CN112226457B - Application of rice osa-miR5504 gene in rice dwarf breeding - Google Patents
Application of rice osa-miR5504 gene in rice dwarf breeding Download PDFInfo
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Abstract
The invention relates to the technical field of plant genetic engineering, in particular to application of a rice osa-miR5504 gene in rice dwarf breeding, wherein the rice osa-miR5504 gene is knocked out or inhibited to express through a genetic engineering technology, and a rice variety with short stems is bred, wherein the mature sequence of the rice osa-miR5504 gene is shown as SEQ ID No.1, and the hairpin structure sequence is shown as SEQ ID No. 2. According to the invention, the expression of osa-miR5504 is inhibited by using a genetic engineering technology, the fact that the osa-miR5504 deletion expression can obviously reduce the plant height of rice and increase the effective tiller number of a single rice plant is found for the first time, a new molecular breeding approach is provided for developing rice dwarf breeding by further using the osa-miR5504 gene, and the application prospect is wide.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of osa-miR5504 in rice dwarf breeding, wherein molecular breeding of a new dwarf and lodging-resistant rice variety is created by inhibiting expression of osa-miR 5504.
Background
Rice lives nearly half of the world population. Therefore, how to guarantee the yield and the quality of the rice is very important for solving the world food safety problem. In the rice production process, due to the influence of various artificial or natural factors such as typhoons, hurricanes, fertilizer and water management and the like, rice plants often fall down to different degrees, so that the yield and the quality of rice are obviously reduced, and the world grain safety faces severe examination.
Under normal conditions, high-stalk rice varieties are easy to lodging, and the lodging resistance of low-stalk rice varieties is obviously enhanced; therefore, the cultivation of excellent dwarf rice varieties by using modern genetic engineering means to reduce the lodging rate of the rice varieties and improve or guarantee the yield and quality of the rice varieties is one of the focus problems of modern rice molecular breeding.
miRNA (MicroRNA) is a 20-24nt endogenous small molecule non-coding RNA of a type generally existing in organisms. Research shows that miRNA mainly influences plant growth and development and stress adaptation through controlling the expression level of target genes which directly act on miRNA. It is now known that at least 592 miRNA precursors exist in rice, producing at least 713 mature mirnas. At present, some miRNAs related to the regulation of characters such as rice plant height and the like are discovered. For example, miR159 can positively regulate various Plant type traits of rice, including length and thickness of stem nodes, etc. by affecting the expression of its target genes, OsGAMYB and OsGAMYBL1 [ Zhao et al: suppression of microRNA159 imprints multiple agronomic traits in rice (Oryza sativa L.). BMC Plant Biology,2017,17:215 ]; the microRNA172 can influence the stem length of rice by regulating the expression of SUI4/SNB gene [ Ji et al. mutations in the binding site of microRNA172 binding site of SUPERNUERARY BRACT (SNB) compression interaction in rice, Rice,2019,12:62 ]. The patent discloses a method for creating a dwarfing rice variety by inhibiting the expression of osa-miR5504 for the first time, and the method is not reported at present, so that a new molecular breeding approach is provided for cultivating a lodging-resistant excellent dwarfing rice variety by using the osa-miR 5504.
Disclosure of Invention
The invention aims to provide application of osa-miR5504 in rice dwarf breeding. The gene engineering technology is applied to inhibit the expression of the osa-miR5504 gene, and a novel molecular breeding method is provided for culturing excellent dwarf rice varieties by using the osa-miR5504 gene.
In order to achieve the purpose, the invention adopts the technical scheme that:
the application of the rice osa-miR5504 gene in rice dwarf breeding is realized by knocking out or inhibiting the expression of the rice osa-miR5504 gene through a genetic engineering technology to breed a rice variety with short stems, wherein the mature sequence of the rice osa-miR5504 gene is shown as SEQ ID No.1, and the hairpin structure sequence of the rice osa-miR5504 gene is shown as SEQ ID No. 2.
