CN108517356A - A method of avoiding transgenic paddy rice breeding abortion - Google Patents

A method of avoiding transgenic paddy rice breeding abortion Download PDF

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CN108517356A
CN108517356A CN201810336990.4A CN201810336990A CN108517356A CN 108517356 A CN108517356 A CN 108517356A CN 201810336990 A CN201810336990 A CN 201810336990A CN 108517356 A CN108517356 A CN 108517356A
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abortion
rice
paddy rice
transgenic paddy
avoiding
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CN108517356B (en
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崔素娟
赵红桃
祁东梅
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Hebei Normal University
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Abstract

The invention discloses a kind of methods avoiding transgenic paddy rice breeding abortion, with SEQ ID NO：Nucleotide sequence shown in 1 is that purpose gene carries out quantitative PCR respectively for transgenic paddy rice to be measured and wild rice, is then directed to pcr amplification product and carries out gene sequencing, while passing through 2‑△△CtAlgorithm is analyzed, and judges the abortion possibility of transgenic paddy rice to be measured.The present invention is based on rice paddy seed develop molecular mechanism result of study, have developed a set of method for avoiding transgenic paddy rice breeding abortion, can determine and rejecting there are the possible transgenic rice lines of abortion.

Description

A method of avoiding transgenic paddy rice breeding abortion
Technical field
The present invention relates to field of plant genetic, more particularly to the related application of control rice paddy seed development gene Technology.
Background technology
Rice is main cereal crops, has more than the population of one third in the world based on rice, the yield of rice The quality of height and its quality is even more closely bound up with the life of the people.China is large agricultural country, there is data statistics, in China's water The cultivated area of rice accounts for about the 1/3 of the plant cultivation grain gross area, and yield is with rice close to the 1/2 of total output of grain The number of staple food accounts for the 2/3 of total population.Rice paddy seed is the depot organ of nutriment, including carbohydrate, protein and lipid.By This is as it can be seen that the regulated and control network in research seed development is very necessary for the yield for understanding plant reproductive growth and raising cereal.
The seed morphology of different plants is different, but the structure of seed is almost the same, typically by embryo, endosperm and kind skin Three parts form, and a small amount of seed also has perisperm.The development of vegetable seeds is influenced by various, such as environmental factor, in addition The factors such as also extraneous and endogenous hormonal readiness and cell cycle.At present it has been reported that crossing some in vegetable seeds development The gene to play a significant role.
Cell cycle is a critically important process in the development of vegetable seeds, in the early development of seed, endosperm The adjusting of middle cell cycle can determine the loading of the size and starch of seed.The protein kinase relied on as cyclin The repressor of CDKOsKRP1When overexpression the loading of rice paddy seed endosperm can reduce；Cell cycle-related genesOrysa; CycB1;1A type B cell cyclin is encoded, it is impacted that deletion mutant shows as cellularization, endosperm missing, embryo Become larger；In cereal,OsCCS52A Enter endoreduplication by adjusting cell in mitosis, and then maintains the normal of seed Size.
There are many transcription factors of type in the growth course of rice paddy seed：APETALA2 classes are the special transcriptions of plant The factor, including ABI4, ANT, AtERF38, DRN/ ESR1, DRNL/ESR2, OsEBP-89, PLT1, OsNF-YB1 and OsMADS6 etc..The mitotic activity of the number of cell and cell is also adjusted by various hormones in paddy endosperm.BR is synthesized or letter Number transmit existing defects mutation body embryo and endosperm all become smaller.Transcriptome analysis rice paddy seed finds to believe with ABA in endosperm development Number approach and relevant 28 genes of response have expression.
The regulatory mechanism of epigenetics also plays a significant role in rice paddy seed development, research shows that transmethylase 1 (methyltransferase1, MET1) is methylated by mediated dna influences the table of some related genes in embryonic development It reaches, and plays a crucial role in normal embryonic development.In rice, PRC2 complex member OsFIE1, mainly in endosperm Expression, under conditions of heat shock, seed reduces when OsFIE1 is overexpressed, and cellularised process can shift to an earlier date.SUVH, which is used as, contains SET- The encoding gene of domain, timing under one of member SDG728, seed morphology are abnormal.Non-coding RNA, such as miR397, During miR398, miR408 and miR528 participate in cereal filling by regulation and control expression of target gene level.In addition exist In rice, the unwindase ENL1 of chromatin remodeling compound SNF2 families is also vital, mutation to the development of endosperm Body chromatin in the mitosis of after fertilization endosperm nucleus, which cannot be detached normally, causes endosperm nucleus to become larger, endosperm missing.CHR729 As a member of CHD3 families, can be participated in by GA signal paths in the growth course of rice paddy seed.
