CN112226458B - Method for improving rice yield by using rice osa-miR5511 gene - Google Patents
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Abstract
The invention relates to the technical field of plant genetic engineering, in particular to a method for improving rice yield by using a rice osa-miR5511 gene. The method inhibits the expression of osa-miR5511 gene by means of genetic engineering, wherein the mature sequence of the rice osa-miR5511 gene is shown as SEQ ID NO.1, and the hairpin structure sequence is shown as SEQ ID NO. 2. The invention discovers that the spike length of osa-miR5511 deletion expression rice is remarkably increased by 7.2cm, the grain number of each spike is remarkably increased by 23.7 grains, the thousand seed weight is remarkably increased by 3.0 g, the yield of a single plant is remarkably increased by 3.6 g, the yield of the single plant is increased by 24.2%, a new molecular breeding way is provided for culturing high-yield rice fine varieties, and the application prospect is wide.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a method for improving rice yield by using a rice osa-miR5511 gene.
Background
Rice is the main food crop on the earth, and approximately half of the population on the earth is cultivated. In recent years, the world population has increased year by year, the environment has gradually worsened, and the effective cultivated land area has also gradually decreased. Therefore, how to increase the yield of rice to ensure the world grain safety becomes one of the focus of attention of government departments and researchers in various countries in the world. The molecular design breeding by utilizing a genetic engineering means is an important means for improving the yield of rice and guaranteeing the world food safety.
The yield of each rice plant is mainly determined by the characters such as the effective spike number, the grain number per spike, the thousand kernel weight and the like, the grain number per spike is determined by the characters such as the spike length, the primary branch stalk, the secondary branch stalk, the grain density and the like, and the thousand kernel weight is directly determined by the grain length, the grain width, the grain thickness, the grain filling degree and the like. Generally, the rice yield formation related traits are usually quantitative traits regulated by multiple genes, and the genetic basis is complex; besides the coding genes of structural proteins, transcription factors and the like, non-coding RNA genes such as miRNA and the like also have very important regulation and control functions in the formation of rice yield; therefore, mining miRNA genes having important roles in the formation of rice yield traits and analyzing the action mechanism of the miRNA genes are also important aspects in the development of molecular design breeding by using genetic engineering means to culture new high-yield rice varieties in the future.
miRNA is an endogenous non-coding RNA with the size of 20-24nt, which is widely existed in a plant body, and the growth and development of plant individuals or the adaptability to a stress environment are regulated by regulating the expression of target genes of the miRNA. It has been reported that there are at least 592 miRNA precursors in the rice genome and that 713 or more mature miRNAs can be produced [ Kozomara and Griffiths-Jones.miRBase: inactive high confidence microRNAs using deep sequencing data. nucleic Acids Research,2014,42: D68-D73 ]. At present, some miRNAs related to the development and control of rice yield traits have been discovered. For example, miR156 can influence the formation of traits such as tiller number, grain number, thousand grain weight, single plant yield and the like of rice by regulating the expression of an ideal plant type IPA1/OsSPL14 gene [ Jiano et al regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice in Nature Genetics,2010,42:541-544.Miura et al OsSPL14 proteins company and highher plant growth in rice Nature Genetics,2010,42: 545. 549 ]. The overexpression OsmiR397 can target and control the expression level of OsLAC gene to promote the branching of rice panicle, increase the size of rice grains and finally improve the single plant yield of rice [ Zhang et al. overexpression of microRNA OsmiR397 improves rice seed by creating grain size and promoting rice panicle branching. Nature Biotechnology,2013,31:848-852 ]. miR159 can influence the growth of yield traits such as grain size of rice by regulating the expression levels of target genes OsGAYBL 1 and OsGAYB [ ZHao et al. However, the improvement of rice yield by inhibiting the expression of osa-miR5511 has not been reported at present.
Disclosure of Invention
The invention aims to provide a method for improving rice yield by using a rice osa-miR5511 gene. According to the method, the expression of the osa-miR5511 gene is inhibited by a genetic engineering means, and the fact that the osa-miR5511 deletion expression can obviously increase the ear length, the grain number per ear, the grain weight and the single plant yield of rice in the mature period is found for the first time. Provides a new molecular breeding method for cultivating high-yield rice fine varieties.
In order to realize the purpose, the invention adopts the technical scheme that:
the invention provides a method for improving rice yield by using a rice osa-miR5511 gene, which is characterized in that the rice osa-miR5511 gene is knocked out or expression of the rice osa-miR5511 gene is inhibited by a genetic engineering technology, a mature sequence of the rice osa-miR5511 gene is shown as SEQ ID No.1, and a hairpin structure sequence of the rice osa-miR5511 gene is shown as SEQ ID No. 2.
