A primer for detecting specific InDel marker of rice OsSTP14 gene and its application

Abstract

The invention discloses a primer for detecting a specific InDel marker of rice OsSTP14 gene and its application, belonging to the technical field of plant molecular biology. The specific InDel marker primer of the invention comprises a forward primer OsSTP14-iF: CGCAATTATTTGACACGG and a reverse primer OsSTP14-iR: CGAAGTAGAGGGAGGAGGT. The specific InDel marker primer can be used to accurately screen rice germplasm with a high fruiting rate through ordinary PCR, which is of great significance for promoting high-yield rice breeding and accelerating the breeding process.

Description

Technical Field

[0001] The invention relates to a primer for detecting a specific InDel marker of rice OsSTP14 gene and application thereof, belonging to the technical field of plant molecular biology. Background Art

[0002] Sugars are important sources of carbon and energy for organisms, as well as important signaling molecules, and mainly include monosaccharides, disaccharides, and polysaccharides. Among them, monosaccharides are the main energy source and metabolic intermediate of living cells. Plants can produce monosaccharides through photosynthesis, and then, with the assistance of monosaccharide transporters (MSTs), they are transported to "sink" organs for absorption and utilization (Doidy et al., 2012).

[0003] The proteins that mediate the transmembrane transport of various monosaccharides in plants are collectively called monosaccharide transporters (MSTs). Among them, hexose transporters (STPs) are the main members of the monosaccharide transporter family. They have a broad spectrum of substrate sugar absorption characteristics and have transport functions for hexoses such as glucose, fructose, pentose, ribose and galactose (Julius et al., 2017; Johnson and Thomas, 2007).

[0004] At present, a total of 28 STPs genes have been identified in rice, OsSTP1-OsSTP28 (Geiger, 2020). The reported OsSTPs genes are specifically expressed in various sink organs of rice, mainly by regulating the rate and efficiency of sugar absorption and utilization in rice sink organs, affecting the distribution of sugars in heterotrophic organs such as rice flower organs, seeds, and roots (Toyofuku et al., 2000; Li et al., 2024; Ngampanya et al., 2003). Among them, OsSTP14, as an important member of the OsSTPs gene family, is mainly highly expressed in the panicle during the filling period (milky stage), has a regulatory effect on the transmembrane transport of monosaccharides during grain development, and is an important determinant of rice grain fullness and fruit setting rate (Kong et al., 2019). Therefore, the OsSTP14 gene is closely related to the fruit setting rate of rice. At the same time, OsSTP14 and its orthologous genes are also important candidate genes in plant breeding for various resistances such as cold resistance and drought resistance (Deng et al., 2019).

[0005] In recent years, with the population growth and land degradation, the issue of food security has become increasingly prominent, and high-yield rice breeding is still an inevitable trend in the development of modern agriculture. At present, the research on the regulatory genes and mechanisms of yield traits such as rice grain weight and effective panicle number has been relatively in-depth. However, as an important component of yield, due to its complex regulatory network, research on fruit set rate is still in urgent need of advancement. In traditional rice breeding, the investigation and breeding of high and low fruit set rates can only be conducted through seed testing statistics, and can only be carried out during the rice maturity period, resulting in high breeding time and labor costs. However, by discovering and developing molecular markers related to rice fruit set rate, molecular marker screening can be performed at any period and part of rice growth, which greatly improves the efficiency of breeding rice varieties with high fruit set rate, and also provides a theoretical basis for discovering and cloning more fruit set rate-related genes and analyzing their genetic mechanisms, which is of great significance to high-yield rice breeding.

[0006] The present invention is based on the discovery of a 287bp InDel sequence in the second intron region of the OsSTP14 gene in the study of the OsSTP14 gene, and found that the OsSTP14 gene of the rice material containing the 287bp fragment has a relatively high relative expression level in the panicle (milky stage), a high glucose content in the panicle, and a relatively high fruit setting rate; on the contrary, the OsSTP14 gene of the rice material lacking the 287bp fragment has a relatively low relative expression level in the panicle (milky stage), and the glucose content and fruit setting rate of the panicle are also relatively low. Therefore, the OsSTP14 gene sequence is searched through the NCBI (https: / / www.ncbi.nlm.nih.gov / ) rice genome database, and the specific primers OsSTP14-iF and OsSTP14-iR that can amplify the InDel sequence fragment are designed using the software Primer 5.0. Then, the InDel marker is amplified using PCR technology, so that the fruit setting rate of the rice material can be accurately and quickly identified.

