CN116789785B - High-yield and high-light-efficiency gene FarL a of long stamen wild rice and application thereof - Google Patents
High-yield and high-light-efficiency gene FarL a of long stamen wild rice and application thereof Download PDFInfo
- Publication number
- CN116789785B CN116789785B CN202310877712.0A CN202310877712A CN116789785B CN 116789785 B CN116789785 B CN 116789785B CN 202310877712 A CN202310877712 A CN 202310877712A CN 116789785 B CN116789785 B CN 116789785B
- Authority
- CN
- China
- Prior art keywords
- rice
- farl
- gene
- application
- yield
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 80
- 241000746966 Zizania Species 0.000 title claims abstract description 21
- 235000002636 Zizania aquatica Nutrition 0.000 title claims abstract description 21
- 241000209094 Oryza Species 0.000 claims abstract description 77
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 76
- 235000009566 rice Nutrition 0.000 claims abstract description 76
- 230000001965 increasing effect Effects 0.000 claims abstract description 12
- 239000013598 vector Substances 0.000 claims description 18
- 230000009261 transgenic effect Effects 0.000 claims description 17
- 230000014509 gene expression Effects 0.000 claims description 13
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 238000009396 hybridization Methods 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 210000005069 ears Anatomy 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 239000013604 expression vector Substances 0.000 claims description 5
- 238000012546 transfer Methods 0.000 claims description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 240000002582 Oryza sativa Indica Group Species 0.000 claims description 3
- 230000000243 photosynthetic effect Effects 0.000 claims description 3
- 240000008467 Oryza sativa Japonica Group Species 0.000 claims description 2
- 235000013339 cereals Nutrition 0.000 abstract description 22
- 230000029553 photosynthesis Effects 0.000 abstract description 17
- 238000010672 photosynthesis Methods 0.000 abstract description 17
- 238000011161 development Methods 0.000 abstract description 9
- 240000003010 Oryza longistaminata Species 0.000 abstract description 3
- 235000007189 Oryza longistaminata Nutrition 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 230000001105 regulatory effect Effects 0.000 abstract description 3
- 241000196324 Embryophyta Species 0.000 description 30
- 230000018109 developmental process Effects 0.000 description 8
- 241000589158 Agrobacterium Species 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 238000009395 breeding Methods 0.000 description 5
- 230000001488 breeding effect Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000001737 promoting effect Effects 0.000 description 5
- 108090000848 Ubiquitin Proteins 0.000 description 4
- 102000044159 Ubiquitin Human genes 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000003208 gene overexpression Methods 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- 230000008595 infiltration Effects 0.000 description 4
- 238000001764 infiltration Methods 0.000 description 4
- 230000002018 overexpression Effects 0.000 description 4
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 230000001568 sexual effect Effects 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 241000701489 Cauliflower mosaic virus Species 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 2
- 108010033040 Histones Proteins 0.000 description 2
- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 description 2
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000012364 cultivation method Methods 0.000 description 2
- 230000009025 developmental regulation Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000003147 molecular marker Substances 0.000 description 2
- 230000009456 molecular mechanism Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 108020005345 3' Untranslated Regions Proteins 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- XZWYTXMRWQJBGX-VXBMVYAYSA-N FLAG peptide Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 XZWYTXMRWQJBGX-VXBMVYAYSA-N 0.000 description 1
- 102100022366 Fatty acyl-CoA reductase 1 Human genes 0.000 description 1
- 101150066002 GFP gene Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- 101000824458 Homo sapiens Fatty acyl-CoA reductase 1 Proteins 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 240000006394 Sorghum bicolor Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 238000010459 TALEN Methods 0.000 description 1
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 108010028230 Trp-Ser- His-Pro-Gln-Phe-Glu-Lys Proteins 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 101150054900 gus gene Proteins 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000011869 shoot development Effects 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a novel gene FarL from stamen wild rice (Oryza longistaminata) and application thereof, belonging to the technical fields of crop genetic engineering and molecular biology. The invention leads FarL gene into near isogenic line NIL-FarL of rice or over-expresses 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 show that FarL gene can raise rice yield by regulating photosynthesis and secondary branch development of rice to raise the number of spike and grain. Therefore, farL gene has great application prospect in cultivating new rice variety 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 FarL 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 FarL of long stamen wild rice, and further provides application of FarL gene in improving rice yield 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 FarL gene into rice or expresses FarL gene in rice body through rice sexual hybridization to find: after FarL gene is introduced into near isogenic line NIL-FarL of rice or FarL1 is expressed in rice variety 9311, its photosynthesis efficiency, number of secondary branches and stems of mature stage and number of grains per ear are obviously higher than 9311. This indicates FarL that the photosynthesis efficiency, the number of secondary branches and the number of ears and grains of rice are directly regulated by FarL 1. FarL1 is a long stamen wild rice specific gene which can run through interaction with histone H3K27Me2/3 demethylase, while histone H3K27Me2/3 demethylase and functional domains thereof are highly conserved in other important grain crops (sorghum, corn and the like), which shows that the gene may have a similar molecular mechanism in terms of regulating the yield of other crops. Therefore FarL1 is likely to regulate the yield of other monocotyledonous gramineous crops also by similar molecular mechanisms.
