CN116240236A - Method for regulating rice plant type by editing promoter of OsD gene of rice - Google Patents
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Abstract
The invention belongs to the technical field of plant biology, and particularly relates to a method for regulating and controlling rice plant types by editing a promoter of a OsD gene of rice. The invention aims to provide a new choice for improving the plant type of rice. The technical scheme of the invention is a method for regulating and controlling rice plant type by a promoter for editing a OsD gene of rice, s1, constructing a Cas gene editing expression vector for expressing guide RNA of the promoter for editing a OsD gene of rice; s2, transforming rice with the expression vector obtained in the step s1, and obtaining a transformed plant by using a CRISPR-Cas gene editing system; and s3, collecting seeds of the transformed plants, and screening out the seeds of the gene editing mutants to obtain the gene editing rice with improved plant types.
Description
Technical Field
The invention belongs to the technical field of plant biology, and particularly relates to a method for regulating and controlling rice plant types by editing a promoter of a OsD gene of rice.
Background
The plant type is a key factor for determining the crop yield, and the ideal rice plant type has the characteristics of moderate plant height, compact plant type, less tillering, no ineffective tillering, large grain size, thick stalks and the like. The most important event in the history of rice variety improvement and fine variety application in China is dwarf breeding starting in 1956, namely the first green revolution of rice. The rice local variety is basically of a high-stalk type, has poor fertility resistance, is easy to lodge, and causes the problem of stable yield. Therefore, the development of dwarf germplasm resources and the cultivation of dwarf varieties become very important. In 1956, the first dwarf early indica variety-dwarf southern special (short square) of China is cultivated by a breeder in China, and the dwarf variety is mainly characterized by dwarf, more ears, developed root systems and compact plant types, and has excellent fertilizer resistance and lodging resistance, and the harvest index is also greatly improved. In 1966, the International Rice institute (IRRI) hybridized Taiwan (Dee-geo-woo-gen) with Pitai (Peta) by using Taiwan local variety low-foot Wujian (Dee-geo-woo-gen) to develop semi-dwarf variety IRS, and then high-yield peculiar trace was created. Dwarf breeding in China is 10 years earlier than international whole breeding. Cultivation of 'Square short' and introduction of IRS promote rice breeding in China to enter the first 'green revolution' era. The gene determining this rice dwarf breeding is SD1 gene. SD1 alone has been used up to now in breeding. The genetic foundation is narrow, and the screening and the development of new plant type genes with breeding value have important significance for guaranteeing the grain safety of China.
Disclosure of Invention
The invention aims to provide a new choice for improving the plant type of rice.
The technical scheme of the invention is a method for editing a promoter of a OsD gene of rice to regulate a rice plant type, which comprises the following steps:
a. constructing a Cas gene editing expression vector for expressing a guide RNA of a promoter for editing the rice OsD gene;
b, transforming the rice by using the expression vector obtained in the step a, and obtaining a transformed plant by using a CRISPR-Cas gene editing system;
c. and collecting seeds of the transformed plants, and screening out the seeds of the gene editing mutants to obtain the gene editing rice with improved plant types.
Further, the guide RNA is at least one of crRNA01 (SEQ ID No. 1), crRNA03 (SEQ ID No. 3), crRNA05 (SEQ ID No. 5), crRNA07 (SEQ ID No. 7), crRNA09 (SEQ ID No. 9), crRNA11 (SEQ ID No. 11), crRNA02 (SEQ ID No. 2), crRNA04 (SEQ ID No. 4), crRNA06 (SEQ ID No. 6), crRNA08 (SEQ ID No. 8), crRNA10 (SEQ ID No. 10) or crRNA12 (SEQ ID No. 12).
Preferably, the guide RNA is crRNA01 (SEQ ID No. 1), crRNA03 (SEQ ID No. 3), crRNA05 (SEQ ID No. 5), crRNA07 (SEQ ID No. 7), crRNA09 (SEQ ID No. 9) and crRNA11 (SEQ ID No. 11); or crRNA02 (SEQ ID No. 2), crRNA04 (SEQ ID No. 4), crRNA06 (SEQ ID No. 6), crRNA08 (SEQ ID No. 8), crRNA10 (SEQ ID No. 10) and crRNA12 (SEQ ID No. 12).
Wherein the framework of the Cas gene editing expression vector is pTX377.
Specifically, the Cas gene editing expression vector is a CRISPR-Cas12a gene editing expression vector.
Further, the CRISPR-Cas12a gene editing expression vector comprises a Cas12a expression unit started by a corn ubiquitin promoter ZmUbi1 and a crRNA-scanfold expression unit started by a rice ubiquitin promoter OsUbi 1.