[0007] References

[0008] Deng X,An B,Zhong H,et al.ANovel Insight into Functional Divergenceofthe MST Gene Family in Rice Based on Comprehensive Expression Patterns[J].Genes(Basel),2019,10(3):239.

[0009] Doidy J,Grace E,Kühn C,et al.Sugar transporters in plants and intheir interactions with fungi[J].Trends Plant Sci,2012,17(7):413-422.

[0010] Geiger D.Plant glucose transporter structure and function[J].PflugersArch,2020,472(9):1111-1128.

[0011] Johnson DA,Thomas MA.The monosaccharide transporter gene family inArabidopsis and rice:a history of duplications,adaptive evolution,andfunctional divergence[J].Mol Biol Evol,2007,24(11):2412-2423.

[0012] Julius BT,Leach KA,Tran TM,et al.Sugar Transporters in Plants:NewInsights andDiscoveries[J].Plant Cell Physiol,2017,58(9):1442-1460.

[0013] Kong W,An B,Zhang Y,et al.Sugar Transporter Proteins(STPs)inGramineae Crops:Comparative Analysis,Phylogeny,Evolution,and ExpressionProfiling[J].Cells,2019,8(6):560.

[0014] Ngampanya B,SobolewskaA,Takeda T,et al.Characterization of ricefunctional monosaccharide transporter,OsMST5[J].Biosci Biotechnol Biochem,2003,67(3):556-562.

[0015] Li M, Li H, Zhu Q, et al. Knockout of the sugar transporter OsSTP15enhances grain yield by improving tiller number due to increased sugarcontent in the shoot base of rice (Oryza sativa L.) [J]. New Phytol, 2024, 241(3): 1250-1265.

[0016] Toyofuku K, Kasahara M, Yamaguchi J. Characterization and expression of monosaccharide transporters (OsMSTs) in rice [J]. Plant Cell Physiol, 2000, 41(8):940-947. Summary of the invention

[0017] The present invention provides a PCR method and specific primers thereof, which are simple to operate, require less sample, and can quickly and accurately identify the high and low fruiting rates of rice germplasm materials at any stage of rice growth and development. By this method, rice germplasm with a high fruiting rate can be accurately screened, which is of great significance for promoting high-yield rice breeding and accelerating the breeding process.

[0018] The primers provided by the present invention for detecting the specific InDel marker of the rice OsSTP14 gene include a forward primer OsSTP14-iF and a reverse primer OsSTP14-iR of the following sequence:

[0019] Forward primer OsSTP14-iF: CGCAATTATTTGACACGG

[0020] Reverse primer OsSTP14-iR: CGAAGTAGAGGGAGGAGGT.

[0021] The present invention also provides an application of the specific InDel molecular marker primer in screening rice with a high fruit-setting rate.

[0022] The specific operation steps of the above application are as follows:

[0023] (1) Extract the total DNA of the rice germplasm to be tested.

[0024] (2) Using the genomic DNA obtained in step (1) as a template, PCR amplification is performed using forward primer OsSTP14-iF and reverse primer OsSTP14-iR to obtain a PCR product.

[0025] (3) Detect the PCR product obtained in step (2) using agarose gel electrophoresis.

[0026] If the PCR amplification product of the rice germplasm to be tested is a 1346 bp band, it indicates that the seed setting rate of the germplasm is high; if the PCR amplification product of the rice germplasm to be tested is a 1059 bp band, it indicates that the seed setting rate of the germplasm is low.

[0027] Preferably, the PCR reaction system in step (2) is: 0.5 μL forward primer, 0.5 μL reverse primer, 7.5 μL 2×Taq PCR Master Mix, 5.5 μL ddH2O, and 1 μL total DNA with a concentration of 10 ng / μL.

[0028] Preferably, the PCR amplification program in step (2) is: pre-denaturation at 94°C for 4 min; then denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 45 s, 30 cycles; extension at 72°C for 5 min; and storage at 10°C.

[0029] Preferably, 5 μL of the PCR product in step (3) is electrophoresed on a 1.5% agarose gel at a voltage of 110 V for 35 min, then stained with ethidium bromide (EB) and photographed under a gel imaging system.

[0030] Compared with the prior art, the main innovations and beneficial effects of the present invention are:

[0031] (1) The detection of rice fruit set rate using this method is not limited by the rice growth period. At any period, conventional PCR technology and trace DNA samples can be used to accurately identify the high or low fruit set rate of rice germplasm materials, quickly screen out rice germplasm with high fruit set rate, and accelerate the efficiency of high-yield variety breeding.

