CN114085847A - Rice OsSUT3 gene mutant and molecular identification method and application thereof - Google Patents

Abstract

A rice OsSUT3 gene mutant and a molecular identification method and application thereof are provided, the rice OsSUT3 gene mutant belongs to natural mutation, and the nucleotide sequence is shown as SEQ ID NO: shown at 11. Constructing a near-allelic gene system NIL-OsSUT3 of rice OsSUT3 mutant gene by using cold paddy containing rice OsSUT3 gene mutant as a donor parent^MThe method creates an important research material for deeply exploring the biological functions of the OsSUT3 gene of the rice, and has good application prospect in transformation of new germplasm and improvement of rice varieties. The molecular identification method and the specific primer thereof can quickly and accurately screen or detect the rice pollen sterility character, and provide technical support for the cultivation of sterile materials for hybrid seed production.

Description

Rice OsSUT3 gene mutant and molecular identification method and application thereof

Technical Field

The invention belongs to the field of crop genetic breeding, and particularly relates to an OsSUT3 gene mutant with low rice pollen fertility and low fruiting rate, and a molecular identification method and application thereof.

Background

In higher plants, nearly 80% of the photosynthetic products are transported from the "source" organ to the "sink" organ of heterotrophic tissue such as roots, stems, flowers, seeds^[1]. Sucrose is the major form of carbohydrate transport over long distances between organs in the source reservoir. Studies in various plants have found that sucrose transport across membranes and its distribution in plants require sucrose transporters (SUTs) for mediation. SUT is a transmembrane binding protein with sucrose transport activity, transporting sucrose/H by co-operation⁺Enter phloem and then are transported to the reservoir organs through the conduit of xylem for a long distance, thereby realizing the reasonable distribution of sucrose among the source reservoir organs^[2]. The source substances required for plant organ development and energy storage are mainly distributed to various heterotrophic organs by SUT. Both the decline in the rate of SUT transport and the disruption of regulatory mechanisms will therefore affect the uptake of energy by heterotrophic organs, ultimately leading to organ (tissue) dysplasia, hypofunction or loss. In crops, variation of the SUT gene is directly closely related to yield.

In rice, 5 SUT family member genes, OsSUT1-OsSUT5, were identified in total^[3]. Among them, OsSUT1 is the first SUT gene cloned and identified in monocotyledons^[4]. Although the OsSUTs gene is cloned earlier, due to its complex regulatory network, the study of the function of its family genes is not comprehensive and deep enough. Most of reported OsSUTs gene functions influence the characters of rice seed germination, vegetative growth, grain size and the like by coordinating the speed and efficiency of sucrose transport^[5,6,7]. In addition, the OsSUTs gene can enhance the resistance of rice to abiotic stress such as high salt, high temperature and the like^[8,9]Has important effect on the aspects of adaptability to iron deficiency, high aluminum and other environments^[10]。

OsSUT3 as an important member of OsSUTs gene family, and research on OsSUT3 at presentThe expression level of the gene in a plurality of tissues (leaves, roots, stems and ears) of rice is relatively low from the aspect of the expression mode; however, OsSUT3 continuously maintains high expression level from 1-2 days before flowering to 5-7 days after flowering of rice; particularly in the early flowering stage, the OsSUT3 gene is specifically expressed in rice pollen in high quantity^[5](ii) a This shows that the expression of OsSUT3 has temporal and spatial specificity, and high expression specificity in rice pollen, and the gene is presumed to play an important role in late pollen development, pollination and fertilization^[11,12,13]. On the other hand, pollen development is always a hot research problem concerned by breeding workers, and the molecular mechanism of the OsSUT3 gene in the pollen development process is disclosed to be of great significance for analyzing the molecular mechanism of pollen development and improving varieties. However, the homology of OsSUT3 and OsSUT1 gene sequence is as high as 78%^[3]It is very difficult to artificially construct a mutant strain of OsSUT3 gene^[5]No success has been found in previous studies, which has posed some difficulty in studying the function and molecular mechanism of ossit 3.

With the development of molecular biology, near-allelic lines (NIL) are more and more widely applied, and the same or similar genetic backgrounds of NIL differ only in target trait gene segments, so that NIL is an ideal material for genetic research at a gene level. Meanwhile, the completion of the rice whole genome sequencing lays a solid theoretical and technical foundation for establishing an OsSUT3 gene polymorphism screening system.

Reference documents:

[1]Julius B T,Leach K A,Tran T M,et al.Sugar transporters in plants:new insights and discoveries[J].Plant Cell Physiol,2017,58(9):1442-1460.

[2]Kühn C,Grof C P.Sucrose transporters of higher plants[J].Current Opinion in Plant Biology,2010,13(3):287-297.

[3]Aoki N,Hirose T,Scofield G N,et al.The sucrose transporter gene family in rice[J].Plant Cell Physiology,2003,44(3):223-232.

[4]Hirose T,Imaizumi N,Scofield G N,et al.cDNA cloning and tissue specific expression of a gene for sucrose transporter from rice(Oryza sativa L.)[J].Plant Cell Physiology,1997,38(12):1389.

[5]Hirose T,Zhang Z,Miyao A,et al.Disruption of a gene for rice sucrose transporter,OsSUT1,impairs pollen function but pollen maturation is unaffected[J].Journal of Experimental Botany,2010,61(13):3639-3646.

[6]Scofield G N,Aoki N,Hirose T,et al.The role of the sucrose transporter,OsSUT1,in germination and early seedling growth and development of rice plants[J].Journal of Experimental Botany,2007,58(3):483-495.

[7]Zhang J,Li D,Xu X,et al.The potential role of sucrose transport gene expression in the photosynthetic and yield response of rice cultivars to future CO₂ concentration[J].Physiol Plant,2020,168(1):218-226.