FarL 1A protein involved in photosynthesis and secondary branch development regulation of rice is derived from long stamen wild rice (Oryza longistaminata), and the amino acid sequence of the protein is shown as SEQ ID NO. 4.
To facilitate the study and use of FarL protein, a tag as shown in Table1 may be attached at the amino-or carboxy-terminus of the protein sequence.
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 |
A FarL gene involved in photosynthesis and secondary branch development regulation of rice has the nucleotide sequence of any one of the sequences shown in 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.
FarL1 gene has the application of promoting photosynthesis of rice and secondary branch development so as to improve rice yield. Correspondingly, the primer for amplifying the full length of FarL gene, the expression cassette, recombinant vector, transgenic cell line or recombinant bacterium containing FarL gene also has the application of promoting the photosynthesis of rice and secondary branch development so as to improve the yield of rice
The FarL gene can obviously increase the number of the ears per cluster 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 FarL gene full length is applied to cultivation of rice high-yield varieties.
The application of FarL gene-containing expression cassette, recombinant vector, transgenic cell line or recombinant bacteria in cultivating rice variety with high yield.
Improving photosynthesis efficiency, secondary branch number and rice yield of the rice can be realized by introducing FarL1 into a cultivated rice variety through hybridization transfer or transferring FarL1 into the cultivated rice through transgene to enable normal expression or over-expression, and the specific operation method is that FarL1 gene is introduced into other rice varieties through sexual hybridization with other rice varieties through using long stamen wild rice or NIL-FarL1 carrying FarL gene; or through transferring the expression vector into rice body to make FarL a over-express.
Existing crop transformation vectors may be selected for the construction of recombinant vectors containing FarL a 1. 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 utilizing FarL genes, any promoter which is helpful for changing FarL gene expression, such as a cauliflower mosaic virus (CAMV) 35S promoter, a Ubiquitin (Ubiquitin) gene promoter (pUbi) and the like, can be added before a gene start site when constructing a vector, and in addition, 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 FarL protein structure.
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 FarL a can be transformed into crop tissue or cells by various means, such as microinjection, agrobacterium-mediated genetic transformation, by common assay methods such as Ti plasmid, ri plasmid, or viral vector.
The FarL gene itself can also be used as a molecular marker in rice breeding.
The rice includes indica rice, japonica rice, etc.
The invention promotes photosynthesis of rice and development of secondary branches by a method of transferring FarL1 into rice through sexual hybridization gene transfer or transgenic technology among rice varieties, which shows that the gene can be applied to breeding of high-yield crops of rice as improvement of photosynthesis of rice and increase of secondary branches. Therefore, farL genes provide powerful means and tools for breeding rice high-yield new varieties by using molecular marker assisted breeding and using genetic engineering methods, and have 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 pattern of FarL.sup.1 near isogenic line (NIL-FarL 1).
FIG. 3 is a FarL gene structure diagram containing the FAR1 DNA binding domain.
FIG. 4 is a schematic diagram of FarL gene overexpression vector structure.
FIG. 5 is a schematic representation of the FarL gene-encoded protein.
FIG. 6 is a molecular assay of FarL1 in an overexpressing transgenic plant.
FIG. 7 is an analysis of FarL1 expression levels in NIL-FarL1 and FarL1 transgenic lines (over-expressed OE).