[0032] (2) A new method is provided for identifying the high and low fruit set rate of rice, eliminating the previous limitation that the fruit set rate can only be identified through seed testing after the rice is harvested; and when the test population is large, the traditional seed testing method requires a high investment of time and labor costs. This method can be used to test a large number of rice germplasm materials at the same time, which can greatly reduce the workload of germplasm screening and has the advantages of simple operation, accuracy, and speed. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The electrophoresis diagram of the PCR product in Example 1, M is the DNA molecular weight standard.

[0034] Figure 2 This is the sequencing comparison result of the PCR products in Example 1.

[0035] Figure 3 The relative expression level of the OsHXK3 gene in Example 1 is detected, and ** indicates significant difference at the 0.01 level.

[0036] Figure 4 The results of glucose content detection in Example 1 and the corresponding electrophoresis diagram of PCR products, ** indicates significant difference at the 0.01 level; M is a DNA molecular weight standard, and the molecular weights of the two PCR fragments in the figure are 1346 bp (large molecular weight) and 1059 bp (small molecular weight), respectively.

[0037] Figure 5 The main panicle morphology and grains of different rice materials in Example 1 (the upper ones are empty grains, and the lower ones are full grains).

[0038] Specific implementation

[0039] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the technical solutions, method steps or conditions of the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in each example are conventional means well known to those skilled in the art; the reagents used in each example are commercially available.

[0040] 1. Materials used in the test

[0041] Table 1 Tested rice varieties

[0042]

[0043]

[0044] All materials in the table were provided by Rice Research Institute of Yunnan Agricultural University.

[0045] 1.93-11 Public literature: Zhang GH, Li SY, Wang L et al. LSCHL4 from JaponicaCultivar, which is allelic toNAL1, increases yield of indica super rice 93-11[J]. Molecular Plant, 2014, 7(8): 1350-1364.

[0046] 2.Nipponbare open literature: Deng Y, Pan Y, Luo M, Detection and correction of assembly errors of rice Nipponbare reference sequence[J]. Plant Biol(Stuttg),2014,16(3):643-650.

[0047] 3. Hua Er Zao open literature: Wen Guosong, Dai Luguo, Chen Yong, et al. Identification and analysis of the wide compatibility of rice seed resources in Yunnan [J]. Hybrid Rice, 1994, S1:4-5+8.

[0048] 4. Lengshuigu Open Literature: Kou Shuyan, Tan Yaling, Li Jun, et al. Genetic diversity of old rice varieties in Xishuangbanna, China at the fertility restorer gene Rf-1 locus [J]. Molecular Plant Breeding, 2009, 7(4): 653-660.

[0049] 5. Dian Tun 506 Public Document: Liu Ying. Analysis of aroma and taste quality of improved aromatic rice varieties [D]. Yunnan Agricultural University, 2022.

[0050] 6. Niubagu, Beizigu, Langcang Dabaigu, Menglian Xiaohonggu, Huangpinuo. Public literature: Zhang Chunlong. Distribution of 31-bp InDel in 5'UTR of sucrose transport gene OsSUT3 in rice and its expression regulation characteristics[D]. Yunnan Agricultural University, 2023.

[0051] Example 1 Application of a primer for detecting rice OsSTP14 gene-specific InDel marker in screening high-fruiting rate rice germplasm

[0052] A specific InDel marker primer for detecting the rice OsSTP14 gene comprises a forward primer OsSTP14-iF and a reverse primer OsSTP14-iR as shown in Table 2.

[0053] Table 2 Specific InDel marker primers for detecting rice OsSTP14 gene

[0054] Primer name Base sequence OsSTP14-iF CGCAATTATTTGACACGG OsSTP14-iR CGAAGTAGAGGGAGGAGGT

[0055] (1) DNA extraction

[0056] The CTAB method (Rogers and Bendich, 1985) was used to extract the whole genome DNA from the leaves of all rice materials in Table 1. The specific method is as follows:

[0057] A. Take about 0.5g of rice leaves and put them into a 1.5mL centrifuge tube. Add liquid nitrogen to freeze and grind. Add 600μL of 2×CTAB buffer to the ground powder. Put it in a 65℃ water bath for 45min and mix it every 15min.

[0058] B. Take out the centrifuge tube and add an equal volume of chloroform / isoamyl alcohol (24:1), invert to mix, and centrifuge at 10,000 rpm for 10 minutes.