The invention preferably selects CRISPR gene editing technology to knock out or inhibit the expression of the rice osa-miR5504 gene, and comprises the following steps:
(1) construction of CRISPR/Cas9 knockout expression vector of osa-miR 5504: the CRISPR/Cas9 knockout expression vector of osa-miR5504 is obtained by utilizing a CRISPR/Cas9 knockout target primer pair, a rice endogenous U6a promoter and a pYLCRISPR/Cas9 plasmid through restriction enzyme digestion, connection, transformation, colony PCR detection and sequencing analysis of sgRNA sequence;
(2) obtaining an osa-miR5504 deletion expression dwarf rice plant: the constructed CRISPR/Cas9 knockout expression vector of osa-miR5504 is transformed into japonica rice Nipponbare through an agrobacterium-mediated method to obtain a transgenic rice plant, in the subsequent generation separation process, PCR, sequencing analysis, qRT-PCR and Northern-blot hybridization methods can be adopted to identify and obtain an osa-miR5504 deletion expression transgenic homozygous rice plant without T-DNA insertion and off-target phenomena, the transgenic rice plant and wild rice are simultaneously planted in a field, and the plant height and the effective tiller number are compared and analyzed in the late heading stage and the mature stage to determine to obtain the dwarf rice plant with osa-miR5504 deletion expression.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides application of a rice osa-miR5504 gene in rice dwarf breeding, the height of a rice plant is reduced by inhibiting the expression of the osa-miR5504 gene, rice lodging is prevented, the aim of high yield or stable yield is achieved, a new molecular breeding way is provided for culturing an excellent dwarf rice variety by using the osa-miR5504 gene, and the application prospect is wide. The invention can obviously reduce the plant height of the rice and can also obviously increase the effective tiller number of a single plant of the rice.
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 is the sequencing identification of CRISPR/Cas9 knockout target and off-target sites of osa-miR 5504. A: stem-loop structure sequence, mature body sequence (red character sequence) and knockout target sequence (yellow base sequence) of osa-miR 5504; b: sequencing and analyzing the target point mutation type; c: possible off-target sequences and their location on the genome; D. e and F: sequencing results of the 3 most probable non-mutation off-target sequences in the genome of NIP, CR1 and CR2 mutants, namely Os09g0307050, Os11g0171500 and Os12g 0169950. NIP is wild type rice Nipponbare, CR1 is a mutant with 1C base deletion in the osa-miR5504 mature body sequence, and CR2 is a mutant with 12 base deletion in the osa-miR5504 mature body sequence.
FIG. 3 is the expression detection of mature osa-miR5504 in CRISPR/Cas9 transgenic homozygous mutant. A: and (5) identifying the result of Stem loop qRT-PCR. B: and identifying the result of Stem loop semi-quantitative RT-PCR. C: and (3) detecting the result of Northern-Blot hybridization. M: marker DNA; NIP: nipponbare (wild type); CR1 and CR 2: CRISPR/Cas9 knockout homozygous mutants of 2 miR5504, respectively; u6 is an internal reference gene.
FIG. 4 shows the plant height and the effective spike number per plant of osa-miR5504 deletion-expressed rice. A: the plant height of the osa-miR5504 deletion expression mutant at the late heading stage. B: and (5) measuring and counting the plant height of osa-miR5504 in the mature period. C: and measuring the length of each stem node of all effective tillers of each single plant of the osa-miR5504 mutant in the mature period. NIP: nipponbare (wild type); CR1 and CR 2: are osa-miR5504 deletion expression mutants respectively. I. II, III, IV and V: the 1 st, 2 nd, 3 rd, 4 th and 5 th nodes next to the ear. **: very significant differences with p < 0.01.
Detailed Description
The experimental procedures referred to in the following examples are conventional ones unless otherwise specified. In the present invention, both the primer sequence synthesis and DNA sequence sequencing analysis are performed by Shanghai bioengineering technology, Inc.