[8]Siahpoosh M R,Sanchez D H,Schlereth A,et al.Modification of OsSUT1 gene expression modulates the salt response of rice Oryza sativa cv.Taipei 309[J].Plant Science,2012,182:101-111.

[9]Miyazaki M,Araki M,Okamura K,Ishibashi Y,Yuasa T,et al.Assimilate translocation and expression of sucrose transporter,OsSUT1,contribute to high-performan ce ripening under heat stress in the heat-tolerant rice cultivar Genkitsukushi[J].Plant Physiol,2013,170(18):1579-1584.

[10]Chen P F,Chen L,Jiang Z R,et al.Sucrose is involved in the regulation of iron deficiency responses in rice(Oryza sativa L.)[J].Plant Cell Rep,2018,37(5):789-798.

[11]Takeda T,Toyofuku K,Matsukura C,et al.Sugar transporters involved in flowering and grain development of rice[J].Plant Physiology,2001,158(4):465-470.

[12]Ngampanya B,Takeda T,Narangajavana J,et al.Sugar Transporters involved in flowering and grain development of rice[J].Applied Glycoscience,2003,50:237-240.

[13]Li D D,Xu R C,Lv D,et al.Identification of the core pollen-specific regulation in the rice OsSUT3 promoter[J].International Journal of Molecular Sciences,2020,21(6):1909.

[14]Clarke J D.Cetyltrimethyl ammonium bromide(CTAB)DNA miniprep for plant DNA isolation[J].Cold Spring Harb Protoc,2009,3:4.

disclosure of Invention

In order to solve the technical problem that the effect and the function of the OsSUT3 gene of rice are difficult to accurately judge and deeply research in the prior art, the invention provides an OsSUT3 gene mutant with low rice pollen fertility, a molecular identification method and application thereof, and a near allele line NIL-OsSUT3 for constructing the OsSUT3 mutant gene^MThe method of (1).

The technical scheme of the invention is as follows:

the invention provides a rice OsSUT3 gene mutant, the nucleotide sequence of which is shown as SEQ ID NO: shown at 11.

The invention also provides a specific primer PriSUT3-1 for detecting the rice OsSUT3 gene mutant, wherein the specific primer PriSUT3-1 consists of a primer PriSUT3-1F and a primer PriSUT3-1R, and the nucleotide sequence of the primer PriSUT3-1F is shown as SEQ ID NO: 1, and the nucleotide sequence of the primer PriSUT3-1R is shown as SEQ ID NO: 2, respectively.

The invention also provides a molecular identification method of rice OsSUT3 gene mutation, which comprises the steps of carrying out PCR amplification on the DNA of rice by adopting a specific primer PriSUT3-1, analyzing the length of an amplification product: if the amplified product is 756bp, the OsSUT3 genotype of the rice is a wild type, and the pollen grows well; if the amplification product is 485bp, the OsSUT3 genotype of the rice is a mutant, and the pollen is dysplastic; if the amplification product is 756bp and 485bp banding patterns, the OsSUT3 genotype of the rice is a heterozygous genotype, and the pollen development is damaged; the specific primer PriSUT3-1 consists of a primer PriSUT3-1F and a primer PriSUT3-1R, and the nucleotide sequence of the primer PriSUT3-1F is shown as SEQ ID NO: 1, and the nucleotide sequence of the primer PriSUT3-1R is shown as SEQ ID NO: 2, respectively.

In the molecular identification method for OsSUT3 gene mutation of rice, the pollen is well developed, and the pollen fertility is more than or equal to 90%; the pollen dysplasia is that the pollen fertility is more than or equal to 60 percent and less than 80 percent; the pollen development damage is more than or equal to 80 percent and less than 90 percent of pollen fertility.

The invention also provides a molecular identification method for auxiliary screening or detection of rice pollen fertility traits, which comprises the step of carrying out PCR amplification on the DNA of rice by adopting a specific primer PriSUT3-1, wherein the specific primer PriSUT3-1 consists of a primer PriSUT3-1F and a primer PriSUT3-1R, and the nucleotide sequence of the primer PriSUT3-1F is shown as SEQ ID NO: 1, and the nucleotide sequence of the primer PriSUT3-1R is shown as SEQ ID NO: 2, respectively.

According to the molecular identification method for auxiliary screening or detection of rice pollen fertility traits, if the amplified product is 756bp, the OsSUT3 genotype of the rice is a wild type, and the pollen grows well; if the amplification product is 485bp, the OsSUT3 genotype of the rice is a mutant, and the pollen is dysplastic; if the amplification product is 756bp and 485bp banding patterns, the OsSUT3 genotype of the rice is heterozygous genotype, and the pollen development is damaged.

The pollen development is good and the pollen fertility is more than or equal to 90 percent by the molecular identification method for auxiliary screening or detection of the rice pollen fertility character; the pollen dysplasia is more than or equal to 60 percent and the pollen fertility is less than 80 percent; the pollen development damage is more than or equal to 80 percent and less than 90 percent of pollen fertility.

The invention also provides application of the rice OsSUT3 gene mutant in breeding rice pollen sterile materials.

In the application of the rice OsSUT3 gene mutant in breeding of rice pollen sterile materials, a specific primer PriSAT 3-1 is adopted to carry out PCR amplification on the DNA of rice, and if the amplification product is 485bp, the rice is a rice pollen sterile material with poor pollen development; the specific primer PriSUT3-1 consists of a primer PriSUT3-1F and a primer PriSUT3-1R, and the nucleotide sequence of the primer PriSUT3-1F is shown as SEQ ID NO: 1, and the nucleotide sequence of the primer PriSUT3-1R is shown as SEQ ID NO: 2, respectively.