FIG. 8 is the net photosynthetic rate of the sword leaf over-expressed by 9311, NIL-FarL1 and FarL 1.
FIG. 9 is a plant type and ear type of FarL1 over-expressed transgenic lines. FarL1-OE represents FarL1 over-expressed transgenic line.
Detailed Description
The following is how to increase photosynthesis and secondary shoot development of rice by using FarL gene so as to increase the number of grains per ear of rice and further increase 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 FarL1 high-yield near isogenic line (NIL-FarL 1) in 9311 background
1. Hybridization is carried out by using long stamen wild rice and excellent indica rice variety 9311 to obtain F 1, backcrossing is carried out with 9311 twice to obtain BC 2F1, bagging is carried out for selfing for 20 generations, and stable homozygous long stamen wild rice chromosome segment infiltration line population is obtained. 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 FarL.
2. Construction of FarL 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 termed the FarL near isogenic line (NIL-FarL 1). The spike of NIL-FarL1 was enlarged and the number of secondary shoots was significantly increased compared to 9311 (see FIG. 2). Further examination of the basic agronomic traits of NIL-FarL1 revealed 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 increased by 16.34 and 63.56 grains respectively compared with 9311, and the yield of the single plant increased 25.24% compared with 9311 (see Table 2).
Yield trait statistics for Table 2.9311 and near NIL-FarL1 isogenic lines
Example 2: farL1 creation of high-yield transgenic line
1. Acquisition of full-Length fragment of FarL Gene
The cDNA of the long stamen wild rice chromosome fragment introduction system 1762 is used as a template to design a primer pair FarL-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. A schematic diagram of the FarL gene structure is shown in FIG. 3.
TABLE 3 primer sequences
| Primer name | Primer sequence (5 '-3') |
| FarL1-F | ATGGACGAGGACGAAGT |
| FarL1-R | TCACCTCCAGAACACTAGC |
2. Construction of FarL Gene overexpression vector
The product obtained by amplification of primer pair FarL-F/R is inserted into an expression vector pCAMBIA1301 containing a strong promoter (a Ubiquitin promoter) through recombination reaction, and positive clones are screened by using a marker gene on the vector, so that a recombinant expression vector FarL-OE is obtained.
3. Obtaining FarL Gene overexpression transgenic plants
The constructed FarL-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.
Infection of 9311 callus by recombinant Agrobacterium strain containing recombinant plasmid FarL-OE, and dark culture on screening medium containing 50mg/L hygromycin to obtain positive transgenic callus. And differentiating positive callus, rooting and transplanting to obtain T 0 -generation plants. Obtaining the T 1 generation plants through conventional molecular detection and rice cultivation methods. The model structure of FarL gene in over-expression rice line is shown in figure 4, CDS sequence of FarL1 is shown in SEQ ID NO. 3; the amino acid sequence of FarL gene coded protein 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 FarL Gene-Positive Strain
Genomic DNA of FarL1 over-expressed plants and wild plants is obtained by using a conventional CTAB extraction genomic DNA method, and a forward primer (pCAMBIA 1301-F) and a FarL1 gene specific primer (FarL OE-R) on a pCAMBIA1301 over-expression vector are designed (the primer sequences are shown in Table 4); genomic DNA of FarL1 over-expressed plants and genomic DNA of wild plants were amplified using genomic DNA of wild plants as negative control, and all FarL1 amplified bands in over-expressed 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 FarL Gene expression level 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 FarL over-expressed plants and wild type plants was obtained using a conventional RNA extraction method, and corresponding cDNA was obtained using a reverse transcription kit (purchased from Invitrogen). The expression level of FarL1 is detected by qRT-PCR by using FarL RT-F/R primer pairs by taking an action gene as an internal reference (the primer sequence is shown in table 5), and the expression level of a FarL1 gene over-expression plant is obviously improved (shown in 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 FarL gene over-expressed plants 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 (7)
1. A high-yield and high-light-efficiency gene FarL gene of long stamen wild rice is characterized in that: the amino acid sequence of the protein coded by the FarL gene is shown as SEQ ID NO. 4.
2. The FarL gene according to claim 1, wherein: the nucleotide sequence of FarL gene is any one of the sequences shown in SEQ ID NO.1, 2 or 3.
3. Use of the FarL gene according to 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 an expression cassette, recombinant vector, transgenic cell line or recombinant bacterium comprising the FarL 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.
