SD5 gene for regulating and controlling plant height and yield of rice and application of SD5 gene

Abstract

The invention provides an SD5 gene for regulating and controlling the plant height and yield of rice and application of the SD5 gene, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the whole genome of the SD5 gene is as shown in SEQ ID NO. 1, and the nucleotide sequence of the coding region of the SD5 gene is as shown in SEQ ID NO. 2. A transgenic vector is constructed by cloning a promoter sequence of a DEP1 gene and a coding region sequence of an SD5 gene, and transgenic rice of which the SD5 gene is driven by a promoter of the DEP1 gene is obtained. Experimental results show that compared with a transformation receptor ZH17, the plant height of a transgenic line is reduced by 8.1%, the number of grains per spike of a main stem is increased by 9.6%, the thousand seed weight is increased by 4.3%, and the yield of a single plant is increased by 15.9%. Therefore, the SD5 gene plays an important role in regulating and controlling the plant height and the yield of the rice and can be used for cultivating lodging-resistant rice varieties and high-yield rice varieties.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to a gene for regulating plant height and yield of rice and application thereof. SD5 BACKGROUND

[0002] Rice is one of the most important food crops in the world. The yield of rice is mainly determined by two factors, i.e., the number of grains per panicle and the thousand-grain weight. In recent years, a plurality of yield genes related to the number of grains per panicle and the thousand-grain weight have been cloned. However, due to the limitation of materials, the genes affecting the yield traits are cloned by constructing mutants, although the genetic network can be analyzed, the phenotype change of the mutants cannot completely reflect the phenotype change of the genes in the natural population. In particular, some mutant sites may not exist in the natural population, and most of the mutant sites may be related to the yield reduction. Therefore, constructing a natural population to clone and analyze the genes affecting the yield has more important practical significance for further improving the yield of rice.

[0003] Common wild rice is the ancestor of Asian cultivated rice, and has more abundant genetic diversity and gene resources compared with the cultivated rice which has been artificially domesticated. The common wild rice has various types of genetic differentiation, and contains rich genes capable of improving the yield of rice. Therefore, it has important theoretical significance and practical value to explore and utilize the excellent domestication genes lost or weakened in the cultivated rice from the genome of the common wild rice and apply the genes to rice breeding. This is also an effective way to solve the current problem of rice breeding. SUMMARY

[0004] In view of this, the present application aims to provide SD5 application of the gene in dwarfing plant height and improving the yield of rice, the present application drives DEP1 the gene by the promoter of the gene SD5 The transgenic rice of the gene has the characteristics of plant height reduction, improvement of the number of grains per panicle, improvement of the thousand-grain weight, and improvement of the yield per plant.

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

[0006] In order to achieve the above-mentioned application objects, the present application provides the following technical solutions. The present application provides SD5 application of the gene in dwarfing plant height and improving the yield of rice, the transgenic vector is constructed by cloning the promoter sequence of the DEP1 gene and the coding region sequence of the gene SD5 The transgenic rice of the gene is obtained by driving the gene with the promoter of the DEP1 gene. SD5

[0007] Preferably, the gene is DEP1 gene. SD5 ​​The nucleotide sequence of the whole genome of the gene is shown as SEQ ID NO. 1, and the SD5 The nucleotide sequence of the coding region of the gene is shown as SEQ ID NO. 2, and the SD5 The amino acid sequence of the coding protein of the gene is shown as SEQ ID NO. 3.

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

[0009] Preferably, the transgenic rice has a decreased plant height, increased grain number per panicle, increased 1000-grain weight, and increased yield per plant.

[0010] The application also provides a method for dwarfing plant height and increasing rice yield, comprising the following steps: The nucleotide sequence of the whole genome of the gene is shown as SEQ ID NO. 1, and the SD5 The coding region sequence of the gene and the promoter of the DEP1 gene are jointly connected to the overexpression vector pCAMBIA1300 to obtain a recombinant plasmid pDEP1::SD5. The recombinant plasmid pDEP1::SD5 is transformed into an Agrobacterium competent cell, and the Agrobacterium competent cell is used to infect callus of a rice receptor material, and a transgenic rice plant is regenerated.

[0011] Preferably, the transgenic rice plant has a decreased plant height, increased grain number per panicle, increased 1000-grain weight, and increased yield per plant.

[0012] Preferably, the nucleotide sequence of the forward primer for amplifying the coding region sequence of the gene is shown as SEQ ID NO. 14, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 15. SD5 Preferably, the nucleotide sequence of the forward primer for amplifying the promoter of the DEP1 gene is shown as SEQ ID NO. 17, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 18.

