A sd5 gene for regulating rice plant height and yield and application thereof

Abstract

This invention provides a method for regulating rice plant height and yield. SD5 This invention relates to genes and their applications, specifically to the field of plant genetic engineering technology. SD5 The complete genome nucleotide sequence of the gene is shown in SEQ ID NO.1. SD5 The nucleotide sequence of the gene coding region is shown in SEQ ID NO.2. This invention utilizes cloning... DEP1 Gene promoter sequence and SD5 Transgenic vectors were constructed using the coding region sequences of genes to obtain transgenic vectors. DEP1 Gene promoter drive SD5 Transgenic rice. Experimental results showed that, compared with the recipient ZH17, the transgenic line had an 8.1% decrease in plant height, a 9.6% increase in the number of grains per panicle, a 4.3% increase in thousand-grain weight, and a 15.9% increase in yield per plant. SD5 Genes play an important regulatory role in rice plant height and yield, and can be used to breed lodging-resistant and high-yielding rice varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically a method for regulating rice plant height and yield. SD5 Genes and their applications. Background Technology

[0002] Rice is one of the world's most important food crops. Rice yield is primarily determined by two factors: the number of grains per panicle and the thousand-grain weight. In recent years, several yield genes related to these two factors have been cloned. However, due to limitations in available materials, cloning genes affecting yield traits by constructing mutants, while enabling the analysis of their genetic networks, does not fully reflect the phenotypic changes of these mutants in natural populations. In particular, some mutation sites may not exist in natural populations, and most mutation sites may be associated with reduced yield. Therefore, constructing natural populations to clone and analyze genes affecting yield is of greater practical significance for further improving rice yield.

[0003] Common wild rice is the ancestor of cultivated rice in Asia, possessing far greater genetic diversity and gene resources compared to domesticated rice. The diverse genetic differentiation within common wild rice contains a wealth of genes capable of increasing rice yield. Therefore, discovering and utilizing superior domestication genes lost or weakened in cultivated rice from the genome of common wild rice and applying them to rice breeding has significant theoretical and practical value. This is also an effective approach to solving current rice breeding problems. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide SD5 The application of genes in dwarfing plant height and increasing rice yield, this invention uses DEP1 Gene promoter drive SD5 Transgenic rice has the characteristics of reduced plant height, increased number of grains per panicle, increased thousand-grain weight, and increased yield per plant.

[0005] Another object of the present invention is to provide a method for dwarfing plant height and increasing rice yield.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides SD5 The application of the gene in dwarfing plant height and increasing rice yield was achieved by cloning the promoter sequence of the DEP1 gene and... SD5 Transgenic vectors were constructed using the coding region sequence of the gene to obtain a transgenic vector driven by the promoter of the DEP1 gene. SD5 Genetically modified rice.

[0007] Preferably, the SD5The complete genome nucleotide sequence of the gene is shown in SEQ ID NO.1. SD5 The nucleotide sequence of the gene coding region is shown in SEQ ID NO.2. SD5 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO. 3.

[0008] Preferably, the nucleotide sequence of the promoter of the DEP1 gene is shown in SEQ ID NO.16.

[0009] Preferably, the genetically modified rice exhibits decreased plant height, increased number of grains per panicle on the main stem, increased thousand-grain weight, and increased yield per plant.

[0010] The present invention also provides a method for dwarfing plant height and increasing rice yield, comprising the following steps: Using homologous recombination to... SD5 The gene coding region sequence and the promoter of the DEP1 gene were ligated together into the overexpression vector pCAMBIA1300 to obtain the recombinant plasmid pDEP1::SD5; The recombinant plasmid pDEP1::SD5 was transformed into Agrobacterium competent cells and infected with callus tissue of rice recipient material to regenerate transgenic rice plants.

[0011] Preferably, the transgenic rice plant exhibits decreased plant height, increased number of grains per panicle on the main stem, increased thousand-grain weight, and increased yield per plant.

[0012] Preferably, used to amplify the SD5 The nucleotide sequence of the forward primer for the gene is shown in SEQ ID NO.14, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.15.

[0013] Preferably, the nucleotide sequence of the forward primer used to amplify the promoter of the DEP1 gene is shown in SEQ ID NO. 17, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO. 18.

[0014] Preferably, the Agrobacterium is Agrobacterium tumefaciens EHA105.