In particular, the crRNA-scafold expression unit expresses in tandem at least one of crRNA01 (SEQ ID No. 1), crRNA03 (SEQ ID No. 3), crRNA05 (SEQ ID No. 5), crRNA07 (SEQ ID No. 7), crRNA09 (SEQ ID No. 9), crRNA11 (SEQ ID No. 11), crRNA02 (SEQ ID No. 2), crRNA04 (SEQ ID No. 4), crRNA06 (SEQ ID No. 6), crRNA08 (SEQ ID No. 8), crRNA10 (SEQ ID No. 10) or crRNA12 (SEQ ID No. 12).
Wherein, the Cas gene editing expression vector further comprises a hygromycin resistance gene Hyg expression unit which is started by a CaMV35S promoter.
Preferably, the crRNA-scafold expression unit expresses the fragment shown as SEQ ID No.13 or SEQ ID No. 14.
The invention also provides a guide RNA or a nucleic acid molecule complementary thereto, wherein the guide RNA is at least one of crRNA01 (SEQ ID No. 1), crRNA03 (SEQ ID No. 3), crRNA05 (SEQ ID No. 5), crRNA07 (SEQ ID No. 7), crRNA09 (SEQ ID No. 9), crRNA11 (SEQ ID No. 11), crRNA02 (SEQ ID No. 2), crRNA04 (SEQ ID No. 4), crRNA06 (SEQ ID No. 6), crRNA08 (SEQ ID No. 8), crRNA10 (SEQ ID No. 10) or crRNA12 (SEQ ID No. 12).
In particular, the guide RNA is crRNA01 (SEQ ID No. 1), crRNA03 (SEQ ID No. 3), crRNA05 (SEQ ID No. 5), crRNA07 (SEQ ID No. 7), crRNA09 (SEQ ID No. 9) and crRNA11 (SEQ ID No. 11); or crRNA02 (SEQ ID No. 2), crRNA04 (SEQ ID No. 4), crRNA06 (SEQ ID No. 6), crRNA08 (SEQ ID No. 8), crRNA10 (SEQ ID No. 10) and crRNA12 (SEQ ID No. 12).
The invention also provides application of the guide RNA or the nucleic acid molecule which can be complemented with the guide RNA in improving rice plant types.
In particular to the application in reducing the plant height of rice and ensuring the size and weight of rice seeds.
The invention also provides a vector for expressing the guide RNA for editing the rice OsD gene promoter.
In particular, the guide RNA is at least one of crRNA01 (SEQ ID No. 1), crRNA03 (SEQ ID No. 3), crRNA05 (SEQ ID No. 5), crRNA07 (SEQ ID No. 7), crRNA09 (SEQ ID No. 9), crRNA11 (SEQ ID No. 11), crRNA02 (SEQ ID No. 2), crRNA04 (SEQ ID No. 4), crRNA06 (SEQ ID No. 6), crRNA08 (SEQ ID No. 8), crRNA10 (SEQ ID No. 10) or crRNA12 (SEQ ID No. 12).
Preferably, the vector for expressing the guide RNA of the rice OsD gene promoter is a CRISPR-Cas12a gene editing expression vector, and comprises a crRNA-scaffold expression unit started by the rice ubiquitin promoter Osubi 1.
Further, the CRISPR-Cas12a gene editing expression vector comprises a Cas12a expression unit started by a corn ubiquitin promoter ZmUbi1 and a hygromycin resistance gene Hyg expression unit started by a CaMV35S promoter.
Preferably, the crRNA-scafold expression unit expresses the fragment shown as SEQ ID No.13 or SEQ ID No. 14.
The invention also provides application of the vector for expressing the guide RNA editing the OsD gene promoter of the rice in improving rice plant types.
In particular to the application in reducing the plant height of rice and ensuring the size and weight of rice seeds.
Genes regulating plant types are mainly genes involved in hormone signaling pathways (biosynthesis/signaling pathways of gibberellin, brassinolide, strigolactone, etc.). Under the condition of fully consulting and analyzing the path genes, the invention selects the editing material and preferential editing site which can possibly regulate the expression quantity by editing the gene promoter to obtain the ideal plant type. After the genes of D18, D2, D11, SD1 and the like are edited, the gene with the optimal plant height regulated by the promoter editing is obtained by screening, and meanwhile, crRNA (guide RNA) with an ideal editing site is obtained.
The invention has the beneficial effects that: the invention provides a method for improving rice quality traits, which is based on a CRISPR-Cas12a gene editing system to directionally edit OsD gene promoter elements, regulate OsD gene expression, thereby changing plant height and improving rice plant type traits. In the method, 12 specific crRNAs are designed and screened aiming at the rice OsD gene promoter, and 2 CRISPR-Cas12a gene editing vectors for specifically editing rice OsD gene promoter elements are constructed on the basis of the crRNAs. The method can be used for efficiently knocking out OsD gene promoter elements of rice OsD gene promoter elements of rice to obtain new materials with different plant heights (30 mutants in 31 obtained mutants have obviously reduced plant heights, wherein the plant height reduction range is within 10 percent, 17 plants are 10-20 percent, 6 plants are 20-30 percent, and 1 plant height is less than 30 percent), and the size and weight of rice seeds are not changed. The method has simple steps, is easy to operate, and has good prospect in the aspects of plant type and genome function research and application of rice.