5. Use according to claim 3 or 4, characterized in that: the rice includes indica rice and japonica rice.
6. Use according to claim 3 or 4, characterized in that: the application is realized by introducing FarL genes through hybridization transfer or improving the expression quantity of FarL1 through transgenesis.
7. A method for improving rice yield, which is characterized by comprising the following steps: for introducing FarL gene of claim 1 into rice by hybridization transfer or transferring into rice by expression vector, the FarL gene of claim 1 is over-expressed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310877712.0A CN116789785B (en) | 2023-07-18 | 2023-07-18 | High-yield and high-light-efficiency gene FarL a of long stamen wild rice and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310877712.0A CN116789785B (en) | 2023-07-18 | 2023-07-18 | High-yield and high-light-efficiency gene FarL a of long stamen wild rice and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116789785A CN116789785A (en) | 2023-09-22 |
| CN116789785B true CN116789785B (en) | 2024-04-19 |
Family
ID=88039585
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310877712.0A Active CN116789785B (en) | 2023-07-18 | 2023-07-18 | High-yield and high-light-efficiency gene FarL a of long stamen wild rice and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116789785B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2023
- 2023-07-18 CN CN202310877712.0A patent/CN116789785B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| Favorable QTLs from Oryza longistaminata improve rice drought resistance;Shaoying Huang等;BMC Plant Biology;第22卷;文章编号136 * |
| 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;徐高峰等;中国水稻科学;第24卷(第01期);62-66 * |
| 非洲长雄蕊野生稻的遗传特性及其固定杂种优势研究探讨;陈建三等;中国农业科学;第28卷(第05期);22-28 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116789785A (en) | 2023-09-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104293828B (en) | Plant Genome pointed decoration method | |
| US10982225B2 (en) | Flowering time-regulating genes and related constructs and applications thereof | |
| CN110904071B (en) | Application of RAF49 protein and encoding gene thereof in regulation and control of plant drought resistance | |
| WO2023065966A1 (en) | Application of bfne gene in tomato plant type improvement and biological yield increase | |
| WO2019129145A1 (en) | Flowering time-regulating gene cmp1 and related constructs and applications thereof | |
| CN107253980B (en) | Application of OsGRF7 gene in rice plant type regulation | |
| WO2015007241A1 (en) | Molecular marker | |
| CN105647940B (en) | The method and its application of OsGRF6 gene raising rice yield | |
| CN110881367A (en) | Corn event Ttrans-4 and methods of use thereof | |
| CN111662366A (en) | Preparation method of early-flowering high-yield tomato material | |
| CN116891862B (en) | Zoysia japonica salt tolerance gene ZmLA1, protein and application thereof | |
| CN107325161B (en) | Protein related to low-nitrogen stress and high-salt stress resistance as well as encoding gene and application thereof | |
| CN116789785B (en) | High-yield and high-light-efficiency gene FarL a of long stamen wild rice and application thereof | |
| CN114736280A (en) | Application of ZmROA1 protein in regulation and control of plant tolerance | |
| CN105175519B (en) | Applications of the Protein S RL2 in cultivating leaf roll Qushui River rice | |
| CN113416238B (en) | ZmbHLH148 protein and application of coding gene thereof in regulation and control of plant drought resistance | |
| CN109609516B (en) | Application of disease-resistant gene in rice false smut resistance improvement | |
| CN103665129B (en) | One kind of plant associated protein TaMYB72 at heading stage and application thereof | |
| CN101906426B (en) | Method for regulating plant photoperiod by combining soybean gibberellin with protein gene | |
| CN117946232A (en) | Long stamen wild rice large spike high yield gene OlGn8.2 and application thereof | |
| CN111394500B (en) | Method for identifying whether a test plant sample is derived from the SbSNAC1-382 event or a progeny thereof | |
| CN112553224B (en) | Application of histone deacetylase gene OsHDT701 in prolonging life of plant seeds | |
| CN113136388B (en) | Application of rice OsMAPKKK5 gene in aspect of improving plant height and grain type of rice | |
| CN114516906B (en) | Corn and mycorrhizal fungi symbiotic related protein, and coding gene and application thereof | |
| CN117305266B (en) | Gene OsBDG1 related to rice stress resistance and application of coded protein thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |