CN112626106B - Application of rice cytokinin oxidase gene OsCKX4 - Google Patents
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Abstract
The invention discloses a method for utilizing rice cytokinin oxidase geneOsCKX4Method for regulating grain number of rice per ear, driven by specific promoter, and reducing geneOsCKX4The expression level of the rice can be controlled, and the number of grains per ear of rice can be controlled. The invention utilizes the technical strategy of antisense inhibition to construct an antisense inhibition vectorOsCKX4Introduction into the recipient variety Zhonghua 11 antisense inhibition in the Zhonghua 11 backgroundOsCKX4The expression of (3) can obviously reduce the transcription level of the plant, increase the number of grains per ear and leave other main agronomic traits unaffected. The invention firstly proposes to utilize regulationOsCKX4The expression level is used for increasing the grain number per ear, and the method has important significance for improving the yield and character of rice.
Description
Technical Field
The invention relates to application of a rice cytokinin oxidase gene OsCKX4, belonging to the technical field of plant genetic engineering.
Background
The rice is one of the most important grain crops in China, the annual planting area exceeds 4 hundred million acres, and the rice bears the ration of about 60 percent of people. With the increasing shortage of arable area and fresh water resources and the continuous increase of population, the food demand in China is continuously increased. Since 2010, the import and export of rice in China are reversed, the import and export of rice become a net import country of rice, and the protection of grain safety faces a serious challenge (chenghua and Fangfiping, 2019). In rice production, the number of grains per ear is an important constituent of rice yield. Therefore, the research on the related gene for controlling the grain number per ear and the application thereof in the rice has important significance on the genetic improvement practice of the rice yield.
Phytohormones are organic compounds produced in plants in minute amounts and capable of regulating (promoting, inhibiting) own physiological processes. Auxin and cytokinin were found to play important regulatory roles in the development of ear branches. Auxin accumulates in large amounts at the beginning of the axillary meristems and is consumed during the growth of the primordia, while the next site of primordia formation forms a new auxin accumulation (Gallavotti et al, 2008). Thus, auxin is thought to be essential for the initiation of the rice axillary meristem as well as for the secondary shoot specification (Mockaitis and Estelle,2008, mcsteen, 2009). LAX1 regulates the formation of lateral meristems during rice reproductive development (Komatsu et al, 2001. LAX1 and its maize homologous gene, barrenn STALK1 (BA 1), are able to regulate auxin synthesis or transport during inflorescence development (Gallavotti et al, 2008, oikawa and kyozuka, 2009. LAX2 is another regulatory factor in rice that determines initiation of axillary meristems and interacts with LAX1 (Tabuchi et al, 2011). Various phenotypic defects of mutant asp1 may be associated with activation of various genes involved in meristem function, reactivation of growth of axillary buds of asp1 and alterations in phyllotaxis, suggesting that this gene may be closely related to auxin behaviour (Yoshida et al, 2012).
Cytokinin (CTK) is widely applied to agricultural production, and can promote cell division and expansion, lateral branch formation and growth, flower bud fruit setting and material transportation and accumulation. Cytokinin oxidase (CKX) is a key enzyme in the CTK metabolic pathway that regulates plant growth and development and organogenesis by altering endogenous CTK levels (Galuszka et al, 2000). The ZmCKX1 gene of the corn is transferred to obviously reduce the CTK level of flower buds, so that the sterility of florets and the reduction of the seed setting rate are caused, and the fertility can be restored by externally applying CTK (Huang et al, 2003).