Drawings
FIG. 1. Knock-out of a rice OsD gene promoter element using a CRISPR-Cas12a multi-site directed editing system;
A. the rice OsD gene promoter is directed to edit the target site design schematic; ARE, TCA-element, CAT-box, MYC … and the like ARE predicted as cis-regulatory elements; crRNA01-crRNA12 is a promoter editing guide RNA based on Cas12a system; the sgrnas are guide RNAs edited based on the coding region of the Cas9 system.
B. Schematic design of CRISPR-Cas12a multi-site directed knockout vector; lbCAs12a V-type CRISPR-Cas protein from Lachnospiraceae bacterium; pZmUbi1, maize ubiquitin promoter; pOsubi1 rice ubiquitin promoter; hspT, hsp gene terminator; nosT, nos gene terminator; hyg+; HH, hammerhead ribozyme; HDV, hepatitis virus ribozyme; DR:
guide RNA motifs.
FIG. 2-CRISPR-Cas 12a multisite directed knockout mutant T0 identification map (note: light colored number indicates that large fragment deletion occurred for this individual).
FIG. 3 shows the plant height measurement of homozygous mutant T1 for the OsD18 promoter (black spots: representing five replicates; letters: representing differences between the different data sets of the Duncan test).
Fig. 4, part OsD promoter directed editing mutant T1 homozygous plant height (scale = 10 cm); plant height.
FIG. 5 part OsD promoter directed editing mutant T2 generation seed size measurement (black dot: representing duplicate experimental measurements; letter: representing differences between different data sets of the Duncan test). Phenotype of seed length seed length phenotype; phenotype of seed width seed width phenotype; seed length; seed width; 1000-grain weight: thousand grain weight.
Figure 6.Osd2 promoter directed editing mutant T1 homozygous plant height (scale = 10 cm).
FIG. 7 shows graphs of seed size measurements for the OsD2 promoter directed editing mutant T2 generation (black dot: representing duplicate experimental measurements; letter: representing differences between different data sets of the Duncan test). Phenotype of seed length seed length phenotype; phenotype of seed width seed width phenotype; seed length; seed width; 1000-grain weight: thousand grain weight.
Fig. 8.Osd11 promoter directed editing mutant T1 homozygous plant height (scale = 10 cm).
FIG. 9A. OsD11 promoter directed editing mutant T1 generation spike size map; and B, directionally editing a mutant T1 generation set of fruiting rate statistical graph (black original points: representing three repeated experimental measurement values; letters: representing differences among different data sets of the Duncan test) by using the OsD11 promoter. Seed setting rate, setting percentage.
Detailed Description
Based on the analysis of a large amount of research work for controlling ideal plant type genes (OsD, osD, osD, osSD1 and the like) of rice in the early stage, the CRISPR-Cas12a genome editing technology is used for editing a rice OsD18 gene promoter, reducing the expression of the gene, reducing plant heights in different degrees and obtaining an ideal plant type editing material and a preferential editing site.
Firstly, 12 guide RNAs (crRNAs) are designed and screened on the basis of analyzing each element of a rice OsD gene promoter (shown in figure 1A); the crRNA expression vectors described above were constructed and preferably the targeted knockout vectors pZJP078 and pZJP079 (the main expression units are shown in FIG. 1B) were obtained. Wherein the backbone vector of the expression vector is pTX377. In the embodiment of the invention, pTX377 is used as a skeleton carrier, thereby achieving high-efficiency editing effect.
The inventor discovers that different editing mutants of the rice OsD gene promoter can be obtained efficiently by using the gene editing system. And the editing material of the ideal plant type is obtained.
Therefore, the invention provides application of the crRNA or the expression vector in creating OsD gene promoter mutants and reducing rice plant height. The method comprises the following steps:
a. designing and preferably constructing CRRNA and constructing a CRISPR-Cas12 gene editing expression vector of a OsD gene promoter;
b. transforming rice with the expression vector obtained in the step a to obtain a genetically transformed plant;
c. and b, screening and identifying the transformed plant obtained in the step b, and screening out the rice OsD gene promoter editing mutant.
d. And carrying out agronomic character test on the rice OsD gene promoter editing mutant to obtain a result of the mutant with reduced rice plant height.