[0013] Preferably, the Agrobacterium is Agrobacterium tumefaciens EHA105.

[0014] Compared with the prior art, the application has the following beneficial effects:

[0015] The application discloses a gene for dwarfing plant height and increasing rice yield. The application detects a QTL (quantitative trait locus) for regulating plant height and yield on the 5th chromosome by using separate populations constructed by TYIL55 and T5 and 9WIL148 and NA93-11, and finely positions SD5 in a 6.2kb interval, and the interval only has one predicted gene SD5 . SD5 , Semi-dwarf gene on chromosome 5 .LOC_Os05g34854 This invention, through the application of... 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 used t test. DETAILED DESCRIPTION

[0018] The application provides a dwarf plant height and yield-increasing rice SD5 The application selects a strain TYIL55 from a T5 background containing Yunnan Yuanjiang common wild rice chromosome introgression, which is lower in plant height, spike grain number and thousand-grain weight compared with a recurrent parent T5, and finally leads to a significant decrease in single plant yield. Meanwhile, a 9WIL148 strain is selected from a 93-11 (no awn, represented as "NA93-11") background containing Hainan Wenchang common wild rice chromosome introgression, which is lower in plant height, spike grain number and thousand-grain weight compared with a recurrent parent NA93-11, and leads to a significant decrease in single plant yield. The application detects a QTL (quantitative trait locus) for regulating plant height and yield on the 5th chromosome in a separation population constructed by using the TYIL55 and T5 and the 9WIL148 and NA93-11 respectively, and fine-positions the QTL in a 6.2kb interval, and the interval only has one predicted gene SD5 SD5 LOC_Os05g34854 In the application, the whole genome nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the gene is shown as SEQ ID NO. 2. SD5 SD5 ​​​SD5 The amino acid sequence of the protein encoded by the gene is MHASPHPLQIAHDTLLLSLTHTLCTAETGSTIRTGTAMVYISNAQDASKLIVTAKGGGGEADDAAAAAAAVVLDLWRQPAKIPAPFVWPRADVALPPSSPPTGELDVPVVDLAAALRDAAGMRRAVAQVAAACASHGFFQVSGHGVPPSLARAALDGAAGFFRLPPAAKQRARRAPGTVTGYTAAHADRFVDNLPWKETLSFGHRHANAAGNNSSTVADYFSTLGDDFKHLGEVYQEYCEAMEEVTKAIMAVLGESLGVGGGYYREFFEDSSSIMRCNYYPPCPEPERTLGTGPHCDPSALTVLLQDGDVDGLQVLVAGAWRPVRPLPGAFVVNIGDTFMALTNGRYKSCLHRAVVHREQERRSLAFFLCPREDRVVRPPAGAGAGERRLYPDFTWADFMRFTQRHYRADTRTLDAFARWLRPPACSGAAPVVGPPTTATQAATV (SEQ ID NO. 3).

[0019] The application also provides the above-mentioned SD5 application of the gene in regulating the plant height and yield of rice. The yield traits preferably include the grain number of the main stem ear, the thousand-grain weight and the yield per plant. The application constructs a complementary vector containing the about 2.5 kb promoter sequence and the complete gene region sequence of the candidate gene. The recombinant plasmid pCTP-SD5 is transformed into TYIL55 by the Agrobacterium-mediated method, and the expression of the gene in the T2 generation functional complementary lines (CTP1 and CTP2) and TYIL55 is detected by the fluorescent quantitative PCR. SD5 The results show that SD5 The expression of the gene in the complementary lines is significantly higher than that in TYIL55. The phenotype investigation on the obtained T2 complementary lines and the transformation receptor shows that, compared with the control, the complementary lines have the increased expression of the candidate gene, the increased plant height of about 10.9%, the increased grain number of the main stem ear of about 48.9%, the increased thousand-grain weight of about 4.3% due to the longer grain, and the increased yield per plant of about 46.0%.

