag). The PCR procedure was as follows: pre-denaturation at 95 C. for 3 min; 35 cycles of denaturation at 95 C. for 15 sec, annealing at 60 C. for 15 sec, extension at 72 C. for 90 sec; at last holding at 72 C. for 5 min, followed by keeping in constant temperature of 4 C. After sequencing, the CDS sequence of GmGA3ox1 gene of G. max without stop codon was obtained.

(29) 2) Construction of Subcellular Localization Vector

(30) The CDS sequence of GmGA3ox1 gene of G. max without stop codon was inserted into pFGC5941 expression vector containing GFP tag. The pFGC5941 vector had a 35S promoter, which could strongly induce the expression of the target gene GmGA3ox1 in the receptor. Then the constructed vector was transferred into A. tumefaciens strain EHA105 by freeze-thaw method. At the same time, the unloading pFGC5941 vector was also transformed into EHA105 as a control.

(31) 3) Subcellular Localization of GmGA3ox1

(32) The bacterial solution containing 35S: GmGA3ox1-GFP and 35S: GFP were injected into the leaves of Nicotiana benthamiana with the size of 7-8 weeks, respectively. After 36-48 hours of cultivation, the localization of the reporter gene GFP was observed by Leica TCS SP2 laser confocal microscopy. The GFP signal showed that the GmGA3ox1-GFP fusion protein was localized to the cell membrane and cytoplasmic matrix, while the unloading vector was localized to the entire tobacco cells (FIG. 5).

Example 4: Use of Gene GmGA3ox1 in Genetic Engineering

(33) 1) Cloning of Gibberellin 3-Hydroxylase Gene of G. max, GmGA3ox1

(34) The first strand of cDNA was synthesized by using total RNA of G. max variety Nannong 1138-2 leaves as a template for reverse transcription, and then amplified by PCR. The primer sequences were set forth in SEQ ID NO: 17 (gtcgacgta tegataagct tatgccttct ctctctccgaag cc), SEQ ID NO: 18 (cgctctagaaa ctagtggatc cctaaatatc tagattgaag cccac). The PCR procedure was as follows: pre-denaturation at 95 C. for 3 min; 35 cycles of denaturation at 95 C. for 15 sec, annealing at 60 C. for 15 sec, extension at 72 C. for 90 sec; and at last holding at 72 C. for 5 min, followed by keeping in constant temperature of 4 C. After sequencing, the CDS sequence of GmGA3ox1 gene of G. max with a complete coding region length of 1137 bp was obtained. In addition, the first strand of cDNA was synthesized by using the total RNA of the leaves of the local variety of G. max Jurong Flat Green Bean as a template for reverse transcription, and then amplified by PCR. The primer sequences were set forth in SEQ ID NO: 19 (ggatatgttg aaactttcct ttgc) and SEQ ID NO: 20 (atgcatgatc aatcatttac). The PCR procedure was as follows: pre-denaturation at 95 C. for 3 mins; 35 cycles of denaturation at 95 C. for 15 sec, annealing at 60 C. for 15 sec, extension at 72 C. for 120 sec; and at last holding at 72 C. for 5 min, followed by keeping in constant temperature of 4 C. After sequencing, GmGA3ox1 genome sequence of G. max with length of 4187 bp was obtained.

(35) 2) Construction of Plant Expression Vector

(36) When constructing overexpression vector for transforming A. thaliana, the CDS sequence of GmGA3ox1 gene amplified from Nannong 1138-2 leaves was inserted into pBI121 expression vector to obtain pBI121-GmGA3ox1 plant overexpression vector. The plant transformation vector pBI121 contained a 35S strong promoter, which could strongly induce the expression of the target gene GimGA3ox1 in the receptor. Then the vector was transferred into A. tumefaciens strain EHA105 by freeze-thaw method.

(37) When constructing the overexpression vector of G. max, the GmGA3ox1 genomic sequence with length of 4187 bp was amplified from the G. max Jurong Flat Green Bean. The 4187 bp fragment included 1554 bp promoter and 5UTR region, 1899 bp intron and exon region and 539 bp 3UTR region. The 4187 bp fragment was ligated into pCAMBIA3301 vector without CaMV 35S promoter to obtain pCAMBIA3301-GmGA3ox1 plant overexpression vector. The obtained vector was transformed into A. tumefaciens strain EHA105.