[0015] Compared with the prior art, the present invention has the following advantages: This invention discloses a method for dwarfing plant height and increasing rice yield. SD5 In this invention, segregating populations constructed using TYIL55 and T5, and 9WIL148 and NA93-11 respectively, all showed a QTL regulating plant height and yield on chromosome 5. SD5 , Semi-dwarf gene on chromosome 5 ), and finely mapped SD5 to a 6.2kb region, which contains only one predicted gene. LOC_Os05g34854 This invention, through the... SD5 Validation of gene function revealed that in candidate genes SD5 While the expression level increased, the rice plant height, number of grains per panicle, thousand-grain weight, and yield per plant all increased. To further obtain lodging-resistant and high-yielding rice varieties, this invention utilizes a promoter highly expressed in the young panicle. pDEP1 drive SD5 High expression was observed in young spikelets. The recombinant plasmid was transferred into ZH17 using a constructed vector. Young spikelets and internodes from T3-positive transgenic lines and the transformation recipient (ZH17) were selected. SD5 Quantitative analysis showed that, compared to the transformation receptor ZH17, SD5 The expression level was increased in the young panicles of the transgenic lines, and decreased somewhat in the internodes. Further phenotypic identification revealed that, compared to the recipient line, the transgenic lines had an 8.1% decrease in plant height, an increased number of panicle branches leading to a 9.6% increase in the number of grains per panicle, larger grains resulting in a 4.3% increase in thousand-grain weight, no significant difference in the number of effective panicles and seed setting rate, ultimately leading to a 15.9% increase in yield per plant. These results indicate that in cultivated rice, optimization... SD5 The expression pattern can further increase output. Attached Figure Description

[0016] Figure 1 This figure compares the plant height, number of grains per panicle, thousand-grain weight, and yield per plant of TYIL55 and T5, and 9WIL148 and NA9311. Figure a shows the distribution of introgression fragments in TYIL55; gray background indicates chromosome fragments from T5, and red background indicates chromosome fragments from common wild rice from Yuanjiang, Yunnan. Figure b shows the plant type comparison, with a scale bar of 20cm. Figure c shows the statistical results of plant height data for TYIL55 and T5. Figure d shows the statistical results of the number of grains per panicle on the main stem for TYIL55 and T5. Figure e shows the statistical results of the thousand-grain weight for TYIL55 and T5. Figure f shows the statistical results of the statistical results of yield per plant for TYIL55 and T5. Figure cf... P <0.01, n=20, analysis adopted t Tests; g represents the distribution of infiltrated fragments from 9WIL148, with gray backgrounds indicating chromosome fragments from NA93-11 and red backgrounds indicating chromosome fragments from common wild rice from Wenchang, Hainan; h represents the comparison of plant architecture among different rice lines, with a scale bar of 20cm; i represents the statistical results of plant height data for 9WIL148 and NA9311; j represents the statistical results of the number of grains per panicle on the main stem for 9WIL148 and NA9311; k represents the statistical results of the thousand-grain weight data for 9WIL148 and NA9311; l represents the statistical results of the yield per plant for 9WIL148 and NA9311; il represents the yield data for 9WIL148 and NA9311. P<0.01, n=20, analysis adopted t Test; m represents the fine mapping of SD5 in the NA93-11 background, assuming that the location of chromosome recombination in the exchanged plants is between the two markers, and the number between the two markers represents the number of exchanged plants in that interval; the direction indicated by the unidirectional red arrow is the SD5 mapping direction; phenotypic data on plant height and number of grains per ear on the main stem of the mapping parents and each homozygous recombination line were statistically analyzed, ns represents no significant difference. P <0.01, n=15, analysis adopted t test.

