CN116789785A - High-yield and high-light-efficiency gene FarL1 of long stamen wild rice and application thereof - Google Patents
High-yield and high-light-efficiency gene FarL1 of long stamen wild rice and application thereof Download PDFInfo
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Abstract
The invention discloses a novel gene FarL1 from long stamen wild rice (Oryza longistaminata) and application thereof, belonging to the technical fields of crop genetic engineering and molecular biology. The invention leads the FarL1 gene into near isogenic line NIL-FarL1 of rice or over-expression in rice 9311, and the photosynthesis efficiency, the number of secondary branches and stems of spike and the number of grains per spike are obviously increased. These results indicate that the FarL1 gene can obviously increase the number of ears per ear of grain by regulating photosynthesis and secondary branch development of rice, thereby improving the yield of the rice. Therefore, the FarL1 gene has great application prospect in cultivating new rice varieties with high yield.
Description
Technical Field
The invention belongs to the technical fields of crop genetic engineering and molecular biology, and particularly relates to a high-yield and high-light-efficiency gene FarL1 of long stamen wild rice and application thereof.
Background
Rice is one of three food crops in the world, and the population taking rice as a staple food occupies more than half of the population in the world. With the increase of population number, the development of society and the continuous improvement of human living standard, the supply gap of rice is continuously enlarged, so that the global rice yield is increased to fill the gap, and the method is important for ensuring the grain safety of the 21 st century. The excavation of good genes related to yield is an important way for improving rice yield. The long stamen wild rice (Oryza longistaminata) is old wild rice from Africa, contains rich genetic variation, has numerous excellent characteristics of strong stalks, lodging resistance, large grains, strong disease and insect resistance, and the like, and is an important gene resource library for genetic improvement of rice. The development and utilization of related genes such as high yield, disease and pest resistance in long stamen wild rice has important significance for promoting the improvement of high yield of rice and guaranteeing the grain safety.
The rice yield is mainly composed of three factors: effective spike, spike grain number and thousand grain weight. Cloning these yield trait genes and applying these favorable alleles to actual rice breeding work will undoubtedly promote the cultivation of high yield rice varieties, thereby alleviating the grain crisis due to rapid population growth.
The Far (Far-red-impaired response) transcription factor family is a gene family with relatively conservative genetic functions in plants such as rice, arabidopsis and the like, and a great deal of researches show that the gene family is involved in biological processes such as growth and development of plants, photoperiod regulation, stress resistance and the like, and has quite a plurality of biological functions.
Disclosure of Invention
The invention is based on a Long stamen wild rice large ear infiltration system material 1762 (Long et al, crop Journal,2023, doi: 10.1016/j.cj.2023.03.017), and hybridizes with a Long stamen wild rice infiltration system receptor parent 9311, backcrossing, constructing a near isogenic line, and separating a high-yield gene FarL1 for controlling the grain number of each ear in the material by using a map-based cloning method. The gene is a specific gene of long stamen wild rice, has the functions of promoting photosynthesis and secondary branch development of the rice, and increases the grain number of spikes, thereby improving the yield of the rice, and can be widely applied to cultivation of high-yield new rice varieties.
The invention aims to provide a high-yield gene FarL1 of long stamen wild rice, and further provides application of the gene FarL1 in improving the yield of the rice according to the relation between the gene and photosynthesis and secondary branch development of the rice.
The aim of the invention can be achieved by the following scheme:
the invention introduces the FarL1 gene into rice by rice sexual hybridization or expresses the FarL1 gene in the rice body to find that: after the FarL1 gene is introduced into near isogenic line NIL-FarL1 of rice or the FarL1 is expressed in the body of rice variety 9311, the photosynthesis efficiency, the number of secondary branches and stems of mature stage spike and the number of grains per spike are obviously higher than 9311. This suggests that FarL1 directly regulates the photosynthetic efficiency, secondary shoot number and spike-grain number per ear of rice. FarL1 is a long stamen wild rice specific gene which can function through interaction with histone H3K27Me2/3 demethylase, while histone H3K27Me2/3 demethylase and its functional domain are highly conserved in other important grain crops (sorghum, maize, etc.), indicating that the gene may have similar molecular mechanisms in terms of other crop yield regulation. Thus, farL1 is likely to also regulate the yield of other monocotyledonous gramineous crops through similar molecular mechanisms.