The rice cytokinin oxidase family has 11 family members which are OsCKX1-11 in sequence, and the functions of partial members are reported. The expression quantity of the OsCKX2 gene is reduced to cause tillering, grain number per ear and grain weight increase of rice, and the rice yield is obviously improved. Overexpression of the OsCKX4 gene causes the level of the cytokinin of the mutant to be reduced, the root length is obviously lengthened, and the number of crown roots is increased. The OsCKX9 gene can be induced and expressed by strigolactone to regulate the level of cytokinin, and the gene function deletion mutant has the phenotype of increased tillering, short plant height and reduced spike. The function of other rice cytokinin oxidases has not been elucidated. Of 11 members of the rice cytokinin oxidase family, only OsCKX2 is reported to be related to the regulation of panicle number, the functions of other 10 members are not analyzed or related to the regulation of the panicle number, and most of the OsCKX functions are not reported. Therefore, it is urgent to solve the above problems.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide the application of the rice cytokinin oxidase gene OsCKX4, an antisense inhibition vector is constructed by utilizing an antisense inhibition technical strategy, and OsCKX4 is introduced into a receptor variety flower 11, so that a thought is provided for cultivating a new high-yield variety.
In order to solve the technical problems, the invention provides a method for regulating the number of grains per ear of rice by using a rice cytokinin oxidase gene OsCKX4, which is driven by a specific promoter to reduce the expression level of the gene OsCKX4 and control the number of grains per ear of rice.
Furthermore, the nucleotide sequence of the gene OsCKX4 is shown in SEQ ID NO. 1.
Furthermore, the specific promoter is a LOG gene promoter, and the nucleotide sequence of the specific promoter is shown as SEQ ID NO. 2.
Further, the method specifically comprises the following steps:
amplifying a LOG gene promoter region and an OsCKX4 coding region by a PCR method, and connecting the recovered product into a vector pCAMBIA1300 by homologous recombination;
cloning the 1.7kb of LOG promoter region and the total length of the coding region of OsCKX4 into the vector;
the recombinant vector is transformed into agrobacterium strain EHA105, and then the agrobacterium-mediated transformation method is adopted to transform the flower 11 embryogenic callus in the receptor material.
Further, the nucleotide sequence of the primer used for amplifying the LOG gene promoter region is as follows:
PLOGF1：AATTCGAGCTCGGTACCGAGGTTATCGTCTCCCTGCG
PLOGR1：AAAATTGCTTGCCACCCGTC。
further, the nucleotide sequence of the primer adopted for amplifying the OsCKX4 coding region is as follows:
ckx4F1：GACGGGTGGCAAGCAATTTTCAAGGACATAGGTAGTGAT
ckx4R1：CATGCCTGCAGGTCGAC ATGCGGGGAGCCATGAAGCCG。
the invention provides application of a rice cytokinin oxidase gene OsCKX4 in regulation of grain number per ear of rice.
The invention achieves the following beneficial effects: the invention constructs an antisense suppression vector by using an antisense suppression technical strategy, introduces the OsCKX4 into a receptor variety Zhonghua 11, and can remarkably reduce the transcription level and increase the grain number per ear by inhibiting the expression of the OsCKX4 in the background of the Zhonghua 11, wherein other main agronomic characters are not influenced. The invention provides the method for increasing the grain number per ear by regulating the expression level of OsCKX4 for the first time, and has important significance for improving the yield and character of rice.
Drawings
FIG. 1 shows the expression levels of OsCKX4 gene in control material ZH11 and two antisense suppression transformation materials proLOG: aCKX4-2 and proLOG: aCKX 4-3;
FIG. 2 is a direct view of the strain formation stage expression and single plant yield of control material ZH11 and two antisense transformation inhibiting materials proLOG: aCKX4-2 and proLOG: aCKX 4-3;
FIG. 3 is a visual representation of the ear morphology of control material ZH11 and two antisense transformation-inhibiting materials proLOG: aCKX4-2 and proLOG: aCKX 4-3;
FIG. 4 is a graph comparing the number of grains per spike for control material ZH11 and two antisense transformation-inhibiting materials proLOG:: aCKX4-2 and proLOG:: aCKX 4-3.
Detailed Description
The present invention will be further described with reference to the following examples. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
Example 1
The japonica rice variety ZH11 is the own variety of the research institute of rice in China.
Amplifying a LOG gene promoter region and an OsCKX4 coding region by a PCR method, comprising the following steps of:
DNA extraction
Placing rice leaves about 1cm long in a mortar; adding 800μ l 1.5 × CTAB, grind the leaf to homogenate and pour back into a 1.5mL centrifuge tube; water bath at 65 deg.C for 20-30min, and mixing by reversing every 5min; adding an equal volume of 24; 10 Centrifuging at 000rpm for 10min; sucking 400 supernate into a new centrifugal tube, adding 2 times volume of iced 95% ethanol, and icing at-20 ℃ for 20min;12 Centrifuging at 000rpm for 15min; discarding the supernatant, adding 500 μ l of 75% ethanol, and centrifuging at 12,000 rpm for 5min; discarding the supernatant, drying in a clean bench or air drying, adding 80 μ l ddH 2 Dissolving O for later use.
RNA extraction and reverse transcription
Weighing ZH11 tender leaf tissue 100mg, cutting, and rapidly grinding with liquid nitrogen to powder; adding 1mL of TRIzol into the mortar, slightly grinding the mixture to be brown and uniform slurry, and transferring the mixture to an eppendorf tube with the specification of 2 mL; adding 200 mu L of chloroform into 1mL of TRIzol correspondingly, violently reversing and mixing uniformly for 15s, standing at room temperature for 5min, centrifuging at 12000rpm/min at 4 ℃, and centrifuging for 15min; sucking 500 mu L of supernatant liquid into another 1.5mL centrifuge tube, and adding precooled iso-propanol; slightly turning upside down, mixing, standing on ice for 10min, centrifuging at 4 deg.C at 12000rpm/min for 10min, and removing supernatant; adding 75% of precooling DEPC glacial ethanol (1 mL), suspending and precipitating by using a vortex instrument, centrifuging at 4 ℃,12000rpm/min, and 5min; once the air-separation is carried out, the liquid at the bottom is sucked out by a sterile sucker, and the white flaky RNA sediment is not required to be carried out; air drying time is less than 5min, and adding 30 μ L DEPC water for use. Reverse transcription was performed with reference to Promega reverse transcription kit.
PCR amplification of target fragments
The PCR amplification conditions were as follows: the total PCR reaction was 50. Mu.L: wherein 100 ng/. Mu.L rice genomic DNA 2. Mu.L, 10 XPCR Buffer 5. Mu.L, 2mM dNTP 5. Mu.L, 10uM primer 1.5. Mu.L, GXL DNA Polymerase 1. Mu.L, ddH2O to 50. Mu.L. The PCR amplification conditions are specifically: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 deg.C for 10 s, annealing at 60 deg.C for 15s, extension at 68 deg.C for 70 s, 30 cycles, electrophoresis in 1% agarose gel, cutting and recovering. The 2.0kb amplification primer of the LOG promoter is PLGF 1/PLGR 1; the 1.5kb amplification primer of the OsCKX4 coding region is ckx4F1/ckx4R1.
PLOGF1：AATTCGAGCTCGGTACC GAGGTTATCGTCTCCCTGCG(SEQ ID NO:3)
PLOGR1：AAAATTGCTTGCCACCCGTC(SEQ ID NO:4)
ckx4F1：GACGGGTGGCAAGCAATTTTCAAGGACATAGGTAGTGAT(SEQ ID NO:5)
ckx4R1：CATGCCTGCAGGTCGAC ATGCGGGGAGCCATGAAGCCG(SEQ ID NO:6)
Example 2
The LOG gene promoter region and the OsCKX4 coding region were amplified by the PCR method of example 1, and the products were recovered and ligated into the vector pCAMBIA1300 by infusion. Mu.g of pCAMBIA1300 vector was subjected to linearized cleavage using Takara quick-cutting enzymes BamHI and KpnI. The linear vector is recovered and then subjected to infusion connection with the double fragments obtained in example 1, the reagent used for connection is a multi-fragment one-step cloning kit from Nanjing next Heppon company, and the connecting system is 20 mu L. Transforming the ligation product into escherichia coli DH5a, screening by coating a kanamycin-resistant plate, selecting a single clone for sequencing verification, and after the sequencing verification is error-free, transferring the plasmid into an Agrobacterium tumefaciens strain EHA105 by an electric shock method to perform subsequent genetic transformation.
Example 3
The agrobacterium-mediated rice genetic transformation mainly comprises two parts, namely induction culture of an embryonic callus of a receptor system and preparation of agrobacterium liquid containing an expression vector. The method comprises the following steps: 1) Selecting plump Nipponbare seeds, and grinding into brown rice; 2) Sterilizing the surface of the brown rice with 70% anhydrous ethanol for 3min, discarding the residual liquid, treating with commercially available blue moon brand 84 disinfectant for 15min (shaking slowly to sterilize thoroughly), washing with sterile water until no obvious smell of sodium hypochlorite exists, sucking water on the seed surface with sterile filter paper, and inoculating on 2,4-D callus induction culture medium; 3) After 12-15 days, cutting the callus and subculturing twice, each round for 15 days; 4) Selecting golden yellow granular embryogenic callus, placing in a sterilized regeneration bottle (preferably not more than one third of the volume of the bottle for callus), and 5) adding centrifugally collected target Agrobacterium tumefaciens solution containing 200 μ M Acetosyringone (AS) AA liquid culture medium suspension (AA-AS for short) into the callus; 6) Placing the embryogenic callus in the Agrobacterium AA-AS suspension for 20min for infection, and continuously shaking during the infection; 7) Pouring out the bacteria solution, uniformly spreading the callus in a culture dish filled with multiple layers of sterilized absorbent paper, and standing on an aseptic operating platform for 10-15min to make the surface of the callus dry and non-adhesive; 8) Transferring the callus to a co-culture medium with sterilized absorbent paper on the surface, and co-culturing for no more than 55 hours in the dark with the temperature controlled at 28 ℃; 9) Transferring the co-cultured callus to MS bacteria inhibiting culture medium containing 2,4-D in 2.0mg/L and cefuroxime in 500mg/L for 3-4 days; 10 ) the callus was transferred to MS selection medium containing 2.0 mg/L2, 4-D, 500mg/L cefuroxime and 65mg/L hygromycin for 3-4 subcultures; 11 The resistant callus suitable for plant regeneration can be obtained by subculturing for 2-3 times without adding the cefuroxime in the culture medium; 12 Transferring the resistant callus to a regeneration medium for alternate cultivation of dark and light (16 h of light and 8 h of dark), and obtaining a regeneration plant about one month; 13 The regenerated seedlings were subjected to hydroponic transition culture with nutrient solution and then transplanted into greenhouse soil culture for study.
Example 4
Ear branch and ear grain number examination:
two lines proLOG:: aCKX4-2 and proLOG:: aCKX4-3 of T1 generation materials and flower 11 (ZH 11) in a reference material are examined, and 5-7 lines of each line material are selected for examination. Compared with the control material, the expression levels of target genes in proLOG:: aCKX4-2 and proLOG:: aCKX4-3 in the two transformation materials are extremely reduced (figure 1), and no obvious difference exists in plant morphology and single plant yield (figure 2). However, the number of branches, particularly secondary branches, was significantly increased (FIG. 3, table 1), thereby increasing the number of grains per spike (FIG. 4). The second grade branch takes more than two flowers (including more than two flowers) as the standard.
TABLE 1 ZH11 comparison of the major agronomic traits of the ackx4 conversion Material with proLOG
* The significant difference P is less than or equal to 0.05; * Significant difference P is less than or equal to 0.01
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> institute of Rice research in China
Application of <120> rice cytokinin oxidase gene OsCKX4
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1590
<212> DNA
<213> Rice (Oryza sativa L.)
<400> 1
atgcggggag ccatgaagcc gtcgatcgtg cactgcctca agctgctcat gctgctggcg 60
ctcggcgggg tcaccatgca cgtccccgac gaggacgacg tggtcgcgtc gctcggggcg 120
ctgcgcctcg acggccattt cagcttcgac gacgcccacg ccgccgcccg ggacttcggc 180
aaccggtgca gcctcctgcc ggcggccgtg ctccaccctg gctcggtgtc cgacgtcgcc 240
gccaccgtca ggcgcgtgtt ccagctgggc aggagctcgc cgctcaccgt cgcggcgcgc 300
gggcacggcc actcgctcct cggccagtcc caggccgccg gcgggatcgt cgtcaagatg 360
gagtccctcg ccgccgccgc agccagggcg gtgcgggtgc acggcggcgc gtcgccccac 420
gtggacgccc cgggcggcga gctctggatc aacgtgctgc atgagacgct caagcacggg 480
ctggcgccca ggtcatggac cgactacctc catctcacag tcggtggcac cttgtcgaat 540
gcgggggtca gcgggcaggc gttccggcat ggaccgcagg tcagcaatgt caaccaactg 600
gagattgtga cagggagggg agaagttgtc acctgctcgc acgaggtgaa ctctgatctc 660
ttctacgctg ctcttggcgg cctgggccag tttgggatca tcaccagggc tcggattgct 720
cttgaacctg ctccaaagat ggtgcggtgg atacgtgttc tctactcgga ctttgagacc 780
ttcaccgagg accaggagaa gctgatcgcg tctgagaaga ccttcgacta catcgaaggg 840
tttgtgatca taaacaggac aggcatcctc aacaactgga ggacgtcgtt caagccacag 900
gacccagtgc aggcaagcca gttccagtcg gatggaagag tgctatactg ccttgagctg 960
acgatgaact tcaaccacga tgaggctgac atcatggaac aggaagttgg tgcgctgcta 1020
tctcgactca gatacatatc gtccactcta ttctacaccg atgtcacata cctggagttc 1080
ttggacaggg tgcacacttc tgagctgaag ctgagggctc aaggcctctg ggaagtccca 1140
cacccgtggc tgaatcttct gatcccaagg agcacagtcc acaaatttgc aaaggaagtc 1200
ttcggcaaga tcctaaaaga tagcaacaat ggtcccatac tgctttaccc agtgaacaga 1260
accaagtggg acaacagaac atcagtggtc ataccagatg aagaaatttt ctacctggtt 1320
gggttcctat cttcagcacc atcatcctca ggtcatggta gtgtcgaaca tgcaatgaac 1380
ctgaacaaca aaatagtgga cttctgtgaa aagaatggtg ttgggatgaa acagtatcta 1440
gcaccctaca ctacacagaa gcagtggaaa gcccacttcg gagcaaggtg ggagacattt 1500
gaacggagga aacacacgta cgatccccta gcaatcctag ctccagggca gagaatattt 1560
ccaaaggcat cactacctat gtccttgtga 1590
<210> 2
<211> 1800
<212> DNA
<213> Rice (Oryza sativa L.)
<400> 2
tccagggttt accttaccgt tttacaggag gttatcgtct ccctgcggta attggtaacc 60
gccgaaaacc atgtgaattt cgtcttaaaa aatttgaatt aaaaattttg gcggtttcac 120
ggttaccatg cggtttgtcg cggtcatcgc atggtttgac gcggtaacgc gatatttgca 180
cgctaaacgc gatatttgca gaaagcatga aaaaaaaatg aaaagaagct aaaaaaaaga 240
aaaatcactt aaatgtgtgt agaaattggg gaaggatata taggacactt caaatctagc 300
ttgtggatgt aaaatacaaa aatgaagccc aatacatttt ttcttttttc tatttcctaa 360
acaattatcg gttttgccct catatttgat attatcttac tctttttcaa aaattttctt 420
caccatctac cattaaaaat caagaactac atccatgatt ttttttattt ttttccaaaa 480
atttgaattt gctcaaaatt tctcaaaccc taaccaccgg taaccgaccc cctccccggc 540
agtaagcgca ataaccgcac gataacgtga accttgctat catctcgaca catcatagtg 600
aaaataaatt tcagcatatg gacttttaac cttattatgc ctcgtgccac agtctagggt 660
gctcaaagtc tgggtccatc cttttcggag aattaggttt ttcccaaata agggtgaaaa 720
cggattagat tatatcgatc caatttgctc tgaatccgtt caaaacgagg ataaggtatg 780
ggtttttaga aatccggctg atatgaatgc gaatgcgaat gcgaatggta tggtgtggat 840
tcagatttgg atgtggtatc tataaaatct ggcggatatg gattatatga tatttctatt 900
ggattatcta gataatctga aattatcaac ccatataatc actctatcta ttgcatataa 960
catgagtcga cccattttaa tgacctatat tatcaattaa cctaatggtc tgtttggttg 1020
ggggagtgtt taggaggcat tgggaattta gagggatgaa gctgttaact gttttatttg 1080
gttggaagac atgtgaattt tggtgggatg tggatgggga attgaggaat acctgggtac 1140
tgggtggtaa ttgagaatga attcctcctt tactttgcct aaacaattct caaccatctg 1200
ttttcattaa tgacctaatc tccaactaaa ctccctatca atttgtacca tgtgtactaa 1260
acaagatatt gggattaaaa atcaaattcc catcttaatc tcactatcaa ttccctcatc 1320
taaacttcta atccccttcc tcaagttacc aaacaagctg taaactcctt tgttcgagga 1380
ttttatcgtc ttatctcata cttacgtagt tgcgttttca tataatggat attatttatt 1440
catgttatta gcaatggatt attcttatga gtcgttattg ttgtcccctg tattgctaaa 1500
tcaatatcgt cccttgtata tatacataac atccaataat tttaatcctt ttccgaacca 1560
agtccgcacc gctccgcacc catttccgat attcgaaaca cgctccgcat ccgcacccgt 1620
ttttttaaaa aatatccgtt ttcaccccta ttcccaaacg tgtgatgtgt gatggaaaaa 1680
cctgatcgtt ttcagataac gtttgggtga gtgagaggga ggtcgacggg tggcaagcaa 1740
ttttcgcaca agcgcgagag gaaaagaaaa caaaaatgct actaacacac acaaacacaa 1800
<210> 3
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
aattcgagct cggtaccgag gttatcgtct ccctgcg 37
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aaaattgctt gccacccgtc 20
<210> 5
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gacgggtggc aagcaatttt caaggacata ggtagtgat 39
<210> 6
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
catgcctgca ggtcgacatg cggggagcca tgaagccg 38
Claims (4)
1. Use of rice cytokinin oxidase geneOsCKX4Method for regulating the number of grains per ear of rice, characterized in that the gene is reduced by driving with a specific promoterOsCKX4Increasing the number of grains per ear of rice, the geneOsCKX4The nucleotide sequence of (A) is shown as SEQ ID NO. 1, and the specific promoter isLOGThe nucleotide sequence of the gene promoter is shown as SEQ ID NO. 2.
2. The method according to claim 1, comprising in particular:
amplification by PCRLOGPromoter region of gene andOsCKX4coding region, product is recovered and connected into the vector pCAMBIA1300 through homologous recombination;
will be provided withLOGPromoter region 1.7kb andOsCKX4the full length of the coding region of (a) is cloned into the above vector;
the recombinant vector is transformed into agrobacterium strain EHA105, and then the recipient material embryonic callus is transformed by adopting an agrobacterium-mediated transformation method.
3. The method of claim 2, wherein the amplification is performed byLOGThe nucleotide sequence of the primer adopted in the gene promoter region is as follows:
PLOGF1：AATTCGAGCTCGGTACCGAGGTTATCGTCTCCCTGCG
PLOGR1：AAAATTGCTTGCCACCCGTC。
4. the method of claim 2, wherein the amplification is performed byOsCKX4The nucleotide sequence of the coding region adopting the primer is as follows:
ckx4F1：GACGGGTGGCAAGCAATTTTCAAGGACATAGGTAGTGAT
ckx4R1：CATGCCTGCAGGTCGAC ATGCGGGGAGCCATGAAGCCG。
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