[0059] C. Pipette the upper aqueous phase into a new 1.5 mL centrifuge tube, add 600 μL of chloroform / isoamyl alcohol (24:1), invert to mix, and centrifuge at 10,000 rpm for 10 min.

[0060] D. Carefully transfer the upper aqueous phase to a new centrifuge tube, add 2-3 times the volume of ice-cold anhydrous ethanol, and mix by inverting until a flocculent precipitate is formed.

[0061] E. Use a sterilized toothpick to pick out the flocculent precipitate and wash it twice with 70% ethanol. Use sterilized filter paper to absorb the ethanol and dry it naturally to allow the ethanol to evaporate completely.

[0062] F. Add 100 μL of TE solution to dissolve the DNA, take a portion and test it by agarose gel electrophoresis, then store it in a -20℃ refrigerator for later use.

[0063] (2) PCR amplification

[0064] The whole genome DNA of different rice varieties stored in step (1) was used as a template, and PCR amplification was performed using the primer pairs shown in Table 2 to obtain PCR products. The PCR reaction system is shown in Table 3, and the PCR reaction procedure is shown in Table 4.

[0065] Table 3 15 μL PCR reaction system

[0066] Reagents Volume (μL) 2×TaqPCRMasterMix 7.5 OsSTP14-iF (10pM / μL) 0.5 OsSTP14-iR (10pM / μL) 0.5 Whole genome DNA (10ng / μL) 1 <![CDATA[ddH2O]]> 5.5

[0067] Table 4 PCR amplification program

[0068] step Temperature(℃) time ① 94 4min ② 94 30s ③ 57 30s ④to②30cycle 72 45s ⑤ 72 5min

[0069] (3) Agarose gel electrophoresis and detection

[0070] Take 5 μL of PCR product, use 1.5% agarose gel, voltage 110V electrophoresis for 35 minutes, then ethidium bromide (EB) staining, and take pictures under the gel imaging system.

[0071] The electrophoresis results are as follows Figure 1 As shown in the figure, it can be seen that the molecular weight of the amplified products of 93-11, Diantun 506, Beizigu, Nipponbare and Huangpinuo is larger, while the molecular weight of the amplified products of Niubagu, Langcang Dabaigu, Lengshuigu, Huaerzao and Menglian Xiaohonggu is smaller.

[0072] (4) PCR product sequencing

[0073] Subsequently, the PCR amplification products with different molecular weights in step (3) were sequenced at Shanghai Shenggong Biotechnology Technology Service Co., Ltd., and the comparison results were as follows: Figure 2 As shown, it was found that 93-11, Diantun 506 and Nipponbare had a 287bp InDel fragment compared with Langcang Dabai Valley, Lengshui Valley and Menglian Xiaohong Valley. Among them, the PCR amplification product of OsSTP14-i in 93-11, Diantun 506 and Nipponbare had a larger molecular weight of 1346bp; the PCR amplification product of OsSTP14-i in Langcang Dabai Valley, Lengshui Valley and Menglian Xiaohong Valley had a smaller molecular weight of 1059bp ( Figure 1 ).

[0074] (5) Detection of relative expression of OsSTP14 gene

[0075] The relative expression levels of the OsSTP14 gene in the panicles of six rice materials (93-11, Diantun 506, Nipponbare, Langcang Dabaigu, Lengshuigu and Menglian Xiaohonggu) at the milky stage were detected. The specific steps are as follows:

[0076] A. Total RNA extraction was performed according to the instructions of the GenStar Total RNA Extraction Kit (P118).

[0077] B. Reverse transcribe the total RNA extracted in step A to obtain cDNA. The specific operation is carried out according to the instructions of StarScript III Reverse Transcription Kit (A232-10) of GenStar Company.

[0078] The β-Actin gene was used as the internal reference gene, and the specific primers used for fluorescence quantification are shown in Table 5.

[0079] Table 5 qRT-PCR primer sequences

[0080]

[0081] PCR reaction system: refer to GenStar's 2× premixed real-time fluorescence quantitative rapid PCR reaction system kit (A304).

[0082] PCR amplification conditions: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing at 61°C for 1 min, and 40 cycles.

[0083] The final result is obtained using 2 -ΔΔCt The calculation and analysis are as follows Figure 3 As shown in the figure, it can be seen that the relative expression level of OsSTP14 gene in the ear of 93-11, Diantun 506 and Nipponbare at the milky stage is higher, and is significantly higher than the relative expression level of OsSTP14 gene in Langcang Dabai Valley, Lengshui Valley and Menglian Xiaohong Valley.