The invention preferably inhibits or knocks out the expression of the osa-miR5511 gene of rice by a CRISPR gene editing technology, and comprises the following steps:
1) construction of CRISPR knockout expression vector of osa-miR 5511: the CRISPR knockout target primer pair, a rice U6a promoter and a pYLCRISPR/Cas9 plasmid are used for obtaining a CRISPR knockout expression vector of osa-miR5511 through enzyme digestion, connection, transformation of escherichia coli competent cells, positive colony PCR detection and sequencing analysis;
2) obtaining high-yield rice plants with osa-miR5511 deletion expression: directly transforming rice callus/cells by using the CRISPR knockout expression vector for constructing osa-miR5511 through Ti plasmids, plant virus vectors, microinjection, protoplasts or DNA, culturing to obtain transgenic rice plants, identifying by using PCR (polymerase chain reaction), sequencing analysis, qRT-PCR (quantitative reverse transcription-polymerase chain reaction) and Northern-blot methods to obtain transgenic rice with osa-miR5511 deletion expression, planting the transgenic rice in a field seed together with wild rice, and performing seed test comparison analysis in a mature period to determine to obtain high-yield rice plants with osa-miR5511 deletion expression.
The invention provides application of a rice osa-miR5511 gene in rice yield improvement, which inhibits expression of the rice osa-miR5511 gene and is used for breeding rice varieties with rice yield improvement.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for improving the rice yield by using the osa-miR5511 gene for the first time, the method improves the rice yield by inhibiting the expression of the osa-miR5511 gene, provides a new molecular breeding way for culturing high-yield rice fine varieties, and has wide application prospect. The invention can obviously increase the ear length, the grain number per ear, the grain weight and the single plant yield of rice plants.
Drawings
FIG. 1 is a schematic diagram of the structure of pYLCRISPR/Cas9 plasmid. Wherein, P35SThe 35S promoter of cauliflower mosaic virus; pubiIs a maize ubiquitin protein gene promoter; ccdB is a ccdB lethal gene, and is replaced by an sgRNA expression cassette after being digested by BsaI so as to complete construction of a knockout vector; tnos is a terminator; RB is the right border; LB is the left border; kan (Chinese character)RIs a kanamycin resistance gene; HPT is hygromycin resistance gene; pBR322 ori is the replication initiation site of pBR 322; cas9p is a Cas9 protein gene; SP-L1 and SP-R are colony PCR primer pairs and sequencing primers.
Fig. 2 shows molecular identification of CRISPR knockout deletion expression mutants of osa-miR 5511. A: stem loop, mature (red) and target (yellow background) sequences of osa-miR 5511; b: sequencing and analyzing the target point mutation type; c: Northern-Blot hybridization was performed to detect the expression of mature osa-miR 5511. NIP: nipponbare (wild type); CR: the CRISPR knockout deletion of osa-miR5511 expresses the mutant miR 5511. U6 is the reference gene for Northern-Blot hybridization.
FIG. 3 shows that deletion of osa-miR5511 expression results in spike enlargement in rice. A: overall spike phenotype at maturity; b: ear length in mature period; c: total grains per ear in the maturation period; d: thousand grain weight in the mature period; e: yield per plant at maturity. NIP: nipponbare; CR: the osa-miR5511 deletion expression mutant miR 5511. **: very significant differences at p <0.01 levels.
Detailed Description
The experimental procedures used in the following examples are conventional ones unless otherwise specified. In the present invention, the synthesis of primers or the determination of DNA sequences are carried out by Shanghai bioengineering technology, Inc.
Example 1: construction of CRISPR knockout vector of osa-miR5511 and rice genetic transformation
Obtaining a mature sequence SEQ ID NO.1 and a hairpin structure sequence SEQ ID NO.2 of rice osa-miR5511 from a miRBase database (http:// www.mirbase.org /), and designing an on-line software CRISPR-P2.0(http:// CRISPR. hzau. edu. cn/CRISPR2/) to obtain a CRISPR knockout target primer pair SEQ ID NO.3 of the osa-miR 5511:GCCGCATGCATATCCCAGCTGTTT (Bsa I restriction site underlined) and SEQ ID NO. 4:AAACAAACAGCTGGGATATGCATG (Bsa I cleavage site underlined); then, the recombinant plasmid is connected with a rice U6 promoter through denaturation annealing to form a sgRNA expression cassette, and then the expression cassette is connected into a pYLCRISPR/Cas9 plasmid through enzyme digestion to obtain a CRISPR knockout vector of miR5511, and further refer to the method of the predecessor [ Hiei et al, effective transformation of rice (Oryza sativa L.), mediated by Agrobacterium and sequence analysis of the cloning of the T-DNA plant J,1994,6(2):271-]The method comprises the steps of utilizing an agrobacterium-mediated transformation method to transform japonica rice Nipponbare (NIP), carrying out callus induction, callus subculture, pre-culture, co-culture, resistant callus screening, pre-differentiation, rooting culture, seedling hardening, transplanting and the like to obtain 24 genetically transformed rice plants in total, extracting genome DNA of the genetically transformed rice plants based on a CTAB method, and utilizing hygromycin gene specific inductionPCR identification is carried out on the plant, all obtained plants are determined to be transgenic positive rice plants, and the sequence of a specific primer pair for hygromycin gene amplification is as follows:
HPT-F：5’-CTGAACTCACCGCGACGTCTGTC-3'(SEQ ID NO.5)；
HPT-R：5’-TAGCGCGTCTGCTGCTCCATACA-3’(SEQ ID NO.6)；
the PCR amplification conditions were: 4min at 94 ℃; [94 ℃ 30sec, 62 ℃ 30sec, 72 ℃ 50sec ], 32 cycles; 7min at 72 ℃.