[0020] The application also provides SD5 application of the gene in dwarfing the plant height and improving the yield of rice, and the transgenic vector is constructed by cloning the promoter sequence of the gene and DEP1 the coding region sequence of the gene, so that the rice with the dwarfed plant height and the improved yield is obtained. SD5 The application also providesDEP1 The promoter of the gene drives SD5 The transgenic rice of the gene. The transgenic rice has decreased plant height, increased grain number per panicle, increased 1000-grain weight and increased yield per plant. The present application utilizes the promoter with high expression in young panicle pDEP1 drive SD5 High expression in young panicle, the recombinant plasmid is transformed into ZH17 by constructing a related vector, the young panicle and internode pairs in the T3 positive transgenic line and the transformation receptor (ZH17) are selected SD5 Quantitative analysis, the results show that compared with the transformation receptor ZH17, SD5 The expression amount in the young panicle of the transgenic line is improved, and the expression amount in the internode is reduced to a certain extent. Further phenotype identification finds that compared with the transformation receptor, the plant height of the transgenic line is decreased by 8.1%, the increase of the number of panicle branches leads to the increase of the grain number per panicle of the main stem by 9.6%, the larger grain leads to the increase of the 1000-grain weight by 4.3%, the effective panicle number and the seed setting rate have no significant difference, and finally the yield per plant is increased by 15.9%.

[0021] The present application provides a method for dwarfing plant height and improving rice yield, preferably comprising the following steps: The coding region sequence of the gene and the promoter of the gene are connected to the overexpression vector pCAMBIA1300 by the method of homologous recombination to obtain a recombinant plasmid pDEP1::SD5. SD5 DEP1 The recombinant plasmid pDEP1::SD5 is transformed into the competent cells of agrobacterium, and the callus of the rice receptor material is infected to regenerate the transgenic rice plant.

[0022] In the present application, the coding region sequence of the gene and the promoter of the gene are connected to the overexpression vector pCAMBIA1300 by the method of homologous recombination to obtain a recombinant plasmid pDEP1::SD5. DEP1 ​

[0023] In the present application, the nucleotide sequence of the forward primer for amplifying the coding region sequence of the gene is GAGGGGTGGTTCTGAGTTGGATGCATGCATCTCCTCACCCAT (SEQ ID NO. 14), and the nucleotide sequence of the reverse primer is GCCAAGCTTGCATGCCTGCAGCTAGACGGTGGCCGCCTGGGTC (SEQ ID NO. 15). SD5 In the present application, the nucleotide sequence of the forward primer for amplifying the coding region sequence of the gene is GAGGGGTGGTTCTGAGTTGGATGCATGCATCTCCTCACCCAT (SEQ ID NO. 14), and the nucleotide sequence of the reverse primer is GCCAAGCTTGCATGCCTGCAGCTAGACGGTGGCCGCCTGGGTC (SEQ ID NO. 15). DEP1 In the present application, the nucleotide sequence of the forward primer for amplifying the promoter sequence of the gene is GAATTCGAGCTCGGTACCCGGGGATCCGTGAATCCGCCACGCTAACC (SEQ ID NO. 17), and the nucleotide sequence of the reverse primer is ATGGGTGAGGAGATGCATGCATCCAACTCAGAACCACCCCTC (SEQ ID NO. 18).

[0024] In the present application, preferably, the restriction enzyme sites of the complementary vector are selected from the group consisting of H Ⅰ, and Bam H Ⅰ and Pst Ⅰ as the restriction enzyme sites of the complementary vector to cleave the vector pCAMBIA1300, and the cDNA of NA93-11 is used as the template to perform PCR amplification with TKS high-fidelity enzyme to obtain a 1338bp DNA fragment A; the DNA of ZH17 is used as the template to amplify the promoter sequence with TKS high-fidelity enzyme to obtain a DNA fragment B of about 2000bp. The DNA fragment A and the DNA fragment B are connected to the overexpression vector pCAMBIA1300 in a way of homologous recombination to obtain the recombinant plasmid pDEP1::SD5. DEP1 In the present application, the total volume of the reaction system of the connection is preferably 10µL, which respectively contains: 100ng·µL -1 pCAMBIA1300 1µL, 50ng·µL -1 fragment A 1µL, 50ng·µL -1 fragment B 1µL, 2×Assembly MasterMix recombinase 5μL, and double-distilled water 2μL.