[0017] Figure 2 This figure compares the plant height, number of grains per spike on the main stem, thousand-grain weight, and yield per plant of the T2 generation homozygous complementary line and TYIL55. Figure a shows the plant type comparison between TYIL55 and CTP-1 (scale bar: 20cm); b shows the grain type comparison between TYIL55 and CTP-1 (scale bar: 5mm); c shows the spike type comparison between TYIL55 and CTP-1 (scale bar: 5cm); d shows the young spikes of TYIL55, CTP-1, and CTP-2. SD5 Gene expression analysis, n=3; e represents the plant height data of TYIL55, CTP-1, and CTP-2; f represents the number of grains per spike on the main stem of TYIL55, CTP-1, and CTP-2; g represents the thousand-grain weight data of TYIL55, CTP-1, and CTP-2; h represents the yield per plant of TYIL55, CTP-1, and CTP-2; eh... P <0.01, n=20, CTP-1 and CTP-2 are independent complementary transgenic lines, and the analysis uses t test; Figure 3 The promoter highly expressed in ZH17 and young spikelets pDEP1 drive SD5 Transformation of gene expression SD5 The comparison results of plant height, number of grains per panicle, thousand-grain weight, and yield per plant of genetically modified rice are shown in the figure. Figure a shows plant type comparison (scale bar: 20cm); b shows grain type comparison (scale bar: 5mm); c shows panicle type comparison (scale bar: 5cm); d shows young panicles and internodes of ZH17 and SD5 genetically modified rice. SD5 Expression level analysis, n=3; e represents ZH17 and transgenic expression. SD5 Statistical results of plant height data for genetically modified rice; f represents ZH17 and transgenic rice. SD5 Statistical results of the number of grains per panicle on the main stem of genetically modified rice; g represents ZH17 and transgenic rice. SD5 Statistical results of the thousand-grain weight data of genetically modified rice; h represents ZH17 and transgenic rice. SD5 Statistical results of single-plant yield data for genetically modified rice; eh P<0.05, P<0.01, n=20, analysis using t test. Detailed Implementation

[0018] This invention provides a method for dwarfing plant height and increasing rice yield. SD5 This invention selected the line TYIL55, a line with reduced plant height, number of grains per panicle, and thousand-grain weight, ultimately leading to a significant decrease in yield per plant, from an introgression line with T5 as the background and chromosome introgression from common wild rice from Yuanjiang, Yunnan. Simultaneously, from an introgression line preserved by Sun Chuanqing's research group at the College of Agriculture, China Agricultural University, with 93-11 (awnless, denoted as "NA93-11") as the background and chromosome introgression from common wild rice from Wenchang, Hainan, a line 9WIL148, with reduced plant height, number of grains per panicle, and thousand-grain weight, and a significant decrease in yield per plant, was selected compared to the recurrent parent NA93-11. This invention utilized segregating populations constructed from TYIL55 and T5, and from 9WIL148 and NA93-11, respectively, and detected a QTL (Quality Transmission Traceability) regulating plant height and yield on chromosome 5 in both cases. SD5 ), and will SD5 Fine mapping was performed within a 6.2kb interval, which contained only one predicted gene. LOC_Os05g34854 In this invention, the... SD5 The complete genome nucleotide sequence of the gene is shown in SEQ ID NO.1. SD5 SD5 The amino acid sequence of the protein encoded by the gene is MHASPHPLQIAHDTLLLSLTHTLCTAETGSTIRTGTAMVYISNAQDASKLIVTAKGGGGEADDAAAAAAAVVLDLWRQPAKIPAPFVWPRADVALPPSSPPTGELDVPVVDLAAALRDAAGMRRAVAQVAAACASHGFFQVSGHGVPPSLARAALDGAAGFFRLPPAAKQRARRAPGTVTGYTAAHADRFVDNLPWKETLSFGHRHANAAGNNSS TVADYFSTLGDDFKHLGEVYQEYCEAMEEVTKAIMAVLGESLGVGGGYYREFFEDSSSIMRCNYYPPCPEPERTLGTGPHCDPSALTVLLQDGDVDGLQVLVAGAWRPVRPLPGA FVVNIGDTFMALTNGRYKSCLHRAVVHREQERRSLAFFLCPREDRVVRPPAGAGAGERRLYPDFTWADFMRFTQRHYRADTRTLDAFARWLRPPACSGAAPVVGPPTTATQAATV (SEQ ID NO.3).

[0019] The present invention also provides the aforementioned SD5 Application of genes in regulating rice plant height and yield. The yield traits preferably include the number of grains per panicle, thousand-grain weight, and yield per plant. This invention constructs a complementary vector containing a candidate gene promoter sequence of approximately 2.5 kb and the complete gene region sequence. The recombinant plasmid pCTP-SD5 was transformed into TYIL55 using Agrobacterium-mediated transformation. Quantitative real-time PCR was used to detect the T2 generation functionally complementary lines (CTP1 and CTP2) and TYIL55. SD5 Gene expression, results SD5 The expression levels of the gene were significantly higher in the complementary lines than in TYIL55. Phenotypic investigations were conducted on the obtained T2 complementary lines and the transformation recipient. Statistical analysis showed that, compared with the control, the complementary lines, while exhibiting increased candidate gene expression, also showed an increase in plant height of approximately 10.9%, an increase in the number of grains per ear on the main stem of approximately 48.9%, an increase in thousand-grain weight due to longer grains of approximately 4.3%, and an increase in yield per plant of approximately 46.0%.