Specifically, the method for creating the rice OsD gene promoter mutant in the technical scheme comprises the following specific steps:
(1) Selection of crRNA target sites
The rice OsD gene (LOC_Os01g10040) is located on chromosome 1 of the genome. Target sites were designed according to the recognition and cleavage rules of the CRISPR-Cas12 system for target sites. The target site was designed at the promoter region (-2000 bp-0 bp) of the OsD gene (see FIG. 1A). The crRNA sequences are shown as SEQ ID No.1-SEQ ID No.12, and each crRNA has a length of 23nt. PAM site is TTTV.
(2) Construction of OsD gene promoter directional editing expression vector
crRNA01, crRNA03, crRNA05, crRNA07, crRNA09, and crRNA11 construct a vector, crRNA02, crRNA04, crRNA06, crRNA08, crRNA10, and crRNA12 construct a vector (see fig. 1B). Two crRNA fragments to be expressed are respectively synthesized in biological company, and the sequences of the crRNA fragments are shown as SEQ ID No.13 and SEQ ID No. 14.
SEQ ID No.13 and SEQ ID No.14 were assembled onto vector pTX377 (Tang X, lowder LG, zhang T, malzahn A, zheng X, voytas DF, zhong Z, chen Y, ren Q, li Q, kirkland ER, zhang Y, qi Y.2017.ACRISPR-Cpf1 system for efficient genome editing and transcriptional repression in Plants. Nature Plants, 3:17018) by Golden gate respectively, and the constructed recombinant vector was transformed into E.coli DH 5. Alpha. Competent cells, and the monoclonal was picked up for PCR detection to obtain the directionally-edited vectors pZJP078 and pZJP079 for the rice OsD gene promoters, the main expression units of which are shown in FIG. 1B.
(3) Genetic transformation and genotyping
The rice OsD gene promoter is used for directionally editing expression vectors pZJP078 and pZJP079, and regeneration transformed plants are obtained through agrobacterium-mediated rice genetic transformation and screening. Extracting genome DNA of rice regenerated seedling, amplifying target fragment by using specific primer Intron-F1 (primer sequence is shown as SEQ ID No. 15) and downstream primer ZY010-R1 (primer sequence is shown as SEQ ID No. 16), and detecting transgene positive. And extracting single plant DNA for positive identification. PCR is carried out by using designed specific primers OsD-sscp-F1 (the primer sequence is shown as SEQ ID No. 17) and OsD-sscp-R1 (the primer sequence is shown as SEQ ID No. 18), mutants with large fragment deletion are screened, and the directional knockout mutants with large fragment deletion are obtained through Sanger sequencing verification. For mutants without large fragment deletions, homozygous mutants were screened for sequencing analysis in the T1 generation.
(4) Analysis of agronomic traits for mutants
The agronomic traits (plant height, thousand kernel weight, seed size, etc.) of the OsD gene promoter directed editing material were determined. The invention is illustrated more specifically by the following detailed description of examples.
The nucleotides referred to in the following examples:
the crRNA01 sequence designed by SEQ ID No. 1: uucaccaauuaauucucucuuuu
crRNA02 sequence designed by SEQ ID No. 2: cucuccaaaucuauuaauuaaug
The crRNA03 sequence designed by SEQ ID No. 3: uguuguguagcuaagcugguggc
The crRNA04 sequence designed by SEQ ID No. 4: cuauaacauaacuuaauuuguau
The crRNA05 sequence designed by SEQ ID No. 5: acaggaacaguagcguccuccgu
The crRNA06 sequence designed by SEQ ID No. 6: cuaagaagacuaaccuuauauua
The crRNA07 sequence designed by SEQ ID No. 7: cucguggcugcagacuagugcug
The crRNA08 sequence designed by SEQ ID No. 8: uaauguauuagcaaccauucaug
The crRNA09 sequence designed by SEQ ID No. 9: ggcuacguacggcgccgcccugg
The crRNA10 sequence designed by SEQ ID No. 10: aauauaaacaaauaauaaugcgg
The crRNA11 sequence designed by SEQ ID No. 11: gcauaauaaucaacauuauuuug
The crRNA12 sequence designed by SEQ ID No. 12: uguucucuuuguugagaugugag
SEQ ID No.13 was designed to synthesize a DNA fragment for the construction of the vector pZJP 078:
caccggtctcAAGATttcaccaattaattctctcttttTAATTTCTACTAAGTGTAGATtgttgtgtagctaagctggtggcTAATTTCTACTAAGTGTAGATacaggaacagtagcgtcctccgtTAATTTCTACTAAGTGTAGATctcgtggctgcagactagtgctgTAATTTCTACTAAGTGTAGATggctacgtacggcgccgccctggTAATTTCTACTAAGTGTAGATgcataataatcaacattattttgggccgGAGACCATCG
SEQ ID No.14 was designed to synthesize a DNA fragment for the construction of the vector pZJP 079:
caccggtctcAAGATctctccaaatctattaattaatgTAATTTCTACTAAGTGTAGATctataacataacttaatttgtatTAATTTCTACT
AAGTGTAGATctaagaagactaaccttatattaTAATTTCTACTAAGTGTAGATtaatgtattagcaaccattcatgTAATTTCTACTA
AGTGTAGATaatataaacaaataataatgcggTAATTTCTACTAAGTGTAGATtgttctctttgttgagatgtgagggccgGAGACCAT
CG
SEQ ID No.15 used as upstream primer for transgene positive detection Intron-F1: TTCTGATCCTCTCCGTTCCT
SEQ ID No.16 used as downstream primer ZY010-R1: AAGACCGGCAACAGGATTC for transgene positive detection
Upstream primer sequence OsD-sscp-F1: gacaacgcactaggggtgatg of SEQ ID No.17 for mutant detection
Downstream primer sequence OsD-sscp-R1: gtggaatgagtagtaaagtgag of SEQ ID No.18 for mutant detection
Example 1 construction of Rice OsD18 promoter-editing vector
(1) CrRNA design
The coding region of the rice OsD gene and (-2000 bp-0 bp) promoter sequence were retrieved and downloaded from the database website NCBI (https:// www.ncbi.nlm.nih.gov /) alignment. The cis-elements were then analyzed via the website (http:// bioinformation. Psb. Ugent. Be/webtools/plantacare/html /). Then, designing crRNA according to the recognition and shearing rule of the CRISPR-Cas12 system to the target site; and then predicting and selecting the optimal 12 crRNAs (shown in figure 1A, SEQ ID No.1-SEQ ID No. 12) for the mismatch rate and the off-target site of the crRNAs by utilizing CRISPR RGEN Tools website online (http:// www.rgenome.net/cas-offinder /). Design crRNA01, crRNA03, crRNA05, crRNA07, crRNA09, crRNA11 construct a vector, crRNA02, crRNA04, crRNA06, crRNA08, crRNA10, crRNA12 construct a vector (see fig. 1B). Then respectively synthesizing two crRNA fragments to be expressed in biological company, according to the knockout vector pTX377 enzyme cutting site, adding BsaI enzyme cutting sites at two ends, its sequence is shown as SEQ ID No.13 and SEQ ID No.14, and making them pass through the Chengdu engine biological company to synthesize.
(2) Ligation reaction
The synthesized fragments SEQ ID No.12 and SEQ ID No.13 were assembled onto the backbone vector pTX377 by means of Golden gate, respectively. The Golden gate reaction system is: 1. Mu.L of T4 DNA ligase, 2. Mu.L of T4 DNA ligase buffer (10X), 1. Mu.L of pTX377 backbone vector plasmid (100 ng/. Mu.L), 1. Mu.L of restriction enzyme BsaI, 2. Mu.L of synthetic fragment, ddH 2 O13. Mu.L. The Golden gate reaction procedure is: (5 min at 37 ℃ C., 10min at 16 ℃) for 15 cycles, 5min at 37 ℃ C., 10min at 85 ℃ C.).
(3) Plasmid transformation E.coli competence
Adding 10 mu L of the connection product into DH5 alpha competence, gently mixing, placing on ice for 20min, performing heat shock for 60-90 s at 42 ℃, then placing on ice bath for 4min, adding 350 mu L of LB, and placing on a shaking table with the constant temperature of 37 ℃ and 200rmp for shake culture for about 40 min. After the completion of the cultivation, 4000rmp was centrifuged for 5min, and after the centrifugation, the cells were suspended with the remaining supernatant, and the bacterial liquid was spread on LB medium and cultured overnight in a constant temperature incubator at 37 ℃.
(4) Colony PCR
The monoclonal on LB plate was picked up with sterile toothpick and placed in water containing 50. Mu.L ddH2O5uL of the bacterial liquid is taken as a template for PCR amplification. A25 uL system was used as follows: 2X Taq DNA Polymerase Mix. Mu.L, intron-F1.5. Mu.L (SEQ ID No. 15), ZY 010-R1.5. Mu.L (SEQ ID No. 16) 0.5. Mu.L, bacterial liquid Template 5. Mu.L, ddH 2 O9. Mu.L. The PCR procedure was: 95℃for 3 min- & gt (95℃for 30 s- & gt 58℃for 30 s- & gt 72℃for 30 s) 35 cycles- & gt 72℃for 5 min- & gt 12℃for 10min (Taq DNA enzyme, dNTP, etc. are available from Tiangen biosystems). After the PCR was completed, the sample was electrophoretically detected in a 1% agarose gel at 130V for 30 min.