[0025] In the present application, the E. coli is transformed after the connection is completed. The specific steps are preferably as follows: the DH5a competence is placed on ice to be thawed; the recombination system liquid is sucked into the competence in the super-clean table, and is inserted into ice for 15-20 min; the competence with the recombination system liquid is placed into a 42℃ water bath, and is heat shocked for 90 s, and then is inserted into ice for 2-3 min; 600 μL of LB liquid medium is added into the competence in the super-clean table, and is placed into a shaker, 37℃, 220 rpm, and is shaken for 45-60 min; the shaken competence is placed into a centrifuge, 12000 rpm, and is centrifuged for 30-60 s, and the supernatant is sucked into a tube in the super-clean table to reserve 100 μL of liquid; the remaining 100 μL of liquid is sucked and beaten to be mixed, and is plated, and is darkly cultured at 37℃ for 12-14 h; a single colony is picked up with a gun head into LB liquid medium, 37℃, 220 rpm, and is shaken for 6-8 h, and the shaken bacterial liquid is sent to a company for sequencing of the target fragment; through sequencing analysis, 100 μL of the connected successful bacterial liquid is sucked into LB liquid medium to shake bacteria for 6-8 h, and the connected successful bacterial liquid is preserved with 50% glycerol in the super-clean table, and is placed into a -80℃ refrigerator.

[0026] In the present application, the successfully constructed recombination plasmid pDEP1::SD5 is transformed into Agrobacterium tumefaciens, and the specific steps are preferably as follows: the EHA105 competence is placed on ice to be thawed; the recombination plasmid pDEP1::SD5 is sucked into the competence in the super-clean table, and is inserted into ice for 30 min, and then is quickly frozen in liquid nitrogen for 2 min, and then is transferred into a 37℃ water bath, and is heat shocked for 5 min, and then is inserted into ice for 5 min; 600 μL of LB liquid medium is added into the competence in the super-clean table, and is placed into a shaker, 28℃, 220 rpm, and is shaken for 3-6 h; the shaken competence is placed into a centrifuge, 12000 rpm, and is centrifuged for 30-60 s, and the supernatant is sucked into a tube in the super-clean table to reserve 100 μL of liquid; the remaining 100 μL of liquid is sucked and beaten to be mixed, and is plated, and is darkly cultured at 28℃ for 1-2 days, and the plate is shaken to shake bacteria, and then 50% glycerol is used to preserve bacteria and is placed into a -80℃ refrigerator.

[0027] In the present application, the callus of the rice receptor material infected by the above-mentioned Agrobacterium is regenerated, subcultured to obtain a transgenic rice plant, and the transgenic rice plant has the characteristics of decreased plant height, increased grain number of the main stem ear, increased thousand-grain weight, and increased yield per plant.

[0028] The technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limitations to the scope of protection of the present application.

[0029] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all commercially available products unless otherwise specified.

[0030] Example 1 SD5 Cloning of genes 1. Test materials The elite indica rice line T5 was provided by the Rice Research Institute of Anhui Academy of Agricultural Sciences. The dwarf and low-yield introgression line TYIL55 with T5 as the background was constructed by the Sun Chuanqing research group of China Agricultural University. The elite super hybrid rice parent NA93-11 and the dwarf and low-yield introgression line 9WIL148 were also from the Sun Chuanqing research group of China Agricultural University. The segregation populations were constructed by backcrossing and selfing TYIL55 and 9WIL148 with T5 and NA93-11, respectively, to fine map the QTLs regulating plant height, panicle grain number, and 1000-grain weight. The japonica rice material Zhonghua 17 (ZH17) was selected as the transformation receptor of the transgenic vector.

[0031] 2. Population construction The ideal line TYIL55 was selected from the introgression line containing the Yunnan Yuanjiang common wild rice chromosome fragment introgressed into the background of T5. The F2 mapping population was constructed by further crossing and selfing TYIL55 with T5 for QTL analysis. Heterozygous single plants with only the wild rice chromosome fragment introgressed near the QTL site and the rest of the positions as the recurrent parent genetic background as much as possible were selected from the F2 population. The population was further enlarged by selecting single plants for crossing. The QTL was fine mapped while constructing near iso-genic lines (NILs) by combining the genotypes of the previous generation single plants and the phenotypic values of the next generation lines. NA93-11 and 9WIL148 were used as the mapping parents to analyze the QTLs of the F2 mapping population constructed by crossing and selfing. The QTL was fine mapped while constructing near iso-genic lines by using a similar method.