[0020] The present invention also provides SD5 The application of genes in dwarfing plant height and increasing rice yield: This invention utilizes cloning... DEP1 Gene promoter sequence and SD5 Transgenic vectors were constructed using the coding region sequences of genes to obtain transgenic vectors. DEP1 Gene promoter drive SD5 Transgenic rice with a gene. The transgenic rice exhibits decreased plant height, increased grain number per panicle, increased thousand-grain weight, and increased yield per plant. This invention utilizes a promoter highly expressed in young panicles. pDEP1 drive SD5 High expression was observed in young spikelets. The recombinant plasmid was transferred into ZH17 using a constructed vector. Young spikelets and internodes from T3-positive transgenic lines and the transformation recipient (ZH17) were selected. SD5 Quantitative analysis showed that, compared to the transformation receptor ZH17, SD5 The expression level was increased in the young spikelets of the transgenic lines and decreased to some extent in the internodes. Further phenotypic identification revealed that, compared with the recipient, the transgenic lines had a plant height that decreased by 8.1%, an increase in the number of spikelet branches that led to a 9.6% increase in the number of grains per spikelet on the main stem, and larger grains that led to a 4.3% increase in the thousand-grain weight. There was no significant difference in the number of effective spikelets and the seed setting rate, ultimately resulting in a 15.9% increase in yield per plant.

[0021] This invention provides a method for dwarfing plant height and increasing rice yield, the method preferably comprising the following steps: Using homologous recombination to... SD5 Gene coding region sequence, DEP1 The promoters of the genes were ligated together into the overexpression vector pCAMBIA1300 to obtain the recombinant plasmid pDEP1::SD5; the recombinant plasmid pDEP1::SD5 was transformed into Agrobacterium competent cells and infected into the callus tissue of rice recipient material to regenerate transgenic rice plants.

[0022] In this invention, the DEP1

[0023] In this invention, the amplification of the... SD5 The nucleotide sequence of the forward primer for the gene coding region sequence is GAGGGGTGGTTCTGAGTTGGATGCATGCATCTCCTCACCCAT (SEQ ID NO.14), and the nucleotide sequence of the reverse primer is GCCAAGCTTGCATGCCTGCAGCTAGACGGTGGCCGCCTGGGTC (SEQ ID NO.15). This is used to amplify the gene coding region sequence. DEP1 The nucleotide sequence of the forward primer for the gene promoter is GAATTCGAGCTCGGTACCCGGGGATCCGTGAATCCGCCACGCTAACC (SEQ ID NO.17), and the nucleotide sequence of the reverse primer is ATGGGTGAGGAGATGCATGCATCCAACTCAGAACCACCCCTC (SEQ ID NO.18).

[0024] In this invention, preferably selected Bam H Ⅰ and Pst I. The restriction enzyme site of the complementary vector pCAMBIA1300 was digested, and the cDNA of NA93-11 was used as a template for PCR amplification with TKS high-fidelity enzyme to obtain a 1338 bp DNA fragment A; the DNA of ZH17 was used as a template for amplification with TKS high-fidelity enzyme. DEP1 The promoter sequence was used to obtain DNA fragment B, approximately 2000 bp in length. DNA fragments A and B were ligated into the overexpression vector pCAMBIA1300 using homologous recombination to obtain the recombinant plasmid pDEP1::SD5. In this invention, the total volume of the ligation reaction system is preferably 10 µL, containing 100 ng·µL of each fragment. -1 pCAMBIA1300 1µL, 50ng·µL -1 Fragment A 1µL, 50ng·µL -1 Fragment B 1µL, 2×Assembly MasterMix recombinase 5µL, double-distilled water 2µL. In this invention, the preferred ligation reaction conditions are 48-55℃ for 18-25 min, more preferably 50℃ for 20 min. In this invention, the homologous recombination kit is preferably purchased from Sino-American Taihe Pharmaceutical Co., Ltd., catalog number c5891-50.

[0025] In this invention, E. coli is transformed after ligation. The preferred specific steps are: placing DH5α competent cells on ice to thaw; aspirating the recombinant system solution into the competent cells in a clean bench and immersing them in ice for 15-20 minutes; placing the competent cells containing the recombinant system solution in a 42°C water bath for heat shock for 90 seconds, then immersing them in ice for 2-3 minutes; and adding 600 μL of recombinant system solution to the competent cells in a clean bench. LB liquid medium was placed in a shaker and incubated at 37°C and 220 rpm for 45-60 min. The competent cells were then centrifuged at 12000 rpm for 30-60 s. The supernatant was removed in a clean bench, leaving 100 μL of liquid in a tube. The remaining 100 μL of liquid was mixed and plated. The cells were then incubated upside down in the dark at 37°C for 12-14 h. Single colonies were picked up with a pipette tip and added to LB liquid medium. The cells were incubated at 37°C and 220 rpm for 6-8 h. The incubated bacterial culture was then sent to the company for sequencing of the target fragment. After sequencing analysis, 100 μL of the successfully ligated bacterial culture was added to LB liquid medium and incubated for 6-8 h. The successfully ligated bacterial culture was then preserved in 50% glycerol in a clean bench and stored at -80°C.

[0026] In this invention, the successfully constructed recombinant plasmid pDEP1::SD5 is transformed into Agrobacterium tumefaciens. The preferred steps are as follows: EHA105 competent cells are placed on ice to thaw; in a clean bench, the recombinant plasmid pDEP1::SD5 is aspirated into the competent cells, placed on ice for 30 min, then flash-frozen in liquid nitrogen for 2 min, transferred to a 37°C water bath, heat-shocked for 5 min, and then placed on ice for 5 min; 600 μL of LB liquid medium is added to the competent cells in a clean bench, and the cells are placed in a shaker at 28°C and 220 rpm for 3-6 h; the shaken competent cells are placed in a centrifuge at 12000 rpm for 30-60 s, and the supernatant is aspirated into a tube, retaining 100 μL of liquid; the remaining 100 μL of liquid is mixed and plated, incubated upside down in the dark at 28°C for 1-2 days, and after picking and shaking the plates, the bacteria are preserved using 50% glycerol and stored at -80°C.

[0027] In this invention, the callus tissue of rice recipient material infected with Agrobacterium obtained above is regenerated and passaged to obtain transgenic rice plants. The transgenic rice plants have the characteristics of reduced plant height, increased number of grains per panicle on the main stem, increased thousand-grain weight, and increased yield per plant.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0030] Example 1 SD5 Cloning of genes 1. Test materials The superior indica rice line T5 was provided by the Rice Research Institute of Anhui Academy of Agricultural Sciences. The dwarf, low-yielding introgression line TYIL55, based on T5, was constructed by the research group of Sun Chuanqing at the College of Agriculture, China Agricultural University. The superior super hybrid rice parent NA93-11 and the dwarf, low-yielding introgression line 9WIL148 were also from the research group of Sun Chuanqing at the College of Agriculture, China Agricultural University. Using backcrosses and self-pollinations of TYIL55 and 9WIL148 with T5 and NA93-11, respectively, segregating populations were constructed to finely map QTLs regulating plant height, grain number per panicle, and thousand-grain weight. The japonica rice material Zhonghua 17 (ZH17) was selected as the transformation recipient for the transgenic vector.

[0031] 2. Group building The ideal line TYIL55 was selected from the introgression line containing chromosome segments of common wild rice from Yuanjiang, Yunnan, with T5 as the background. This line was further crossed with T5 and self-pollinated to construct an F2 mapping population for QTL analysis. From the F2 population, heterozygous individuals were selected where only wild rice chromosome segments were introgressed near the QTL loci, with other loci exhibiting recurrent parental genetic background as much as possible. The population was further enlarged, and exchanged individuals were selected. The QTL was finely mapped using the genotypes of previous generations and the phenotypic values ​​of subsequent generations, while simultaneously constructing near-isogenic lines (NILs). Using NA93-11 and 9WIL148 as mapping parents, QTL analysis was performed on the F2 mapping population constructed through hybridization and self-pollination. A similar method was used to finely map the QTL while constructing near-isogenic lines.

[0032] 3. Results Analysis The line TYIL55 was selected from introgression lines containing chromosomes of *Oryza sativa* from Yuanjiang, Yunnan, with T5 as the background. TYIL55 contains introgression segments on chromosomes 2, 4, and 5. Figure 1 Compared to T5, plant height decreased by approximately 15 cm, the number of grains per spike on the main stem decreased by approximately 33.9%, and the thousand-grain weight decreased by approximately 7.7%, ultimately resulting in a yield reduction of approximately 37.6% per plant. Figure 1 (bf in the text). QTL analysis was performed on a segregating population constructed by crossing T5 and TYIL55 and self-pollinating. A QTL regulating plant height and the number of grains per spike on the main stem was found at a marker site on chromosome 5. This QTL was named... SD5 ( Semi - dwarf gene on chromosome 5Among introgression lines with chromosomes from Hainan Wenchang common wild rice as a background (NA93-11), the line 9WIL148 was selected, which showed a decrease in plant height and number of grains per panicle compared to NA93-11. 9WIL148 contains introgression segments on chromosomes 3, 5, 6, and 7, particularly on chromosome 5. SD5 Wild rice chromosome fragments also infiltrated at the locus ( Figure 1 Compared to NA93-11, the plant height decreased by approximately 20 cm, the number of grains per spike on the main stem decreased by approximately 28.4%, and the thousand-grain weight decreased by approximately 12.6%, ultimately resulting in a yield reduction of approximately 34.0% per plant. Figure 1 (hl in the middle).