In the application of the rice OsSUT3 gene mutant in breeding of rice pollen sterile materials, the pollen dysplasia is more than or equal to 60% and the pollen fertility is less than 80%.

The invention also provides rice OsSUT3 mutantGene near allele line NIL-OsSUT3^MThe construction method comprises the following steps:

(1) and (3) breeding of receptor parents: selfing and purifying wild indica rice variety 9311 to selfing 10 th generation as 9311F₁₀9311F₁₀As a recipient parent;

(2) breeding a donor parent: selfing with cold water grain for the 5 th generation F₅As a donor parent;

(3) field hybridization and true hybrid identification: with the acceptor parent 9311F₁₀As female parent, cold water grain F of donor parent₅Performing interspecific hybridization as male parent, germinating the interspecific hybrid seed, identifying true hybrid with specific primer PriSAT 3-1, dyeing pollen grains of true hybrid plant with iodine-potassium iodide solution, observing under optical microscope, counting pollen fertility of individual plant, and selecting pollen fertility of 80% or more<Between 90% of the recombinant individuals continue to recurrent parent 9311F₁₀Backcrossing is carried out, and the backcrossing is repeated until 6 th generation to obtain BC₆Namely rice OsSUT3 mutant gene near allele line NIL-OsSUT3^M(ii) a The specific primer PriSUT3-1 consists of a primer PriSUT3-1F and a primer PriSUT3-1R, and the nucleotide sequence of the primer PriSUT3-1F is shown as SEQ ID NO: 1, and the nucleotide sequence of the primer PriSUT3-1R is shown as SEQ ID NO: 2 is shown in the specification;

the cold water cereals in the step (2) are mutant material cold water cereals.

Compared with the prior art, the invention has the beneficial effects that:

(1) the rice OsSUT3 gene mutant belongs to natural mutation, and compared with the OsSUT3 gene of a wild type, the OsSUT3 mutant gene has 271bp deletion, wherein 77bp of the 3 'end of a first exon and 194bp of the 5' end of the first intron are included. The molecular detection can be carried out by adopting agarose gel electrophoresis commonly used in laboratories, the identification of the OsSUT3 mutant gene is realized, special detection technology and method are not needed, and the method has important significance for accurately, efficiently and quickly screening and identifying the rice OsSUT3 gene mutant and accelerating the preparation of the near-isogenic line of the OsSUT3 mutant gene.

(2) Uses cold paddy material containing rice OsSUT3 gene mutant as supplyConstructing a near allele line NIL-OsSUT3 of the rice OsSUT3 mutant gene by using the parent strain^MImportant research materials are created for the deep research of the biological functions of the OsSUT3 gene of the rice; the method has good application prospect in transformation of new germplasm and rice variety improvement, for example, a new flower variety with less pollen amount which is favored by consumers is cultivated, and reference is provided for variety improvement of important agronomic characters such as rice pollen development, seed setting rate and the like.

(3) The molecular identification method for rice OsSUT3 gene mutation and the specific primer thereof can quickly and accurately screen or detect the rice pollen sterility character, and provide technical support for the cultivation of sterile materials for hybrid seed production.

SEQ ID NO: 1 shows the nucleotide sequence of primer PriSUT 3-1F.

SEQ ID NO: 2 shows the nucleotide sequence of primer PriSUT 3-1R.

SEQ ID NO: 3 shows the nucleotide sequence of primer PriSUT 3-2F.

SEQ ID NO: 4 shows the nucleotide sequence of primer PriSUT 3-2R.

SEQ ID NO: shown in FIG. 5 is the nucleotide sequence of primer PriSUT 3-3F.

SEQ ID NO: shown in FIG. 6 is the nucleotide sequence of primer PriSUT 3-3R.

SEQ ID NO: 7 shows the nucleotide sequence of primer PriSUT 3-4F.

SEQ ID NO: shown in FIG. 8 is the nucleotide sequence of primer PriSUT 3-4R.

SEQ ID NO: shown in FIG. 9 is the nucleotide sequence of primer PriSUT 3-5F.

SEQ ID NO: shown in FIG. 10 is the nucleotide sequence of primer PriSUT 3-5R.

SEQ ID NO: 11 shows the nucleotide sequence of the rice OsSUT3 gene mutant.

SEQ ID NO: 12 shows the CDS nucleotide sequence of OsSUT3 gene in wild type rice variety 9311.

Drawings

FIG. 1: and (4) screening the polymorphism of the OsSUT3 gene of the rice. M is DNA molecular weight standard; lanes 1-13 show the bands of PCR products amplified with the specific primer PriSUT3-1 for different rice varieties of DNA, and lanes 1-13 show the numbers: 1: wild type rice variety 9311 (indicated as 9311); 2: nipponbare; 3: eight-treasure rice; 4: yunzi 41; 5: no. 9 Lijing; 6: guanghui 128; 7: cold water cereals; 8: the sixth month of the valley; 9: disorganizing the valley; 10: a locust grain; 11: yunnan miscellaneous 35; 12: china No. 1; 13: tainan No. 11; wherein, the rice germplasm resource materials shown in lanes 1-6 and lanes 8-13 are wild rice varieties: 9311. the PCR amplification products of Nipponbare, eight treasure valley, Yunzi No. 41, Lijing No. 9, Guanghui 128, Liuyue valley, disarmed valley, locust valley, Yunnan miscellaneous 35, China No. 1 and Tainan No. 11 have larger molecular weights, the bands are all 756bp and are OsSUT3 gene wild type bands, the PCR amplification product of Lane 7, namely cold water valley, has smaller molecular weight and the bands are 485bp and are OsSUT3 gene mutant amplification bands.

FIG. 2: the CDS sequence of the OsSUT3 gene of the wild type rice variety 9311 (represented by 9311) is compared with the CDS sequence of the OsSUT3 gene mutant of the rice in the cold paddy (represented by LSM). In FIG. 2, the box represents the first exon of the OsSUT3 gene. The continuous dots within the boxes represent the deleted segment of the first exon of the OsSUT3 mutant gene in the rice cold water trough.

FIG. 3: comparison of the amino acid sequences encoded by the OsSUT3 gene in wild type rice variety 9311 (identified as 9311) and in rice cold paddy (identified as LSM). Wherein the OsSUT3 protein sequence in the wild rice variety 9311 comprises 506 amino acids, and the termination codon TGA is advanced due to the deletion of 77bp fragments of the first exon of OsSUT3 in the OsSUT3 gene mutant of the paddy rice in the cold paddy rice, so that only 56 amino acids can be synthesized.

FIG. 4: iodine-potassium iodide staining of wild type rice variety 9311 pollen and rice cold water glutelin pollen. In fig. 4, the left image is 9311 and the right image is a cold water trough.

FIG. 5: and (3) identifying true hybrids of the rice OsSUT3 mutant gene near isogenic lines. M is DNA molecular weight standard; lane 1: parental wild-type rice variety 9311(756 bp); lane 2: parental cold water cereals (485 bp); swimming device3-12 is a wild type rice variety 9311 crossed with cold water grain F₁Plants were generated, with lanes 5 and 9 being true hybrids (two bands: 756bp and 485bp), lanes 3, 4, 6, 7, 8, 10, 11, 12 being false hybrids, and lane 11 being a single band identical to the cold water trough of the parent mutant material.

FIG. 6: rice OsSUT3 mutant gene near allele line NIL-OsSUT3^MThe construction of (1). In fig. 6, 9311 is a wild type rice variety 9311, cold paddy is a mutant material cold paddy, i.e., a mutant containing rice ossit 3 gene, and the nucleotide sequence of the rice ossit 3 gene mutant is shown in SEQ ID NO: shown at 11.

The cold water trough described in fig. 1-6 is a mutant material cold water trough.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications and substitutions to the technical solutions, method steps or conditions of the present invention, and the like, without departing from the spirit and substance of the present invention, are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; the reagents used in the examples are all commercially available.

Based on the earlier study on OsSUT3 gene phylogeny by the applicant, the invention screens out that OsSUT3 gene of cold paddy of rice variety has natural mutation in 494 parts of rice variety, the first exon segment of the natural mutation is lost, which leads to advanced stop codon, the cold paddy containing OsSUT3 gene mutant (hereinafter, mutant material cold paddy) is hybridized with wild type rice variety 9311, the generated hybrid progeny is backcrossed with recurrent parent 9311 for multiple times, and rice OsSUT3 mutant gene near-allelic gene line NIL-OsSUT3 is obtained^MProvides good materials for the research of rice pollen development and molecular design breeding, and has important significance and potential utilization value for the research of the molecular mechanism of the OsSUT3 gene.

Example 1 obtaining and identification of OsSUT3 Gene mutants in Rice

First, obtaining OsSUT3 gene mutant of rice

The applicant has been on the OsSUT3 geneAccording to the research of phylogenetic evolution, 5 pairs of specific primers (shown in table 1) are designed according to the genome sequence (http:// www.ncbi.nlm.nih.gov /) of the OsSUT3 gene, 494 parts of collected rice germplasm resource materials are germinated, and the total DNA is extracted by a CTAB method in the four-leaf stage (the CTAB method is shown in Clarke, 2009)^[14]) PCR amplification is carried out, the full-length sequence of the CDS of the OsSUT3 gene is amplified, and the polymorphism is screened. The method for extracting the rice leaf genome DNA by adopting the CTAB method comprises the following specific steps: putting rice leaves less than or equal to 0.5g into a 2mL Eppendorf tube, adding liquid nitrogen, grinding, and adding 800 mu L CTAB extraction buffer solution; water bath at 65 ℃ for 40min, during which the mixture is reversed and mixed. Adding 800 μ L chloroform + isoamyl alcohol mixture (chloroform: isoamyl alcohol volume ratio 24:1), and slightly shaking for 10 min; centrifuging at the room temperature of 12000r/min for 10min, sucking the supernatant, transferring the supernatant into a new 1.5mL Eppendorf tube, adding 2 times of ice absolute ethyl alcohol, uniformly mixing, and centrifuging at the speed of 10000r/min for 2 min. The supernatant was decanted, the pellet washed with 75% v/v ethanol, dried and 100. mu.L of sterile ddH was added₂O shaking to dissolve the DNA precipitate.

And (2) carrying out PCR amplification on each rice germplasm resource material by using the 5 pairs of primers respectively in the same PCR reaction system and the same PCR reaction conditions, wherein the renaturation temperature in the circulation is the Tm value (+ -1 ℃) of each primer pair (shown in table 1), carrying out electrophoresis on the reaction product for 40min by using 1.5% agarose gel at the voltage of 120V after the reaction is finished, carrying out Ethidium Bromide (EB) staining, recovering the PCR amplification product by using a DNA gel recovery kit, and sequencing and splicing the sequence to obtain the CDS full length of the rice OsSUT3 gene.

The 5 pairs of primers are respectively as follows: primer PriSUT3-1, primer PriSUT3-2, primer PriSUT3-3, primer PriSUT3-4 and primer PriSUT 3-5. The 5 pairs of primers are used for amplifying the full length of the ORF of the rice OsSUT3 gene and identifying the polymorphism difference of the gene coding region.

The primer PriSUT3-1 consists of a primer PriSUT3-1F (forward primer) and a primer PriSUT3-1R (reverse primer), and the nucleotide sequence of the primer PriSUT3-1F is shown as SEQ ID NO: 1, and the nucleotide sequence of the primer PriSUT3-1R is shown as SEQ ID NO: 2, respectively.

The primer PriSUT3-2 consists of a primer PriSUT3-2F (forward primer) and a primer PriSUT3-2R (reverse primer), and the nucleotide sequence of the primer PriSUT3-2F is shown as SEQ ID NO: 3, the nucleotide sequence of the primer PriSUT3-2R is shown as SEQ ID NO: 4, respectively.

The primer PriSUT3-3 consists of a primer PriSUT3-3F (forward primer) and a primer PriSUT3-3R (reverse primer), and the nucleotide sequence of the primer PriSUT3-3F is shown as SEQ ID NO: 5, the nucleotide sequence of the primer PriSUT3-3R is shown as SEQ ID NO: and 6.

The primer PriSUT3-4 consists of a primer PriSUT3-4F (forward primer) and a primer PriSUT3-4R (reverse primer), and the nucleotide sequence of the primer PriSUT3-4F is shown as SEQ ID NO: 7, the nucleotide sequence of the primer PriSUT3-4R is shown as SEQ ID NO: shown in fig. 8.

The primer PriSUT3-5 consists of a primer PriSUT3-5F (forward primer) and a primer PriSUT3-5R (reverse primer), and the nucleotide sequence of the primer PriSUT3-5F is shown as SEQ ID NO: 9, the nucleotide sequence of the primer PriSUT3-5R is shown as SEQ ID NO: shown at 10.

TABLE 1 primer sequences for amplifying CDS full length of OsSUT3 gene

The PCR reaction system of the PCR amplification comprises: the total volume is 15 mu L, wherein the total volume is 1.5 mu L (TaKaRa) of Taq PCR Master Mix, 1.5 mu L of 10pmol/L forward primer, 1.5 mu L of 10pmol/L reverse primer, 2.5 mu L of sterile water and 2 mu L of template DNA; after the PCR system is prepared, the PCR system is slightly shaken and then put into an Eppendorf PCR instrument for sequence amplification.

And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 40s in a cycle, annealing at 56 ℃ for 1min, renaturation at 72 ℃ for 1min, and extension at 72 ℃ for 10min after 30 cycles, wherein 96 samples can be screened simultaneously each time.

The experimental results are as follows: the 5 pairs of specific primers (table 1) are used for carrying out PCR amplification on CDS regions of 494 parts of rice germplasm resources, and the polymorphism of the CDS of the OsSUT3 gene among rice germplasm is screened, and the result shows that: amplification products of the primers PriSUT3-2, PriSUT3-3, PriSUT3-4 and PriSUT3-5 have no obvious polymorphism difference among 494 rice germplasms, and an amplification band of the primer PriSUT3-1 in cold water paddy of a rice variety is obviously different from that of other 493 rice germplasms. Sequencing, sequence comparison and CDS prediction show that a 77bp fragment of a first exon of an OsSUT3 gene in a cold-water valley of a rice variety is lost, so that a stop codon is advanced, and functional proteins of OsSUT3 cannot be translated normally, namely the cold-water valley is a natural rice mutant with OsSUT3 gene function loss.

The electrophoresis results of some of the PCR products in the above experiments are shown in fig. 1, wherein the rice germplasm resource materials shown in lanes 1-6 and lanes 8-13, i.e., wild type rice variety 9311 (9311 in fig. 1), japan sunny, eight treasure valley, Yunzu 41, Lijing 9, Guanghui 128, Liuyu, disagu, locust valley, Yunnan miscellaneous 35, Zhongyu 1, and Tainan 11, have a relatively large molecular weight of PCR amplification products, which are 756bp, are wild type bands of the oscut 3 gene, and a relatively small molecular weight of PCR amplification products of cold valley in lane 7, which are 485bp, are amplification bands of the rice oscut 3 gene mutant. These biomaterials have been disclosed in the following non-patent literature, where the applicant has a stock available, contact address: the FengYuan route 452 in the area of the dragon, Yunnan province, Yunnan agricultural university (noted: Yunnan agricultural university institute of rice farming), zip code: 650201.

9311: indica rice variety (Wanglan et al, using Nipponbare/9311 recombinant inbred line population to locate rice maturity leaf-shape related trait QTL [ J ]. China Rice science, 2014, 28 (6): 589) 597, p590, third section of left column).

Clear in japan: japonica rice variety (Wanglan et al, using Nipponbare/9311 recombinant inbred line population to locate the rice maturity leaf shape related trait QTL [ J ]. China Rice science, 2014, 28 (6): 589) adulterate 597, p590, third section of left column).

Eight-treasure rice: indica rice variety (Luolong, etc., main agronomic character combining ability and heritability research of germplasm resources of fragrant soft rice [ J ]. southwest agriculture bulletin, 2011, 24 (5): 1625-.

Yunzi 41: japonica rice variety (benghanfen et al, characteristic features of Yunzu 41 and cultivation technical points [ J ]. Yunnan agriculture, 2013, (6)).

No. 9 Lijing: breeding and cultivation technology of japonica rice variety (Yanghong, etc., high cold japonica rice new variety 'Lijing No. 9'. Yunnan agricultural science and technology, 2014, (1): 56-58)

Guanghui 128: testing and matching F of indica type restorer line (Guo Qiang, etc., Guanghui 128 and different types of sterile lines₁Surrogate Performance study [ J]Guangxi agricultural science 2004,35(4): 279-281).

And (4) six months: cultivated rice (Li Ming Qi et al, comparison of photosynthesis and chlorophyll content of wild rice and cultivated rice [ J ]. plant physiology, 1984,10 (4): 333-.

Disorganized valley: indica rice (plum, comparison of genetic diversity of local varieties of farmer protected Yunnan rice collected at different periods [ D ]. Chinese academy of agricultural sciences, 2014, 04, 01, attached Table 1: 601 parts of materials collected at different periods, serial number 346).

Locust grain: royal, et al, protection and sustainable utilization of traditional local varieties of rice in Yuanjiang county, Yunnan province [ J ]. agronomy proceedings, 2019, 9 (11): 1-5, p4, left column 4.

Yunnan miscellaneous 35: japonica rice variety (Li Shi just et al, Dianzi No. 35 seed production yield structure research of highland hybrid japonica rice [ J ]. college of cloud agriculture, 2009,24 (6): 783 + 787).

China No. 1: (Ludelcheng, et al, Breeding of water-saving rice new variety China No. 1 and high-yield cultivation technology [ J ]. Guangdong agricultural science, 2009 (5): 16-18, 30).

Tainan No. 11: (Shiang-Ting Lee and Wen-Lii Huang, cytokine, auxin, and antiscisic acid afficients succinogenes sugar alcohol con to de novo shoootungensis in rice (Oryza sativa L.) pillow. Botanial Studies,2013,54(1),1-11.Abstract)

Cold water: genetic diversity analysis in rice local population, sun Jianchang et al [ J ]. proceedings of northwest agro-forestry science and technology university (natural science edition), 2011,39 (12): 145 vs 152,158, summary.

The OsSUT3 gene in the wild rice variety 9311, namely 9311 has no mutation, the pollen of the wild rice variety is normally developed, the pollen is normally developed, namely the pollen is well developed, and the pollen fertility of the wild rice variety is more than or equal to 90%.

Identification of OsSUT3 gene mutation site in rice OsSUT3 gene mutant

On the basis of the above experiments, the PCR-recovered product was sequenced and subjected to sequence analysis. The obtained wild rice variety 9311 and the PCR recovery product DNA of the mutant material cold water paddy were sequenced in Biotechnology engineering (Shanghai) GmbH. Using common DNA sequence analysis software DNAman 6.0 to splice the bidirectional sequencing result; the CDS full-length nucleotide sequence of OsSUT3 gene in wild type rice variety 9311 is shown as SEQ ID NO: 12, which is consistent with the CDS sequence of OsSUT3 gene published by NCBI database (accession number: LOC4348577, website: https:// www.ncbi.nlm.nih.gov/nuccore/XM _015758288.2), and consists of 1521 bases; the OsSUT3 mutant gene in the mutant material cold paddy is named as rice OsSUT3 gene mutant, and the nucleotide sequence of the mutant gene is shown as SEQ ID NO: 11, consisting of 1444 bases. After comparison of the wild rice variety 9311 with the ORF sequence of the OsSUT3 gene in the mutant material cold water grain, the OsSUT3 gene in the mutant material cold water grain is mutated, and a 77bp fragment is deleted in the first exon (see FIG. 2). The protein sequence analysis and alignment result shows that the deletion of the 77bp fragment causes base frame shift, so that the termination codon TGA is advanced, and finally only 56 amino acids can be synthesized, while the wild type OsSUT3 protein sequence contains 506 amino acids. Therefore, deletion of 77bp in the OFR sequence of the OsSUT3 gene in the cold water trough of the mutant material resulted in premature termination of the synthesis of the OsSUT3 amino acid, resulting in loss of most of the functional domain of the OsSUT3 protein (fig. 3).

The mutant material cold paddy refers to the nucleotide sequence of OsSUT3 gene contained in cold paddy through natural mutation, such as SEQ ID NO: 11 shows that the rice OsSUT3 gene mutant is contained.

The identification method of the mutant material cold cereal comprises the following steps: PCR reaction System for PCR amplification: the total volume is 15 mu L, wherein the total volume is Taq PCR Master Mix 7.5 mu L (TaKaRa), 10pmol/L primer PriSUT3-1F 1.5 mu L, 10pmol/L primer PriSUT3-1R1.5 mu L, sterile water 2.5 mu L and template DNA 2 mu L; after the PCR system is prepared, slightly shaking the PCR system, and putting the PCR system into an Eppendorf PCR instrument for sequence amplification; and (3) PCR reaction conditions: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 40s in cycle, annealing at 56 deg.C for 1min, renaturation at 72 deg.C for 1min, and extension at 72 deg.C for 10min after 30 cycles; if the PCR amplification product band is 485bp band type, the cold water valley is the mutant material cold water valley. Cold water cereal leaf genomic DNA was extracted using the CTAB method described in example 1.

Compared with the wild OsSUT3 gene, the DNA sequence of the rice OsSUT3 gene mutant is totally deleted by 271bp fragments, wherein 77bp are positioned at the 3 'end of a first exon, and the other 194bp are positioned at the 5' end of a first intron of an OsSUT3 gene.

Example 2 Observation of Cold Water Valley pollen fertility

Materials: wild type rice variety 9311 and mutant material cold water cereals.

The mutant material cold water trough was identified in the same manner as in example 1.

The pollen fertility identification method comprises the following steps: the method adopts an iodine-potassium iodide dyeing method to identify pollen fertility, and comprises the following specific operations: just before the wild type rice variety 9311 and the rice ear of the mutant material cold-water paddy bloom, the spikelet is removed, the anther is taken out by using tweezers, the anther is pinched off in a 0.2% iodine-potassium iodide solution, pollen grains are released, and the mature pollen of the wild type rice variety 9311 and the mutant material cold-water paddy is dyed and observed under an optical microscope. Preparation of 0.2% iodine-potassium iodide solution: dissolving 2.000g of potassium iodide in 100mL of distilled water to prepare a saturated potassium iodide solution, adding 0.200g of iodine, and completely dissolving to prepare a 0.2% iodine-potassium iodide solution.