The FarL1, a protein involved in photosynthesis and secondary stem development regulation of rice, is derived from long stamen wild rice (Oryza longistaminata), and has an amino acid sequence shown in SEQ ID NO. 4.
To facilitate research and utilization of the FarL1 protein, the amino-or carboxy-terminal linkage of the protein sequence may be followed by the tags shown in table 1.
TABLE 1 tags and amino acid sequences thereof
| Label (Label) | Residues | Sequence(s) |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tagⅡ | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
The nucleotide sequence of the FarL1 gene involved in photosynthesis and secondary branch development regulation of rice is any one of sequences shown as SEQ ID NO.1, 2 or 3.
The nucleotide sequence shown in SEQ ID NO.1 is a genome base sequence and consists of 2395 bases, and comprises a promoter, a 5'UTR, an exon and a 3' UTR.
The nucleotide sequence shown in SEQ ID NO.2 is a cDNA coding sequence.
The nucleotide sequence shown in SEQ ID NO.3 is CDS sequence.
The FarL1 gene has the application of promoting photosynthesis of rice and secondary branch development so as to improve the yield of the rice. Correspondingly, the primer for amplifying the full length of the FarL1 gene, the expression cassette, the recombinant vector, the transgenic cell line or the recombinant bacterium containing the FarL1 gene also has the application of promoting the photosynthesis of rice and the development of secondary branches so as to improve the yield of the rice
The FarL1 gene can obviously increase the number of ears per ear of rice by promoting photosynthesis of rice and increasing the number of secondary branches, and has important application value in cultivating high-yield rice varieties.
The primer for amplifying the full length of the FarL1 gene is applied to cultivation of rice high-yield varieties.
The application of the expression cassette, the recombinant vector, the transgenic cell line or the recombinant bacteria containing the FarL1 gene in the cultivation of rice high-yield varieties.
The photosynthesis efficiency, the number of secondary branches and stems and the rice yield of the rice can be improved by introducing the FarL1 into a cultivated rice variety through hybridization transfer, or transferring the FarL1 into the cultivated rice through transgenosis to enable the FarL1 to be normally expressed or overexpressed, and the specific operation method is that the FarL1 gene is introduced into other rice varieties through sexual hybridization of long stamen wild rice or NIL-FarL1 carrying the FarL1 gene and the other rice varieties; or transferring the expression vector into rice to over-express the FarL1.
Existing crop transformation vectors can be selected for the construction of recombinant vectors containing FarL1. The crop transformation vector comprises a binary agrobacterium vector, a vector which can be used for crop microprojectile bombardment and the like, such as pCAMBIA3301, pYLCRISPR/Cas9Pubi-B, pYLCRISPR/Cas9P35S-H, pYLCRISPR/Cas9P35S-N, pCAMBIA2301, pH7WG2D or other editing technology related vectors, such as TALENs, ZFNs and the like.
In order to achieve the purpose of improving the rice yield by using the FarL1 gene, any promoter which is helpful for changing the expression of the FarL1 gene, such as a cauliflower mosaic virus (CAMV) 35S promoter, a Ubiquitin (Ubiquitin) gene promoter (pUbi) and the like, can be added before the gene start site when constructing the vector, and the purpose of enhancing the expression can be achieved by adding an enhancer. Whatever the way it is, it is necessary to ensure the correctness of the coding sequence in order to obtain the correct structure of the FarL1 protein.
Those containing marker genes such as: the vectors of GUS gene, GFP gene, hygromycin resistance gene, herbicide resistance gene and the like construct a recombinant vector, which is more beneficial to experimental operation and later transformant screening.
The recombinant vector containing FarL1 can be transformed into crop tissues or cells by various means, such as microinjection, agrobacterium-mediated genetic transformation, and common test methods such as Ti plasmid, ri plasmid or viral vector.
The FarL1 gene can also be used as a molecular marker for rice breeding.
The rice includes indica rice, japonica rice, etc.