(5) Plasmid extraction, sequencing-by-sequencing verification
The colony PCR was used to verify correct monoclonal, 50. Mu.L of the bacterial solution was inoculated into LB containing 50mg/LKan and shaken for 12 to 16 hours, and plasmids were extracted. The extraction of plasmid DNA was performed according to the AXYGEN AxyPrepTM Plasmid Miniprep Kit instructions. The extracted plasmid was sent to the department of biotechnology, inc. for sequencing verification. The expression vectors pZJP078 and pZJP079 for the rice OsD gene promoter orientation editing were obtained, and the T-DNA region structure schematic diagram thereof is shown in FIG. 1B.
EXAMPLE 2 Agrobacterium-mediated genetic transformation of Rice
Vectors pZJP078 and pZJP079 were transformed into agrobacterium tumefaciens EHA105 competent, respectively. The method comprises the following specific steps: the vectors pZJP078 and pZJP079 are respectively added into the competence of agrobacterium tumefaciens EHA105, kept stand on ice for 30min, quickly frozen by liquid nitrogen for 5min, placed in a constant temperature water bath at 37 ℃ for 5min, placed in ice for 5min, finally added with 1mL of LB liquid medium, 220rmp and 28 ℃ for 120-150min, coated on LB solid medium containing 50mg/L of rifampicin and 50mg/L of kana, and cultured for 2d at 28 ℃. After the cultivation is completed, monoclonal is selected and colony PCR positive verification is carried out (the method is the same as in example 1), and then the agrobacterium liquid is subjected to expansion cultivation and is preserved for standby.
Agrobacterium-mediated transformation of Rice reference (Tang X, ren Q, yang L, bao Y, zhong Z, he Y, liu S, qi C, liu B, wang Y, srenovic S, zhang Y, zheng X, zhang T, qi Y, zhang Y.2019.Single transcript unit CRISPR 2.0systems for robust Cas9 and Cas12a mediated plant genome editing.Plant Biotechnol J17,1431-1445.).
The genetic transformation steps of the rice are specifically as follows: removing husk from mature seeds of rice (Nippon Temminck.) and sterilizing; inoculating the sterilized seeds on an N-6-D solid culture medium containing 0.4% gellan gum, and continuously culturing for 1-5 days under illumination at 32 ℃; the cultured seeds are respectively transferred into rice by agrobacterium-mediated transformation method, and the transformed rice seeds are continuously cultured for 2 weeks at 32 ℃ in an induction selection medium; transferring the callus generated by proliferation into RE-III culture medium; transfer of young plants produced from callus to HF medium induces root production. When the obtained resistant regenerated seedlings grow to about 15cm, cleaning root culture medium with clear water, transplanting into nutrient soil, and culturing in a greenhouse.
Example 3 identification of Rice OsD Gene promoter editing mutant
(1) Rice seedling genome DNA extraction
The rice seedling DNA extraction adopts a CTAB method, and the specific operation steps are as follows:
taking fresh rice leaves with the length of about 2-3 cm, placing the fresh rice leaves in a 2mL EP tube, adding liquid nitrogen for quick freezing, vigorously shaking a foam box after completion, crushing the leaves by friction of steel balls and the leaves, adding 600 mu L of CTAB, uniformly mixing, heating in a 65 ℃ water bath kettle for 30-45 min, uniformly mixing with 600 mu L of trichloromethane after completion, centrifuging for 8600rmp for 10min, transferring the supernatant into a 1.5mL EP tube, adding 600 mu L of dimethyl methanol for uniform mixing, placing in a refrigerator at-20 ℃ for 1h, centrifuging for 10min after 12000rmp, removing the supernatant, adding 500 mu L of 75% alcohol, washing for 2 times, standing for blow-drying, and adding 50 mu L of ddH 2 O dissolves DNA and puts it in a refrigerator at-20 ℃ for standby.
(2) Positive detection of rice seedling transgene
The target fragment is amplified by a specific primer Intron-F1 (the primer sequence is shown as SEQ ID No. 15), and a downstream primer ZY010-R1 (the primer sequence is shown as SEQ ID No. 16) to detect the transgene positive. The PCR amplification system and reaction procedure were the same as that used for colony PCR in example 1.
(3) Mutant genotyping
PCR is carried out on the positive plants obtained by detection by using specific primers OsGBSS I-sscp-F1 (the primer sequence is shown as SEQ ID No. 17) and OsGBSS I-sscp-R1 (the primer sequence is shown as SEQ ID No. 18), the PCR products are screened by using 1% agarose gel to obtain large fragment deletion mutants, and Sanger sequencing verification shows that the large fragment deletion directed editing mutants are obtained, wherein the results show that: of the 32 individuals detected, 12 had large fragment deletions with an efficiency of up to 37.5% (FIG. 2). For mutants without large fragment deletions, homozygous mutants were screened for sequencing analysis in the T1 generation.