Normally developed pollen grains (fertile pollen) are round in bluish purple or ellipsoidal in bluish purple, while poorly developed and non-viable pollen grains (sterile pollen) are yellowish-brown malformations. Respectively selecting 5 fields with relatively uniform pollen distribution from the dyed pollen of the wild rice variety 9311 and the mutant material cold water valley, counting the total number of the pollen and the number of the normally developed pollen, calculating the average number, and calculating the pollen fertility of the wild type 9311 and the mutant material cold water valley by using the counting result.

Calculation formula of pollen fertility (%) is as follows: the number of pollen/total number of pollen developed normally is 100%.

Compared with the wild rice variety 9311, the maldeveloped pollen in the mutant material cold water grain is obviously improved by 24.17 percent (the right picture in figure 4); meanwhile, the number of the pollen in the cold water cereals is also obviously reduced and is only 70.43 percent of the number of the pollen in the wild type rice variety 9311 (the left graph and the right graph in fig. 4), the average pollen fertility of the wild type rice variety 9311 is 96.47 percent, and the average pollen fertility of the mutant material cold water cereals is 67.13 percent, and the results show that the mutation of the rice OsSUT3 gene influences the accumulation of starch grains at the later development stage of the rice pollen and has great influence on the number of the pollen, so that the screening of the material with the rice OsSUT3 mutant gene creates an important research material for deeply exploring the biological function of the rice OsSUT3 gene; has good application prospect in transferring new germplasm and improving rice varieties, for example, breeding new flower varieties with less pollen quantity provides reference for the variety improvement of important agronomic characters such as rice pollen development, seed setting rate and the like.

Example 3 Rice OsSUT3 mutant Gene near allele line NIL-OsSUT3^MConstruction of

1. And (3) breeding of receptor parents: selecting wild rice variety 9311 with published genome sequence, selfing and purifying to 10 th generation (F)₁₀) I.e. 9311F₁₀Highly regular inter-line character, 9311F₁₀As the recipient parent, the statistics of the pollen quantity and fertility of the parent are shown in Table 2.

2. Breeding a donor parent: the cold water grain is a mutant material cold water grain subjected to natural mutation, the cold water grain is identified as the mutant material cold water grain according to the mutant material cold water grain identification method described in the embodiment 1, and then the 5 th generation F of the cold water grain is selected for selfing₅I.e. the cold water trough F₅As donor parents, the statistics of pollen quantity and fertility are shown in Table 2.

3. Field hybridization and true hybrid identification: with the acceptor parent 9311F₁₀As female parent, cold water grain F of donor parent₅And (2) interspecific hybridization is carried out as a male parent, the obtained interspecific hybrid seeds germinate, then a leaf CTAB method is adopted to extract DNA, and then a specific primer PriSAT 3-1 (consisting of a primer PriSAT 3-1F and a primer PriSAT 3-1R) is used for PCR amplification, and a PCR reaction system for PCR amplification is adopted: the total volume was 15. mu.L, wherein Taq PCR Master Mix 7.5. mu.L (TaKaRa), 10pmol/L primer PriSUT3-1F 1.5. mu.L, 10pmol/L primer PriSUT3-1R 1.5. mu.L, none2.5 mu L of bacteria water and 2 mu L of template DNA; after the PCR system is prepared, slightly shaking the PCR system, and putting the PCR system into an Eppendorf PCR instrument for sequence amplification; and (3) PCR reaction conditions: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 40s in cycle, annealing at 56 deg.C for 1min, renaturation at 72 deg.C for 1min, and extension at 72 deg.C for 10min after 30 cycles; the PCR product was electrophoresed in 1.5% agarose gel at 120V for 40min, stained with Ethidium Bromide (EB), and observed to show two bands of 756bp and 485bp (FIG. 5), which is a true hybrid. Meanwhile, the pollen iodine-potassium iodide dyeing and statistical method described in the embodiment 2 is adopted to carry out the statistical analysis of single-plant pollen fertility on the mature pollen of the true hybrid plant after the molecular identification, and the pollen fertility which is more than or equal to 80 percent is screened out<90% of recombinant individuals, and continuing to select the selected recombinant individuals and recurrent parent 9311F₁₀Backcrossing is carried out, and the backcrossing is repeated until 6 th generation to obtain BC₆Namely rice OsSUT3 mutant gene near allele line NIL-OsSUT3^M(FIG. 6).

Rice OsSUT3 mutant gene near isogenic line NIL-OsSUT3^MThe near-equality analysis of (1): see Table 2, selecting 13 phenotypic traits, and selecting donor parent mutant material cold paddy, recurrent parent wild type rice variety 9311, rice OsSUT3 mutant gene near allele line NIL-OsSUT3^MA near-isocratic analysis was performed.

Survey and measurement methods: in the analysis of 13 phenotypic/agronomic traits (table 2), 2 non-numerical traits of grain color, glume color were compared using GSB national standard color cards; the glume character adopts a visual method; measuring 7 numerical properties of plant height, leaf length, leaf width, grain length, grain width, grain thickness and stem diameter by using a measuring tape and a vernier caliper, selecting 15 individual plants each time, counting, then taking an average value, repeating for 3 times, and performing difference significance analysis; the seed setting rate is obtained by taking an average value after 15 plants are randomly extracted and counted; statistics of pollen quantity and pollen fertility the iodine-potassium iodide staining method described in example 2 was used, and 5 fields were randomly selected for counting the total pollen quantity and the number of normal developing pollen, and the average was taken. The individual traits are in NIL-OsSUT3^MIndividual plants were randomly selected from among cold water cereals and 9311 lines for investigation and measurement.