The method for directionally transferring the FarL1 into the rice by using the sexual hybridization gene transfer or transgenic technology among rice varieties promotes the photosynthesis of the rice and the development of secondary branches, which shows that the gene can be used for improving the photosynthesis of the rice and increasing the number of the secondary branches to be applied to the breeding of high-yield crops of the rice. Therefore, the FarL1 gene provides powerful means and tools for breeding rice high-yield new varieties by using molecular marker assisted breeding and using a genetic engineering method, and has great application potential.
Drawings
FIG. 1 is a morphological feature of a long stamen wild rice introgression line 1762. A, the plant type and spike type of the introgression line 1762. B, counting the number of branches and the number of spike grains of the infiltration system 1762; PB and SB refer to primary and secondary branches, respectively.
FIG. 2 is a strain and spike of the FarL1 near isogenic line (NIL-FarL 1).
FIG. 3 is a schematic diagram of the gene structure of FarL1, containing the FAR1 DNA binding domain.
FIG. 4 is a schematic diagram of the structure of a FarL1 gene overexpression vector.
FIG. 5 is a schematic representation of the structure of the protein encoded by the FarL1 gene.
FIG. 6 is a molecular assay for FarL1 in overexpressing transgenic plants.
FIG. 7 is an analysis of the expression level of FarL1 in NIL-FarL1 and FarL1 transgenic lines (overexpressing OE).
FIG. 8 is the net photosynthetic rate of 9311, NIL-FarL1 and FarL1 overexpressing lines sword leaf.
FIG. 9 is a plant type and ear type of a FarL1 overexpressing transgenic line. FarL1-OE means a FarL1 overexpressing transgenic line.
Detailed Description
The following is how to increase photosynthesis and secondary shoot development of rice by specifically using the FarL1 gene, thereby increasing the number of grains per ear of rice and further improving the yield of rice. By way of example, the invention is not limited to the following embodiments.
The rice in the following examples was managed according to a conventional cultivation method: firstly, immersing fresh rice seeds to accelerate germination, sowing the seeds in a pre-prepared seedling bed after exposing the seeds to white, transplanting the seedlings to the field in a first period of 4 leaves, then testing the photosynthesis efficiency of sword leaves in a heading period, and examining the phenotype related to the yield in a mature period.
Example 1: creation of a FarL1 high-yielding near isogenic line (NIL-FarL 1) in the 9311 background
1. Hybridization of long stamen wild rice and excellent indica variety 9311 to obtain F 1 Backcrossing 9311 twice to obtain BC 2 F 1 And then bagging and selfing for 20 generations to obtain a stable homozygous long stamen wild rice chromosome fragment introgression line population. In this introgression line population, the number of primary shoots, secondary shoots and ear grains was significantly increased in the introgression line 1762 compared to the recipient parent 9311 (see fig. 1). Therefore, the large-spike introgression line 1762 of long stamen wild rice is selected as a candidate material for cloning the large-spike high-yield gene FarL1.
2. Construction of the farL1 near isogenic line (NIL-farL 1). In order to clone the large spike gene in 1762, the large spike gene is hybridized with 9311 continuously and backcrossed for 3 generations, and then bagging and selfing are carried out, so that a single plant is obtained, the plant type of the single plant is almost the same as 9311, but the number of secondary branches and spikes is obviously increased compared with 9311. It is identical to 9311 in the genome level except that the 8 th chromosome long arm has a small DNA fragment from the stamen wild rice. Gene cloning has shown that this segment contains the large spike gene FarL1 from a long stamen, and is therefore referred to as the FarL1 near isogenic line (NIL-FarL 1). The spike of NIL-FarL1 became larger and the number of secondary shoots was significantly increased compared to 9311 (see FIG. 2). Further examining the basic agronomic characters of NIL-FarL1, it is found that: the four characters of the number of primary branches, thousand seed weight, fruiting rate and effective spikes have no significant difference in the two materials; however, the secondary peduncles and the ear grains per spike were increased by 16.34 and 63.56 grains respectively compared with 9311, and the yield of the individual plants was increased by 25.24% compared with 9311 (see Table 2).