Example 2: identification of CRISPR knockout deletion expression mutant of osa-miR5511 (miR5511)
According to CRISPR knockout target sequence information (figure 2A) of osa-miR5511, a pair of specific primers (SEQ ID NO.7 and SEQ ID NO.8) are designed on a corresponding rice genome, wild type (Nipponbare) and 24 parts of rice genome DNA extracted above are taken as templates, a single target genome DNA fragment containing the knockout target sequence is obtained through amplification, sequencing analysis is carried out, and T with knockout mutation is determined to be obtained0And (3) performing hygromycin gene positive detection and target site sequencing analysis on the transgenic plant in the seedling stage of the separated offspring to obtain an osa-miR5511 knockout mutant without T-DNA insertion. One of the knockout homozygous mutants (CR) without exogenous T-DNA with 1A base inserted in the mature osa-miR5511 sequence (FIG. 2B) was confirmed by Northern-Blot hybridization to show that the mature osa-miR5511 is deleted in the CR mutant (FIG. 2C). The Northern-Blot hybridization probe sequence is SEQ ID NO.9(GGCCAAACAGCTGGGATATG), wherein the nucleotide at the 5 'end is modified with DIG and 3 nucleotides at the 3' end are LNA locked.
Example 3: Osa-miR5511 deletion expression obviously improves single-plant yield of rice
Subsequently, wild-type Nipponica (NIP) and osa-miR5511 deletion expression mutant (CR) were simultaneously planted in the field, and found at the mature stage: the ears of the osa-miR5511 deletion-expressing mutant CR were significantly larger than those of the wild-type NIP (fig. 3A). Then, representative 5 individuals were selected as samples for each of NIP and CR, and the number of grains per ear, the length of ears, the thousand kernel weight, and the yield per plant were examined for effective tillering of each individual plant. The results (FIGS. 3B-E) show that: compared with NIP, the ear length of CR is obviously increased by 7.2cm, the grain number of each ear is obviously increased by 23.7 grains, the thousand grain weight is obviously increased by 3.0 g, the yield of a single plant is obviously increased by 3.6 g, and the yield of the single plant is increased by about 24.2%.
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Claims (4)
1. A method for improving rice yield by using a rice osa-miR5511 gene is characterized in that the rice osa-miR5511 gene is knocked out or expression of the rice osa-miR5511 gene is inhibited by a genetic engineering technology, a mature sequence of the rice osa-miR5511 gene is shown as SEQ ID No.1, and a hairpin structure sequence of the rice osa-miR5511 gene is shown as SEQ ID No. 2.
2. The method for improving rice yield by using the rice osa-miR5511 gene as claimed in claim 1, wherein the genetic engineering technology is CRISPR gene editing technology.
3. The method for improving the yield of the rice by using the rice osa-miR5511 gene as claimed in claim 2, wherein the rice osa-miR5511 gene is knocked out or the expression of the gene is inhibited by a CRISPR gene editing technology, and the method comprises the following steps:
1) construction of CRISPR knockout expression vector of osa-miR 5511: the CRISPR knockout target primer pair, a rice U6a promoter and a pYLCRISPR/Cas9 plasmid are used for obtaining a CRISPR knockout expression vector of osa-miR5511 through enzyme digestion, connection, transformation of escherichia coli competent cells, positive colony PCR detection and sequencing analysis;
2) obtaining high-yield rice plants with osa-miR5511 deletion expression: directly transforming rice callus/cells by using the CRISPR knockout expression vector for constructing osa-miR5511 through Ti plasmids, plant virus vectors, microinjection, protoplasts or DNA, culturing to obtain transgenic rice plants, identifying by using PCR (polymerase chain reaction), sequencing analysis, qRT-PCR (quantitative reverse transcription-polymerase chain reaction) and Northern-blot methods to obtain transgenic rice with osa-miR5511 deletion expression, planting the transgenic rice with wild rice in a field, and performing species test comparison analysis in a mature period to determine to obtain high-yield rice plants with osa-miR5511 deletion expression.
4. The application of the rice osa-miR5511 gene in improving the rice yield is characterized in that the expression of the rice osa-miR5511 gene is inhibited, and a rice variety with the improved rice yield is bred.
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