TABLE 2 detection analysis of 13 phenotypic/agronomic traits between parent and near-allelic lines

The results in table 2 show that of the 13 phenotypic traits, the 2 numerical traits of leaf width and grain thickness are not significantly different in 3 varieties (lines); rice OsSUT3 mutant gene near allele line NIL-OsSUT3^MThe plant height, leaf length, grain width and stem diameter of 5 plants are similar to or basically the same as those of the receptor parent 9311, the seed characters comprise glume color, grain color, glume, pollen quantity, pollen fertility and fructification rate of 6 phenotypic characters/agronomic characters are basically the same as those of the cold paddy of the donor parent, so far, the rice OsSUT3 mutant gene near isogenic line NIL-OsSUT3^MAnd (5) completing construction.

Sequence listing

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Claims (10)

1. The nucleotide sequence of the rice OsSUT3 gene mutant is shown as SEQ ID NO: shown at 11.

2. Specific primer PriSUT3-1 for detecting OsSUT3 gene mutant in rice as claimed in claim 1, wherein: the specific primer PriSUT3-1 consists of a primer PriSUT3-1F and a primer PriSUT3-1R, and the nucleotide sequence of the primer PriSUT3-1F is shown as SEQ ID NO: 1, and the nucleotide sequence of the primer PriSUT3-1R is shown as SEQ ID NO: 2, respectively.

3. A molecular identification method for OsSUT3 gene mutation of rice is characterized by comprising the following steps of carrying out PCR amplification on DNA of rice by adopting a specific primer PriSUT3-1, and analyzing the length of an amplification product: if the amplified product is 756bp, the OsSUT3 genotype of the rice is a wild type, and the pollen grows well; if the amplification product is 485bp, the OsSUT3 genotype of the rice is a mutant, and the pollen is dysplastic; if the amplification product is 756bp and 485bp banding patterns, the OsSUT3 genotype of the rice is a heterozygous genotype, and the pollen development is damaged; the specific primer PriSUT3-1 consists of a primer PriSUT3-1F and a primer PriSUT3-1R, and the nucleotide sequence of the primer PriSUT3-1F is shown as SEQ ID NO: 1, and the nucleotide sequence of the primer PriSUT3-1R is shown as SEQ ID NO: 2, respectively.

4. The method of claim 3, wherein: the good development of the pollen is that the pollen fertility is more than or equal to 90 percent; the pollen dysplasia is that the pollen fertility is more than or equal to 60 percent and less than 80 percent; the pollen development damage is more than or equal to 80 percent and less than 90 percent of pollen fertility.

5. The molecular identification method for auxiliary screening or detecting rice pollen fertility characters is characterized by comprising the following steps: performing PCR amplification on the DNA of the rice by using a specific primer PriSUT3-1, wherein the specific primer PriSUT3-1 consists of a primer PriSUT3-1F and a primer PriSUT3-1R, and the nucleotide sequence of the primer PriSUT3-1F is shown as SEQ ID NO: 1, and the nucleotide sequence of the primer PriSUT3-1R is shown as SEQ ID NO: 2, respectively.

6. The method of claim 5, wherein: if the amplified product is 756bp, the OsSUT3 genotype of the rice is a wild type, and the pollen grows well; if the amplification product is 485bp, the OsSUT3 genotype of the rice is a mutant, and the pollen is dysplastic; if the amplification product is 756bp and 485bp banding patterns, the OsSUT3 genotype of the rice is a heterozygous genotype, and the pollen development is damaged;

the good development of the pollen is that the pollen fertility is more than or equal to 90 percent; the pollen dysplasia is more than or equal to 60 percent and the pollen fertility is less than 80 percent; the pollen development damage is more than or equal to 80 percent and less than 90 percent of pollen fertility.

7. The rice OsSUT3 gene mutant of claim 1 applied to breeding of rice pollen sterile materials.

8. Use according to claim 7, characterized in that: carrying out PCR amplification on the DNA of the rice by adopting a specific primer PriSAT 3-1, wherein if the amplification product is 485bp, the rice is a rice pollen sterile material with poor pollen development; the specific primer PriSUT3-1 consists of a primer PriSUT3-1F and a primer PriSUT3-1R, and the nucleotide sequence of the primer PriSUT3-1F is shown as SEQ ID NO: 1, and the nucleotide sequence of the primer PriSUT3-1R is shown as SEQ ID NO: 2, respectively.

9. Use according to claim 8, characterized in that: the pollen dysplasia is that the pollen fertility is more than or equal to 60 percent and less than 80 percent.

10. Rice OsSUT3 mutant gene near allele line NIL-OsSUT3^MThe method for constructing (a) is characterized by comprising the following steps:

(1) and (3) breeding of receptor parents: selfing and purifying wild indica rice variety 9311 to selfing 10 th generation as 9311F₁₀9311F₁₀As a recipient parent;

(2) breeding a donor parent: selfing with cold water grain for the 5 th generation F₅As a donor parent;

(3) field hybridization and true hybrid identification: with the acceptor parent 9311F₁₀As female parent, cold water grain F of donor parent₅Performing interspecific hybridization as male parent, germinating the interspecific hybrid seed, identifying true hybrid with specific primer PriSAT 3-1, dyeing pollen grains of true hybrid plant with iodine-potassium iodide solution, observing under optical microscope, counting pollen fertility of individual plant, and selecting pollen fertility of 80% or more<Between 90% of the recombinant individuals continue to recurrent parent 9311F₁₀Backcrossing is carried out, and the backcrossing is repeated until 6 th generation to obtain BC₆Namely rice OsSUT3 mutant gene near allele line NIL-OsSUT3^M(ii) a The specific primer PriSUT3-1 consists of a primer PriSUT3-1F and a primer PriSUT3-1R, and the nucleotide sequence of the primer PriSUT3-1F is shown as SEQ ID NO: 1, and the nucleotide sequence of the primer PriSUT3-1R is shown as SEQ ID NO: 2, respectively.

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