Example 4 analysis of agronomic traits for mutants
(1) Mutant strain height determination
The strain height of the mutant was measured at the maturation stage of the T1 generation homozygous mutant, and the results (fig. 3) show: there was a significant reduction in the plant height of 30 of the 31 mutants obtained compared to the control. Wherein, the plant height reduction range is within 10 percent, and the plant height reduction range is within 6, 10 to 20 percent, 17, 20 to 30 percent, 6 and 1 below 30 percent. Meanwhile, taking a photograph of a part of the mutants (FIG. 4), it can be known that the strain height of the OsD18 promoter editing mutant is between that of the wild type and OsD18 coding region mutant d18-1 (OsD coding region is directionally knocked out by Cas9 system) and that of xiaoviei (Hu, S.et al, xiaoviei, a new rice germplasm for large-scale inner research.mol Plant 11,1418-1420 (2018)), and that 3 mutations in the listed OsD18 promoter editing mutant strain are consistent with the strain height of the half-stalk gene OsSD1 coding region mutant (the strain height reduction amplitude is 10% -20% compared with the wild type).
(2) Seed sizing
After harvesting the seeds, agronomic traits such as seed size (length, width, thousand grain weight, etc.) were analyzed. The results (fig. 5) show: compared with the wild type, the mutant seeds have no obvious difference in length, width and thousand grain weight. It was demonstrated that seed size and thousand kernel weight were not affected by editing the OsD gene promoter.
(3) OsD2, osD promoter editing mutant agronomic trait observation
Meanwhile, by adopting a similar method, DNA fragment sequences containing crRNAs and used for constructing vectors are respectively designed and synthesized aiming at promoters of regulatory plant height genes OsD and OsD 11: SEQ ID No.19 (for construction of vector pZJP074, directed against OsD 2): cacttgccaattccattccattaTAATTTCTACTAAGTGTAGATgtggtacctgttgataaataggaTAATTTCTACTAAGTGTAGATa aggatatgtgtggatacaataaTAATTTCTACTAAGTGTAGATgttagtcacttaatatgaaaactTAATTTCTACTAAGTGTAGATt agagtatgtaataatgtaaattTAATTTCTACTAAGTGTAGATgtagaatataaattactttgcat;
SEQ ID No.20 (for constructing vector pZJP075, directed against OsD): tgggcgtgggccctacgcgtgtg TAATTTCTACTAAGTGTAGATataatgtaagagctattggtagtTAATTTCTACTAAGTGTAGATtaataattaatactcctgtacgcT AATTTCTACTAAGTGTAGATtaatacaaccaatgactaggatcTAATTTCTACTAAGTGTAGATtatgagcaggttaaggttgaaat TAATTTCTACTAAGTGTAGATaaattgcatttttaggtccctca;
SEQ ID No.21 (for constructing the vector pZJP077, directed against OsD): aaacaaggccaaagcaaggaaac TAATTTCTACTAAGTGTAGATggaaatgacacagatatgagtcaTAATTTCTACTAAGTGTAGATcttctctcaaatactccatccgt TAATTTCTACTAAGTGTAGATtagtatatatctagttgagttaaTAATTTCTACTAAGTGTAGATtcggtggttttctaccgttagtgT AATTTCTACTAAGTGTAGATgtgaactaaagatggcacaaagc. Through genetic transformation, mutants are screened, and the obtained mutants are observed for agronomic traits. The results showed that although the plant height of the OsD gene promoter editing mutant was reduced (fig. 6), both seed size, thousand kernel weight were significantly reduced (fig. 7). The OsD gene promoter editing mutant also had reduced plant height (fig. 8), but the spike size and fruiting rate were extremely reduced (fig. 9). OsD2, osD are not editing candidate genes for the ideal strain. OsD18 is a candidate gene for the ideal strain.
Claims (17)
1. A method for editing a promoter of a OsD gene of rice to regulate a rice plant type, which is characterized by comprising the following steps:
a. constructing a Cas gene editing expression vector for expressing a guide RNA of a promoter for editing the rice OsD gene;
b. c, transforming rice with the expression vector obtained in the step a, and obtaining a transformed plant by using a CRISPR-Cas gene editing system;
c. and collecting seeds of the transformed plants, and screening out the seeds of the gene editing mutants to obtain the gene editing rice with improved plant types.
2. The method of claim 1, wherein: the guide RNA is at least one of crRNA01 (SEQ ID No. 1), crRNA03 (SEQ ID No. 3), crRNA05 (SEQ ID No. 5), crRNA07 (SEQ ID No. 7), crRNA09 (SEQ ID No. 9), crRNA11 (SEQ ID No. 11), crRNA02 (SEQ ID No. 2), crRNA04 (SEQ ID No. 4), crRNA06 (SEQ ID No. 6), crRNA08 (SEQ ID No. 8), crRNA10 (SEQ ID No. 10) or crRNA12 (SEQ ID No. 12).