[0032] 3. Result analysis The line TYIL55 was selected from the introgression line containing the Yunnan Yuanjiang common wild rice chromosome introgressed into the background of T5. TYIL55 had introgression fragments on the 2nd, 4th, and 5th chromosomes (a in Figure 1 ), and compared with T5, the plant height was reduced by about 15 cm, the panicle grain number of the main stem was decreased by about 33.9%, the 1000-grain weight was reduced by about 7.7%, and finally the yield per plant was decreased by about 37.6% (b-f in Figure 1 ). QTL analysis was performed on the segregation population constructed by crossing and selfing T5 and TYIL55. A QTL regulating plant height and panicle grain number was found at the marker site on the 5th chromosome, which was named SD5 Semi - dwarf gene on chromosome 5 ​Among 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 SEQ ID NO. 16, the primers (the sequence of forward primer is shown as SEQ ID NO. 17, and the sequence of reverse primer is shown as SEQ ID NO. 18) are designed and synthesized, and the DNA of ZH17 is used as a template to amplify by using TKS high-fidelity enzyme DEP1 The promoter sequence, and a DNA fragment B of about 2000 bp is obtained. The DNA fragment A and the DNA fragment B are connected to the overexpression vector pCAMBIA1300 by using the method of homologous recombination to obtain the recombinant plasmid pDEP1::SD5.

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

[0044] 3. T0 generation transformation SD5 Obtaining of the transgenic rice (1) The shelled rice seeds are sterilized by using 70% alcohol and 15% NaClO, and the seeds are washed with sterilized water. After the seeds are dried, the seeds are uniformly covered on the basic medium; (2) The Agrobacterium containing the recombinant plasmid pDEP1::SD5 is transformed into the callus by using the method of immersion and staining, and then the immersion and staining callus is placed on the co-culture medium for dark culture for 3-4 days; (3) The immersion and staining callus after dark culture for 3-4 days is washed with sterile water, and then the immersion and staining callus is washed again with sterile water containing 200 mg / L cefotaxime and 200 mg / L timentin. The immersion and staining callus is placed in a sterilized culture dish with filter paper for drying, and then is transferred to the selection medium for hygromycin selection. The resistant callus with vigorous growth and golden yellow color is obtained by selection; (4) The resistant callus is transferred to the pre-differentiation medium for dark culture for about one week, and then is transferred to the differentiation medium for light culture for about one month. The differentiated seedlings are transferred to the rooting medium for rooting. After growing to a certain period, the leaf samples are taken, and the DNA is extracted for transgenic detection to obtain the T0 generation transgenic rice. SD5

[0045] 4. T3 generation transformation SD5 Obtaining of the transgenic rice and real-time quantitative PCR detection The T3 generation transgenic rice is obtained by continuously self-crossing the transgenic T0 generation plants for three generations. SD5 The T3 generation transgenic rice is named as pDEP1::SD5. The young panicles and internodes of the 2-week seedlings of the T3 generation plants are used as experimental materials. The total RNA is extracted by using the TRIZOL reagent, and then is reversely transcribed by using the Superscript II reverse transcriptase to obtain the T3 generation transgenic rice. SD5 ​cDNA 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 Use of a gene in dwarfing plant height and increasing rice yield, characterized in that, A transgenic rice plant is obtained by cloning DEP1 the promoter sequence of a gene and SD5 the coding region sequence of a gene to construct a transgenic vector, and DEP1 driving the expression of SD5 the gene in the transgenic rice.

2. Use according to claim 1, characterized in that, 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.

3. Use according to claim 1, characterized in that, The DEP1 The nucleotide sequence of the promoter of the gene is shown as SEQ ID NO.

16.

4. Use according to claim 1, characterized in that, The transgenic rice has decreased plant height, increased grain number per panicle, increased 1000-grain weight and increased yield per plant.

5. A method for dwarfing plant height and increasing yield in rice, characterized by, The method comprises the following steps: The gene coding region sequence of claim 1 is connected to the promoter of the gene of claim 2 by homologous recombination to obtain a recombinant plasmid pDEP1::SD5. SD5 gene coding region sequence, DEP1 The promoter of the gene is jointly connected to the overexpression vector pCAMBIA1300 to obtain a recombinant plasmid pDEP1::SD5. The recombinant plasmid pDEP1::SD5 is transformed into the Agrobacterium competent cell, and the callus of the rice recipient material is infected to regenerate the transgenic rice plant.

6. The method of claim 5, wherein, The transgenic rice plant has decreased plant height, increased grain number per panicle, increased 1000-grain weight and increased yield per plant.

7. The method of claim 5, wherein, 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.

8. The method of claim 5, wherein, Used to amplify DEP1 The nucleotide sequence of the forward primer for the gene promoter is shown in SEQ ID NO.17, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

18.

9. The method of claim 5, wherein, The Agrobacterium is Agrobacterium tumefaciens EHA105.

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