Example 1: CRISPR/Cas9 knockout vector construction of osa-miR5504 and rice genetic transformation
Obtaining a mature sequence SEQ ID NO.1 and a hairpin structure sequence SEQ ID NO.2 of rice osa-miR5504 in a miRBase database (http:// www.mirbase.org /), and designing a CRISPR/Cas9 knockout target primer pair SEQ ID NO.3 of the osa-miR5504 by utilizing online software CRISPR-P2.0(http:// CRISPR. hzau. edu. cn/CRISPR2 /):GCCGACAGTGACGGGAGGACTGCA (Bsa I restriction site underlined) and SEQ ID NO. 4:AAACTGCAGTCCTCCCGTCACTGT (underlined as B)sa I cleavage site); then, the recombinant plasmid is connected with a rice U6 promoter through denaturation annealing to form a sgRNA expression cassette, and then is connected into a pYLCRISPR/Cas9 plasmid through enzyme digestion to obtain a CRISPR/Cas9 knockout vector of miR5504, and further refer to the existing method [ Hiei et al, effective transformation of rice (Oryza sativa L.), medium by Agrobacterium and sequence analysis of the nucleic acids of the T-DNA plant J,1994,6(2): 271-282-]The agrobacterium-mediated method is used for transforming japonica rice Nipponbare, the general process can be induction, subculture, pre-culture, co-culture, resistance screening, differentiation, rooting culture, seedling hardening, transplanting and the like, and 33 genetic transformation T strains are obtained in total0The method comprises the following steps of (1) generating rice plants, extracting genome DNA by using a CTAB method, and then carrying out PCR identification on hygromycin gene specific primers to determine that the obtained transgenic rice plants are all positive, wherein the sequence of the hygromycin gene amplified specific primers 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: molecular identification of CRISPR/Cas9 knockout deletion expression mutants of osa-miR5504
According to the CRISPR/Cas9 knockout target sequence information (figure 2A) of osa-miR5504, a pair of specific primers (SEQ ID NO.7 and SEQ ID NO.8) are designed on the corresponding rice genome, wild type (Nipponbare) and 33 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 obtained0Transgenic plants are generated, and hygromycin gene positive detection and target site sequencing analysis are further carried out in the seedling stage of the separated offspring of the plants, so that osa-miR5504 knockout mutants without T-DNA insertion are obtained. Two of the homozygous knockout mutants (CR1 and CR2) (FIG. 2B-F) which have mutation in mature osa-miR5504 sequence, no foreign T-DNA insertion and no off-target phenomenon can be used in subsequent experiments, and are confirmed by RT-PCR, qRT-PCR and Northern-Blot hybridization experiments to obtain the final productMature osa-miR5504 was deletion-expressed in both CR1 and CR2 mutants (fig. 3). The Northern-Blot hybridization probe sequence is SEQ ID NO.9(CTTGCAGTCCTCCCGTCACT), in which the 5 'nucleotide is DIG modified and 3 nucleotides at the 3' end are LNA locked.
Example 3: the osa-miR5504 deletion expression obviously reduces the plant height of rice and increases the effective spike number of a single plant
Subsequently, wild-type Nipponica (NIP) and osa-miR5504 deletion expression mutants (CR1 and CR2) were planted in the field at the same time and found in the late stage of heading: the height of the osa-miR5504 deletion-expressing mutants CR1 and CR2 was significantly shorter than that of the wild-type NIP (fig. 4A). Then, comparative analysis of plant height and effective tiller number of NIP, CR1 and CR2 at mature stage revealed that: compared with NIP, the plant heights of CR1 and CR2 are obviously reduced (figure 4B), and the plant heights of CR1 and CR2 are obviously reduced and are closely related to the obvious shortening of the I and II stem nodes close to the ear (figure 4C); at the same time, the number of effective tillers of each of CR1 and CR2 was significantly increased (fig. 4D).
Sequence listing
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Claims (3)
1. The application of the rice osa-miR5504 gene in rice dwarf breeding is characterized in that the rice osa-miR5504 gene is knocked out or inhibited to express through a genetic engineering technology, and a rice variety with short stems is bred, wherein the mature sequence of the rice osa-miR5504 gene is shown as SEQ ID No.1, and the hairpin structure sequence of the rice osa-miR5504 gene is shown as SEQ ID No. 2.
2. The application of the rice osa-miR5504 gene in rice dwarfing breeding as claimed in claim 1, characterized in that the genetic engineering technology is CRISPR gene editing technology.
3. The application of the rice osa-miR5504 gene in rice dwarfing breeding according to claim 2, which is characterized in that the expression of the rice osa-miR5504 gene is inhibited or knocked out by a CRISPR gene editing technology, and comprises the following steps:
(1) construction of CRISPR/Cas9 knockout expression vector of osa-miR 5504: the CRISPR/Cas9 knockout expression vector of osa-miR5504 is obtained by utilizing a CRISPR/Cas9 knockout target primer pair, a rice endogenous U6a promoter and a pYLCRISPR/Cas9 plasmid through restriction enzyme digestion, connection, transformation, colony PCR detection and sequencing analysis of sgRNA sequence;
(2) obtaining of osa-miR5504 deletion expression dwarf rice plants: and (3) transforming the constructed CRISPR/Cas9 knockout expression vector of osa-miR5504 into japonica rice Nipponbare by an agrobacterium-mediated method to obtain a transgenic rice plant, identifying and obtaining the osa-miR5504 deletion expression transgenic homozygous rice plant without T-DNA insertion and off-target phenomenon by adopting sequencing analysis, qRT-PCR and Northern-blot hybridization methods in the subsequent generation separation process, simultaneously planting the transgenic homozygous rice plant and wild rice in a field, and performing comparative analysis on plant height and effective tillering number at the late heading stage and the mature stage to determine and obtain the dwarf rice plant with osa-miR5504 deletion expression.
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