[0084] (6) Determination of glucose content in ear

[0085] The glucose content in the panicle (milky stage) of the six rice materials (93-11, Diantun 506, Nipponbare, Langcang Dabaigu, Lengshuigu and Menglian Xiaohonggu) in operation (4) was measured using the glucose determination kit (G0504F) produced by Suzhou Grace Biotechnology Co., Ltd., with 5 biological replicates for each material.

[0086] The results of the test are as follows Figure 4 As shown, combined with the detection results of step (3) and step (4), it was found that in different rice germplasms, the rice materials with larger molecular weight fragments (1346bp) obtained by PCR amplification using OsSTP14-i primers had higher glucose content in the panicles; and the rice materials with smaller molecular weight fragments (1059bp) had lower glucose content in the panicles, and there was a very significant difference between the two.

[0087] (7) Fruit setting rate statistics and correlation analysis

[0088] Finally, the fruit setting rate of the rice materials in Table 1 was statistically analyzed, and the glucose content of the panicles of all the rice materials in Table 1 was measured by referring to the method in operation (6). SPSS software was used to perform a correlation analysis between the glucose content of the rice panicles and the fruit setting rate by using the bivariate Spearman test method. The fruit setting rate was calculated according to the formula: The calculation showed that 15 plants were investigated for each material. The statistical and measurement results are shown in Table 6. The main panicle morphology of different rice materials is as follows: Figure 5Table 6 shows that the fruit setting rate and ear glucose content of Niubagu, Langcang Dabaigu, Lengshuigu, Huaerzao and Menglian Xiaohonggu are significantly lower than those of 93-11, Diantun 506, Beizigu, Niponqing and Huangpinuo. The results of the bivariate Spearman test show that the ear glucose content is significantly positively correlated with the fruit setting rate (r = 0.915, P = 0.0002 < 0.01).

[0089] In summary, the relative expression of the OsSTP14 gene in the panicle of the milky stage of the rice material with the amplified product of 1346 bp was higher, the glucose content in the panicle was higher, and the fruit setting rate was also relatively high; on the contrary, the relative expression of the OsSTP14 gene in the panicle of the milky stage of the rice material with the amplified product of 1059 bp was higher, the glucose content in the panicle was lower, and the fruit setting rate was also relatively low. Therefore, the method of the present invention can be used to screen rice germplasm with a high fruit setting rate.

[0090] Table 6 Statistical results of fruit setting rate of tested rice materials

[0091]

[0092] Note: ** indicates significant difference at the 0.01 level.

Claims

1. A method for detecting rice OsSTP14 The application of primers of a gene in screening rice germplasm with high seed setting rate is characterized by: The primers include a forward primer OsSTP14-iF and a reverse primer OsSTP14-iR of the following sequence: Forward primer OsSTP14-iF: CGCAATTATTTGACACGG; Reverse primer OsSTP14-iR: CGAAGTAGAGGGAGGAGGT; The rice germplasms include: 93-11, Diantun 506, Beizigu, Nipponbare, Huangpinuo, Niubagu, Langcang Dabaigu, Lengshuigu, Huaerzao and Menglian Xiaohonggu; If the PCR amplification product of the rice germplasm to be tested is a 1346 bp band, it indicates that the seed setting rate of the germplasm is high; if the PCR amplification product of the rice germplasm to be tested is a 1059 bp band, it indicates that the seed setting rate of the germplasm is low.

2. The use according to claim 1, characterized in that: The specific steps include: (1) Extracting total DNA from the rice germplasm to be tested; (2) using the genomic DNA obtained in step (1) as a template, performing PCR amplification using forward primer OsSTP14-iF and reverse primer OsSTP14-iR to obtain a PCR product; (3) Detect the PCR product obtained in step (2) using agarose gel electrophoresis.

3. The use according to claim 2, characterized in that: The PCR reaction system in step (2) is: 0.5 μL forward primer, 0.5 μL reverse primer, 7.5 μL 2×Taq PCR Master Mix, 5.5 μL ddH2O, and 1 μL total DNA with a concentration of 10 ng / μL.

4. The use according to claim 2, characterized in that: The PCR amplification program in step (2) is: pre-denaturation at 94°C for 4 min; then denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 45 s, 30 cycles; extension at 72°C for 5 min; and storage at 10°C.

5. The use according to claim 2, characterized in that: Take 5 μL of the PCR product in step (3), run it on a 1.5% agarose gel at 110 V for 35 min, then stain it with ethidium bromide (EB) and take a picture under a gel imaging system.

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