[0033] Using 281 F2 individual plants constructed by crossing NA93-11 and 9WIL148 and self-pollinating, phenotypic investigations were conducted on plant height and number of grains per ear on the main stem. SD5 Initial localization was located between markers M2 (forward primer sequence as shown in SEQ ID NO.4, reverse primer sequence as shown in SEQ ID NO.5) and M4 (forward primer sequence as shown in SEQ ID NO.6, reverse primer sequence as shown in SEQ ID NO.7) on chromosome 5. Further narrowing of the localization interval was achieved by selecting exchange plants from 6870 F2 plants constructed by self-pollination of NA93-11 and 9WIL148, ultimately refining the localization of SD5 to approximately 6.2 kb. Figure 1 (m in the original text). By consulting rice genome annotation websites, it was found that there is only one candidate gene within this localization interval, namely... LOC_Os05g34854 Named SD5 Gene. SD5 The complete genome nucleotide sequence of the gene is shown in SEQ ID NO.1. SD5 The nucleotide sequence of the open reading frame of the gene is shown in SEQ ID NO.2, and the protein it encodes is named SD5, whose amino acid sequence is shown in SEQ ID NO.3, consisting of 445 amino acid residues.

[0034] Example 2 SD5 Gene function identification 1. Construction of recombinant plasmid pCTP-SD5 according to SD5 The complete genome sequence of the gene is SEQ ID NO.1. Primers were designed and synthesized (forward primer sequence as shown in SEQ ID NO.8, reverse primer sequence as shown in SEQ ID NO.9). Using T5 DNA as a template, PCR amplification was performed using TKS high-fidelity enzyme, amplifying [the gene containing...]. SD5A total of 10kb of genome fragments, including a promoter sequence of approximately 2.5kb, was amplified and ligated into the complementary expression vector pCAMBIA1300 using homologous recombination to obtain the complementary expression vector pCTP-SD5.

[0035] 2. Agrobacterium-competent transformation The recombinant plasmid pCTP-SD5 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium tumefaciens EHA105 / pCTP-SD5.

[0036] 3. Obtaining T0 generation complementary plants (1) Disinfect the dehulled rice seeds with 70% alcohol and 15% NaClO, wash the seeds with sterile water, dry the seeds and then evenly cover the seeds on the basic culture medium. (2) Agrobacterium containing recombinant plasmid pCTP-SD5 was transferred into the callus by infiltration, and the infiltrated callus was placed on co-culture medium and cultured in the dark for 3-4 days. (3) After dark culture for 3-4 days, the infected callus was rinsed with sterile water and then rinsed again with sterile water containing 200 mg / L cephalosporin and 200 mg / L termethin. The infected callus was placed in a sterile culture dish with filter paper to dry and then transferred to a screening medium for hygromycin screening. Vigorous growth and golden yellow resistant callus were obtained. (4) The resistant callus was transferred to the predifferentiation medium and cultured in the dark for about a week. Then the resistant callus was transferred to the differentiation medium and cultured in the light for about a month. The differentiated seedlings were transferred to the rooting medium to root. After growing to a certain period, leaf samples were taken, DNA was extracted and transgenic detection was performed to obtain T0 generation complementary plants.

[0037] 4. Obtaining T2 generation homozygous complementary strains and real-time quantitative PCR detection Transgenic T0 generation plants were self-crossed for two generations to obtain T2 generation homozygous complementary plants, named CTP-1 and CTP-2, respectively. Two-week-old seedlings of the T2 generation homozygous complementary plants were used as experimental material. Total RNA was extracted using TRIZOL reagent, and then reverse transcribed using SuperScript II reverse transcriptase to obtain cDNA from the two T2 generation homozygous complementary plants.