3. The method of claim 2, wherein the guide RNAs are crRNA01 (SEQ ID No. 1), crRNA03 (SEQ ID No. 3), crRNA05 (SEQ ID No. 5), crRNA07 (SEQ ID No. 7), crRNA09 (SEQ ID No. 9) and crRNA11 (SEQ ID No. 11); or crRNA02 (SEQ ID No. 2), crRNA04 (SEQ ID No. 4), crRNA06 (SEQ ID No. 6), crRNA08 (SEQ ID No. 8), crRNA10 (SEQ ID No. 10) and crRNA12 (SEQ ID No. 12).
4. The method of claim 1, wherein: the Cas gene editing expression vector is a CRISPR-Cas12a gene editing expression vector.
5. The method of claim 4, wherein: the CRISPR-Cas12a gene editing expression vector comprises a Cas12a expression unit started by a corn ubiquitin promoter ZmUbi1 and a crRNA-scaffold expression unit started by a rice ubiquitin promoter OsUbi 1.
6. The method of claim 5, wherein: the crRNA-scafold expression unit expresses at least one of crRNA01 (SEQ ID No. 1), crRNA03 (SEQ ID No. 3), crRNA05 (SEQ ID No. 5), crRNA07 (SEQ ID No. 7), crRNA09 (SEQ ID No. 9), crRNA11 (SEQ ID No. 11), crRNA02 (SEQ ID No. 2), crRNA04 (SEQ ID No. 4), crRNA06 (SEQ ID No. 6), crRNA08 (SEQ ID No. 8), crRNA10 (SEQ ID No. 10), or crRNA12 (SEQ ID No. 12) in tandem.
7. The method of claim 1, wherein: the Cas gene editing expression vector further comprises a hygromycin resistance gene Hyg expression unit which is started by a CaMV35S promoter.
8. The method of claim 6, wherein the crRNA-scafold expression unit expresses a fragment set forth in SEQ ID No.13 or SEQ ID No. 14.
9.A guide RNA or a nucleic acid molecule complementary thereto, characterized in that said guide RNA is at least one of crRNA01 (SEQ ID No. 1), crRNA03 (SEQ ID No. 3), crRNA05 (SEQ ID No. 5), crRNA07 (SEQ ID No. 7), crRNA09 (SEQ ID No. 9), crRNA11 (SEQ ID No. 11), crRNA02 (SEQ ID No. 2), crRNA04 (SEQ ID No. 4), crRNA06 (SEQ ID No. 6), crRNA08 (SEQ ID No. 8), crRNA10 (SEQ ID No. 10) or crRNA12 (SEQ ID No. 12).
10. The guide RNA or a nucleic acid molecule complementary thereto according to claim 9, wherein the guide RNA is crRNA01 (SEQ ID No. 1), crRNA03 (SEQ ID No. 3), crRNA05 (SEQ ID No. 5), crRNA07 (SEQ ID No. 7), crRNA09 (SEQ ID No. 9) and crRNA11 (SEQ ID No. 11); or crRNA02 (SEQ ID No. 2), crRNA04 (SEQ ID No. 4), crRNA06 (SEQ ID No. 6), crRNA08 (SEQ ID No. 8), crRNA10 (SEQ ID No. 10) and crRNA12 (SEQ ID No. 12).
11. A vector for expressing and editing guide RNA of a OsD gene promoter of rice.
12. The carrier of claim 11, wherein: the guide RNA is at least one of crRNA01 (SEQ ID No. 1), crRNA03 (SEQ ID No. 3), crRNA05 (SEQ ID No. 5), crRNA07 (SEQ ID No. 7), crRNA09 (SEQ ID No. 9), crRNA11 (SEQ ID No. 11), crRNA02 (SEQ ID No. 2), crRNA04 (SEQ ID No. 4), crRNA06 (SEQ ID No. 6), crRNA08 (SEQ ID No. 8), crRNA10 (SEQ ID No. 10) or crRNA12 (SEQ ID No. 12).
13. The carrier of claim 11, wherein: the vector for expressing the guide RNA of the rice OsD gene promoter is a CRISPR-Cas12a gene editing expression vector, and comprises a crRNA-scaffold expression unit started by a rice ubiquitin promoter Osubi 1.
14. The carrier of claim 13, wherein: the CRISPR-Cas12a gene editing expression vector comprises a Cas12a expression unit started by a corn ubiquitin promoter ZmUbi1 and a hygromycin resistance gene Hyg expression unit started by a CaMV35S promoter.
15. The carrier of claim 13, wherein: the crRNA-scafold expression unit expresses a fragment shown as SEQ ID No.13 or SEQ ID No. 14.
16. Use of a vector according to any one of claims 11 to 13, a guide RNA according to claim 14 or 15 or a nucleic acid molecule complementary thereto for modifying a rice plant type.
17. The use according to claim 16, wherein: the application of the method in reducing the plant height of rice and ensuring the size and weight of rice seeds.
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