Table 2.9311 and yield trait statistics for near NIL-FarL1 isogenic lines
Example 2: creation of FarL1 high-yield transgenic lines
1. Obtaining full-length fragment of FarL1 Gene
The cDNA of the long stamen wild rice chromosome fragment introduction system 1762 is used as a template to design a primer pair FarL1-F/R (the primer sequence is shown in table 3), and then PCR amplification is carried out, and the nucleotide sequence of the amplified gene fragment is shown as SEQ ID NO. 3. The schematic structure of the FarL1 gene is shown in FIG. 3.
TABLE 3 primer sequences
| Primer name | Primer sequence (5 '-3') |
| FarL1-F | ATGGACGAGGACGAAGT |
| FarL1-R | TCACCTCCAGAACACTAGC |
2. Construction of FarL1 Gene overexpression vector
The product obtained by amplification of the FarL1-F/R by using the primer pair is inserted into an expression vector pCAMBIA1301 containing a strong promoter (a Ubiquitin promoter) through a recombination reaction, and positive clones are screened by using a marker gene on the vector, so that the recombinant expression vector FarL1-OE is obtained.
3. Obtaining of transgenic plants overexpressing the FarL1 Gene
The constructed FarL1-OE transgenic vector can be transferred into EHA105 agrobacterium (Agrobacterium tumefaciens) by an electric transfer or heat shock method, and positive agrobacterium strains which can be used for infecting rice tissues are obtained by screening the vector and the agrobacterium self-characteristics.
The 9311 callus is infected by a recombinant agrobacterium strain containing recombinant plasmid FarL1-OE, and the positive transgenic callus is obtained by dark culture on a screening culture medium containing 50mg/L hygromycin. Differentiation, rooting and transplanting of positive callus to obtain T 0 And (5) replacing plants. Obtaining T through conventional molecular detection and rice cultivation method 1 And (5) replacing plants. The model structure of the FarL1 gene in the over-expression rice line is shown in figure 4, and the CDS sequence of the FarL1 is shown in SEQ ID NO. 3; the amino acid sequence of the protein encoded by the FarL1 gene is shown as SEQ ID NO.4, and the modeling structure of the protein is shown as figure 5.
4. Detection of FarL1 in overexpressing transgenic plants
(1) PCR detection of FarL1 Gene-Positive Strain
Obtaining genome DNA of a FarL1 over-expression plant and a wild plant by using a conventional CTAB extraction genome DNA method, and designing a forward primer (pCAMBIA 1301-F) and a FarL1 gene specific primer (FarL 1 OE-R) on a pCAMBIA1301 over-expression vector (the primer sequences are shown in table 4); genomic DNA of wild plants was used as negative control, and genomic DNA of both the FarL1 overexpressing plants and wild plants was amplified, and all of the amplified bands of the FarL1 in the overexpressing transgenic plants were positive (see FIG. 6).
TABLE 4 FarL1 overexpressing plant positive identification primer sequences
| Primer name | Primer sequence (5 '-3') |
| pCAMBIA1301-F | CCCTGCCTTCATACGCTATT |
| FarL1OE-R | CCTCCAGAACACTAGCTCGG |
(2) Detection of expression level of FarL1 Gene by qRT-PCR
The Ubiquitin promoter is used as a monocotyledonous plant strong promoter, and can improve the expression content of a target gene in a plant body. Total RNA of the FarL1 overexpressing plants and wild-type plants was obtained using conventional RNA extraction methods, and the corresponding cDNA was obtained using a reverse transcription kit (purchased from Invitrogen). The action gene is used as an internal reference, and the expression level of the FarL1 is detected by qRT-PCR by using a FarL1 RT-F/R primer pair (the primer sequence is shown in table 5), so that the expression level of a FarL1 gene over-expression plant is obviously improved (see figure 7).
TABLE 5 expression level detection primer sequences of FarL1 Gene
| Primer name | Primer sequence (5 '-3') |
| FarL1 RT-F | CTTGGAGGAGATGGAGGAGTAT |
| FarL1 RT-R | AGGGTTAGGAAGTCTATGTGAATGT |
| Actin RT-F | GGAAGTACAGTGTCTGGATTGGAG |
| Actin RT-R | TCTTGGCTTAGCATTCTTGGGT |
(3) Observing and counting the number of secondary branches of the transgenic plant
When transgenic lines and 9311 grew to the beginning of heading (about 5% of the ears began to break), the photosynthesis efficiency of the sword leaf was measured (see FIG. 8). Seed collection is carried out when rice plants are mature, then seed examination is carried out on the over-expression transgenic material and the wild type material (9311), and the number of primary branches, the number of secondary branches, the number of grains per ear, the seed setting rate and thousand grain weight are observed and counted. The plant type and spike type of the FarL1 gene over-expression plant are shown in figure 9; the statistics of the number of primary branches, the number of secondary branches, the number of grains per ear, the seed setting rate, thousand grain weight, effective ears and the yield of single plants are shown in Table 6.