[0038] RT-qPCR was used to detect the presence of [a specific virus / organism] in two T2 generation homozygous complementary lines. SD5 Relative gene expression levels (in terms of) UBI (Genes used as internal reference genes). Detection SD5 The primers for the gene are the forward primer SEQ ID NO.10 and the reverse primer SEQ ID NO.11. Detection UBIThe primers for the gene are the forward primer SEQ ID NO.12 and the reverse primer SEQ ID NO.13.

[0039] Following the method described above, the T2 generation homozygous complement was replaced with TYIL55, with all other steps remaining unchanged, to obtain the TYIL55 strain. SD5 The relative expression level of genes.

[0040] 5. Phenotypic identification of T2 generation homozygous complementary plants The rice materials to be tested (TYIL55, T2 generation homozygous complementary plants CTP-1 or CTP-2) were planted at the Beijing Shangzhuang Experimental Station and the Hainan Sanya Experimental Station of China Agricultural University. During the growth and development process, the plant height, number of grains per panicle, thousand-grain weight, and yield per plant of the tested rice were compared and statistically analyzed. The experiment was repeated three times, with 20 plants in each replicate.

[0041] 6. Results Analysis To determine whether the candidate gene is... SD5 Using T5 DNA as a template, a complementary vector containing approximately 2.5 kb of candidate gene promoter sequence and complete gene region sequence was constructed. The recombinant plasmid pCTP-SD5 was transformed into TYIL55 via Agrobacterium-mediated transformation. SD5 gene expression in the T2 generation complementary lines (CTP1 and CTP2) and TYIL55 was detected by quantitative real-time PCR. The results showed that SD5 gene expression was significantly higher in the complementary lines than in TYIL55. Figure 2 (d) Phenotypic investigation was conducted on the obtained T2 complementary lines and transformation recipients. Statistical analysis showed that, compared with the control, the complementary lines, while exhibiting increased candidate gene expression levels, also showed an approximately 10.9% increase in plant height, an approximately 48.9% increase in the number of grains per ear on the main stem, an approximately 4.3% increase in thousand-grain weight due to longer grains, and an approximately 46.0% increase in yield per plant. Figure 2 ac and Figure 2 (eh in the middle).

[0042] Example 3 SD5 Application of genes to increase rice yield while reducing plant height 1. Construction of recombinant plasmid pDEP1::SD5 according to SD5 The sequence of the gene coding region is SEQ ID NO.2. Primers were designed and synthesized (forward primer sequence as shown in SEQ ID NO.14, reverse primer sequence as shown in SEQ ID NO.15). Using NA93-11 cDNA as a template, PCR amplification was performed using TKS high-fidelity enzyme to obtain a 1338 bp DNA fragment A. DFP1The promoter sequence of the gene is SEQ ID NO.16. Primers were designed and synthesized (the forward primer sequence is shown in SEQ ID NO.17, and the reverse primer sequence is shown in SEQ ID NO.18). Using ZH17 DNA as a template, the gene was amplified using TKS high-fidelity enzyme. DEP1 The promoter sequence was obtained, yielding DNA fragment B of approximately 2000 bp. DNA fragments A and B were ligated into the overexpression vector pCAMBIA1300 using homologous recombination to obtain the recombinant plasmid pDEP1::SD5.

[0043] 2. Agrobacterium-competent transformation The recombinant plasmid pDEP1::SD5 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium EHA105 / pDEP1::SD5.

[0044] 3. T0 transfer SD5 The acquisition of genetically modified rice (1) Disinfect the dehulled rice seeds with 70% alcohol and 15% NaClO, wash the seeds with sterile water, dry the seeds and then evenly cover the seeds on the basic culture medium. (2) Agrobacterium containing recombinant plasmid pDEP1::SD5 was transferred into the callus by infiltration, and the infiltrated callus was placed on co-culture medium and cultured in the dark for 3-4 days. (3) After dark culture for 3-4 days, the infected callus was rinsed with sterile water and then rinsed again with sterile water containing 200 mg / L cephalosporin and 200 mg / L termethin. The infected callus was placed in a sterile culture dish with filter paper to dry and then transferred to a screening medium for hygromycin screening. Vigorous growth and golden yellow resistant callus were obtained. (4) The resistant callus was transferred to the predifferentiation medium and cultured in the dark for about one week. Then the resistant callus was transferred to the differentiation medium and cultured in the light for about one month. The differentiated seedlings were transferred to the rooting medium to root. After they grew to a certain stage, leaf samples were taken, DNA was extracted and transgenic detection was performed to obtain T0 generation transgenic seedlings. SD5 Genetically modified rice.