(38) 3) Obtaining Transgenic A. thaliana Plants

(39) The A. thaliana was transformed by the floral-dip method. The flower buds of atga3ox1 A. thaliana mutant were immersed in bacteria solution of A. tumefaciens strain EHA105 containing pBI121-GmGA3ox1 in Step 2) and infected for 30 sec to 1 min. The plants were wrapped with preservative film or fresh-keeping bag to keep the humidity and cultured in dark for 24 h. After that, it could be transformed again after a new batch of inflorescence grew out, depending on the growth status of the plants. A. thaliana was put under normal conditions to continue growing and seeds of which were harvested. Three homozygous T 3 generation 35S: GmGA3ox1/atga3ox1 transgenic A. thaliana lines were selected to investigate the seed weight phenotype (FIGS. 6A-D). The extracted DNA fragments were detected by PCR using specific primers (SEQ ID NO: 21: atgccttctc tctccgaagc ct, SEQ ID NO: 22: aagaceggca acaggattca) to detect whether they were atga3ox1 mutants and whether they were overexpressed GmGA3ox1 transgenic A. thaliana. The PCR detection gel electropherogram was shown in FIGS. 8A-F.

(40) Seeds of Col-0, atga3ox1 mutant and three 35S: GmGA3ox1/atga3ox1 transgenic lines were harvested after maturation and dried in an oven at 37 C. for one week. After seed drying, the 1000-seed weight (N=3) of Col-0, atga3ox1 mutant and three 35S: GmGA3ox1/atga3ox1 transgenic lines were measured. The 1000-seed weight of Col-0 lines was significantly higher than that of atga3ox1 mutant plants (FIG. 6D), which proved that the mutation of AtGA3ox1 gene in A. thaliana could significantly reduce seed weight. In addition, the seed weights of three 35S: GmGA3ox1/atga3ox1 transgenic lines were significantly higher than that of atga3ox1 mutant plants and slightly higher than that of Col-0 plants (FIG. 6D), which proved that overexpression of GmGA3ox1 gene in atga3ox1 mutant could improve seed weight.

(41) 4) Obtaining Transgenic Plants of G. max

(42) The G. max cultivar Williams 82 was transformed by cotyledonary node transformation method. The leaf axil of the G. max seedlings grown for 5 to 6 days were wounded, and the pCAMBIA3301-GmGA3ox1 vectors obtained in step 2) were inoculated into the wounds of the leaf axil. After co-culture at 25 C. for 4-5 days, the G. max was washed with sterilized ultra-pure water and WISH-LIQUID respectively, placed in SIM medium without glufosinate ammonium, and cultured at 26 C. for 15 days to induce buds. After 15 days, the G. max was placed in SIM medium containing 6 mg/L glufosinate ammonium. Subculture was then performed on a 15-day cycle to gradually reduce the dose of glufosinate ammonium. When the buds of the explants grew to about 6 cm, the explants were transferred into the rooting medium and cultured for about 10 days to induce roots. They could be transplanted after the roots grew well. The extracted DNA fragments were detected by PCR using specific primers (SEQ ID NO: 23: agtccacatg tcaagttgt gg, SEQ ID NO: 24: ccggcaacag gattcaatct taa) to detect whether they were transgenic G. max overexpressing GmGA3ox1. The PCR detection gel electropherogram was shown in FIGS. 8A-F.

(43) The obtained transgenic plants of G. max and their controls were sown in flowerpots and placed in a net room under natural conditions. After harvest, the seeds were dried in an oven at 37 C. for one week. After seed drying, 100-seed weight, seed length, width and height (N=10) of W82 and two GmGA3ox1 transgenic G. max lines were measured. The expression levels of GmGA3ox1 in the two transgenic G. max lines were significantly higher than that in the control W82 plants (FIG. 7C) (primers were SEQ ID NO: 23 and SEQ ID NO: 24), which proved that this promoter could effectively drive the expression of GmGA3ox1. In addition, the 100-seed weights and seed lengths of the two GmGA3ox1 transgenic G. max lines were significantly greater than those of W82 plants, and there was no significant difference in seed width and seed height between the two GmGA3ox1 transgenic G. max lines and W82 plants (FIGS. 7A, B, D-G), which proved that overexpression of the excellent haplotype gene of GmGA3ox1 could improve the seed weight and seed length of transgenic G. max.

(44) The above description of Examples is intended to facilitate the convenience of ordinary persons skilled in the art to understand and use the present disclosure. It is clear that those familiar with the technology in the art can easily make various modifications to these Examples and apply the general principles described herein to other embodiments without creative labor. Therefore, the present disclosure is not limited to the above Examples. Improvements and modifications made by those skilled in the art according to the present disclosure are within the claimed scope of the present disclosure.