TABLE 6 agricultural trait statistics for plants overexpressing the FarL1 Gene
The above case is one of the preferred embodiments of the present invention, but the above case is only a part of the embodiments of the present invention. The present invention is not limited by the above embodiments, and other embodiments of the present invention which are changed, modified, edited and arranged based on the spirit and principle of the present invention are all equivalent substitutions, and all belong to a part of the present invention and are protected by the present invention.
Claims (10)
1. A long stamen wild rice high-yield and high-light effect gene FarL1 gene is characterized in that: the amino acid sequence of the protein coded by the FarL1 gene is shown as SEQ ID NO. 4.
2. The FarL1 gene according to claim 1, wherein: the nucleotide sequence of the FarL1 gene is any one of sequences shown as SEQ ID NO.1, 2 or 3.
3. Use of the FarL1 gene of claim 1, wherein: the application includes at least one of the following applications: the application of improving the photosynthetic efficiency of the rice, the application of increasing the number of secondary branches of the rice, the application of increasing the number of rice ears, the application of improving the yield of the rice and the application of cultivating high-yield varieties of the rice.
4. Use of a primer for amplifying the FarL1 gene of claim 1, wherein: the application includes at least one of the following applications: the application of improving the photosynthetic efficiency of the rice, the application of increasing the number of secondary branches of the rice, the application of increasing the number of rice ears, the application of improving the yield of the rice and the application of cultivating high-yield varieties of the rice.
5. Use of an expression cassette, recombinant vector, transgenic cell line or recombinant bacterium comprising the FarL1 gene of claim 1, characterized in that: the application includes at least one of the following applications: the application of improving the photosynthetic efficiency of the rice, the application of increasing the number of secondary branches of the rice, the application of increasing the number of rice ears, the application of improving the yield of the rice and the application of cultivating high-yield varieties of the rice.
6. Use according to any one of claims 3-5, characterized in that: the rice includes indica rice and japonica rice.
7. Use according to any one of claims 3-5, characterized in that: the application is realized by introducing the FarL1 gene through hybridization transfer or improving the expression quantity of the FarL1 through transgenesis.
8. A method for improving rice yield, which is characterized by comprising the following steps: to introduce the FarL1 gene into rice by hybridization transfer or to transfer the gene into rice by an expression vector, the FarL1 gene is overexpressed.
9. The use of the FarL1 gene of claim 1 as a molecular marker in rice breeding.
Use of the farl1 gene for promoting photosynthesis, secondary shoot development, spike number and/or improving yield of monocotyledonous gramineous crops.
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|---|---|---|---|---|
| CN105647940A (en) * | 2014-11-11 | 2016-06-08 | 武汉大学 | Method for improving rice yield through OsGRF6 gene, and applications thereof |
| CN114805508A (en) * | 2021-12-29 | 2022-07-29 | 中国农业科学院作物科学研究所 | Function and application of rice heading stage gene DHD3 |
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| CN105647940A (en) * | 2014-11-11 | 2016-06-08 | 武汉大学 | Method for improving rice yield through OsGRF6 gene, and applications thereof |
| CN114805508A (en) * | 2021-12-29 | 2022-07-29 | 中国农业科学院作物科学研究所 | Function and application of rice heading stage gene DHD3 |
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| SHAOYING HUANG等: "Favorable QTLs from Oryza longistaminata improve rice drought resistance", BMC PLANT BIOLOGY, vol. 22, pages 136 * |
| 徐高峰等: "外源酚酸类物质对一个长雄蕊野生稻材料S37化感效应的调控作用", 中国水稻科学, vol. 24, no. 01, pages 62 - 66 * |
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