[0045] 4. T3 transfer SD5 Obtaining genetically modified rice and detecting it using real-time quantitative PCR Transgenic T0 generation plants were continuously self-crossed for three generations to obtain T3 generation transgenic plants. SD5 The genetically modified rice was named pDEP1::SD5. Using two-week-old panicles and internodes of T3 generation plants as experimental materials, total RNA was extracted using TRIZOL reagent, and then reverse transcribed using SuperScript II reverse transcriptase to obtain T3 generation transgenic rice. SD5cDNA from young panicles and internodes of T3 generation transgenic SD5 rice was detected using RT-qPCR. SD5 Relative gene expression levels (in terms of) UBI (Genes used as internal reference genes). Detection SD5 The primers for the gene are the forward primer SEQ ID NO.10 and the reverse primer SEQ ID NO.11. Detection UBI The primers for the gene are the forward primer SEQ ID NO.12 and the reverse primer SEQ ID NO.13.

[0046] Following the above method, T3 will be converted. SD5 The rice gene was replaced with ZH17, and all other steps remained unchanged to obtain the relative expression level of the SD5 gene in ZH17.

[0047] 5. T3 transfer SD5 Phenotypic identification of genetically modified rice The rice materials to be tested (ZH17, T3 generation) SD5 Genetically modified rice was planted at the Beijing Shangzhuang Experimental Station and the Hainan Sanya Experimental Station of China Agricultural University. During its growth and development, the plant height, number of grains per panicle, thousand-grain weight, and yield per plant were compared and statistically analyzed. The experiment was repeated three times, with 20 plants per replicate.

[0048] 6. Results Analysis Recombinant plasmid pDEP1::SD5 was transferred into cultivated rice variety ZH17 via Agrobacterium-mediated genetic transformation. T3-positive transgenic lines and young panicles and internodes of ZH17 were selected. SD5 Quantitative analysis showed that, compared to the transformation receptor ZH17, SD5 The expression level was increased in the young spikelets of the transgenic lines, and decreased to some extent in the internodes. Figure 3 (d). Further phenotypic identification revealed that, compared to the recipient ZH17, the transgenic line had an 8.1% decrease in plant height, a 9.6% increase in the number of grains per spike on the main stem, a 4.3% increase in thousand-grain weight, and ultimately a 15.9% increase in yield per plant. Figure 3 Chinese AC and Figure 3 (eh). The above results indicate that optimization in cultivated rice... SD5 The expression pattern can further increase output.

[0049] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. SD5 The application of genes in dwarfing plant height and increasing rice yield is characterized by, By cloning DEP1 Gene promoter sequence and SD5 Transgenic vectors were constructed using the coding region sequences of genes to obtain transgenic vectors. DEP1 Gene promoter drive SD5 Genetically modified rice; The SD5 The complete genome nucleotide sequence of the gene is shown in SEQ ID NO.

1. SD5 The nucleotide sequence of the gene coding region is shown in SEQ ID NO.

2. SD5 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.3; The nucleotide sequence of the promoter of the DEP1 gene is shown in SEQ ID NO.

16.

2. The application according to claim 1, characterized in that, The genetically modified rice exhibited decreased plant height, increased number of grains per panicle on the main stem, increased thousand-grain weight, and increased yield per plant.

3. A method for dwarfing plant height and increasing rice yield, characterized in that, Includes the following steps: Using homologous recombination, the method described in claim 1 SD5 Gene coding region sequence, DEP1 The promoters of the genes were ligated together into the overexpression vector pCAMBIA1300 to obtain the recombinant plasmid pDEP1::SD5; The recombinant plasmid pDEP1::SD5 was transformed into Agrobacterium competent cells and infected with callus tissue of rice recipient material to regenerate transgenic rice plants.

4. The method according to claim 3, characterized in that, The genetically modified rice plants exhibited decreased plant height, increased number of grains per panicle on the main stem, increased thousand-grain weight, and increased yield per plant.

5. The method according to claim 3, characterized in that, Used to amplify SD5 The nucleotide sequence of the forward primer for the gene is shown in SEQ ID NO.14, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

15.

6. The method according to claim 3, characterized in that, The nucleotide sequence of the forward primer used to amplify the promoter of the DEP1 gene is shown in SEQ ID NO.17, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

18.

7. The method according to claim 3, characterized in that, The Agrobacterium is Agrobacterium tumefaciens EHA105.

Images