At present it has been reported that the gene of many adjusting and controlling rice seed developments, includes the hair of influence embryo's endosperm or kind skin It educates, but the Effect study of the overwhelming majority or term single gene among these, since the developmental regulation of rice paddy seed is a complexity Process, so using in produce reality and few.
Invention content
The technical problem to be solved by the present invention is to develop the research of molecular mechanism based on inventor team about rice paddy seed As a result, being particularly based on the contact between clone gene and rice paddy seed developmental regulation network, one kind is provided and avoids transgenosis water The method of rice breeding abortion.
In order to solve the above technical problems, the technical solution used in the present invention is as follows.
A method of transgenic paddy rice breeding abortion being avoided, with SEQ ID NO：For the purpose of nucleotide sequence shown in 1 Gene carries out PCR respectively for transgenic paddy rice to be measured and wild rice, is then directed to pcr amplification product and carries out gene survey Sequence, by sequencing result and SEQ ID NO：1 nucleotide alignments, if comparing result shows transgenic paddy rice to be measured There are genetic mutations for correspondence gene, then judging transgenic paddy rice to be measured, there are abortion possibility；If both comparing result displays Nucleotide sequence is consistent, then passes through 2 - △△CtAlgorithm, which is analyzed to obtain target gene, expresses in mutant relative to wild type The multiple of downward, when target gene expresses the multiple value lowered relative to wild type in mutant is less than 0.5, then judgement waits for Surveying transgenic paddy rice, there are abortion possibility.
As a preferred technical solution of the present invention, the PCR reaction systems are：
SYBR Premix Ex Taq 2× 5 μl；
Rox Reference Dye II 50× 0.2 μl；
ddH2O 2.4 μl；
cDNA template 2 μl；
Primer mix(10 μM ) 0.4 μl。
As a preferred technical solution of the present invention, the PCR amplification process is：Using 25s as internal reference, same mould Plate and primer do three repetitions, and at least biology obtains final result after repeating three times；Select 7500 Real-time of ABI PCR instrument is expanded by three-step approach：First stage：95 DEG C of 10 sec, second stage：95 DEG C of 5 sec, 60 DEG C of 34 sec, Cycle 40 times.
As a preferred technical solution of the present invention, described 2 - △△CtAlgorithm carry out calculation formula be：
。
It is using advantageous effect caused by above-mentioned technical proposal：The present invention is based on inventor laboratories about rice seed The result of study of son development molecular mechanism, the contact being particularly based between clone gene and rice paddy seed developmental regulation network, A kind of method avoiding transgenic paddy rice breeding abortion is provided, can determine and rejecting there are the possible transgenic paddy rice product of abortion System.
Description of the drawings
Attached drawing 1 is riceOsLFRGene model and protein pattern figure, in figure, A,OsLFRGene structure and mutation position Point schematic diagram.Oslfr-1It is T-DNA insertion mutation bodies,Oslfr-3It is CRISPR-CAS9 system gene editor's mutant；B, OsLFR protein pattern schematic diagrames.Grey parts indicate that Armodillo-fold structural domains, black portions are BAF250 structural domains.
Attached drawing 2 be embodiment 6 in,OsLFRMutant seeds Phenotypic Observation and molecular level qualification figure, in figure, A, no With the Phenotypic Observation of developmental stage seed.It 1-10 days and reaches maturity after pollination（m）'sOslfrThe form of mutant caryopsis；B, Seed DNA level genotype identification figure.For T-DNA insertion mutation bodiesOslfr-1Identification using DNA-PCR products agar Sugared electrophoresis.Two swimming lanes of each genotype, previous swimming lane areOsLFRThe specific amplified product of segment, latter swimming lane are displaying T- Whether DNA is inserted into；ForOslfr-3Identification using PCR product be sequenced by the way of；WT, versus wild type；DJ, rice T-DNA The background strain Dongjin of insertion mutation；C, mutant rna level qualification figure.DJ is wild type control,Oslfr-1WithOslfr-3For two mutant.
Attached drawing 3 is mature seed Phenotypic Observation and statistical chart in embodiment 6；After rice paddy seed maturation, choose on main tiller Spike of rice carries out Phenotypic Observation and statistics.
Specific implementation mode
The present invention is described in detail in following embodiment.Various raw materials used in the present invention and items of equipment are conventional city Product is sold, can be bought and be directly obtained by market.Experimental method used in following embodiments unless otherwise specified, For conventional method.
Embodiment 1, retrieval and analysis.
Utilize arabidopsis LFR（AtLFR or LFR）The amino acid sequence of albumen, we pass through rice genome information site Blast analysis, had found in rice genomeOsLFR, further analyzeOsLFRThe group of gene intron and exon At analyzing the protein structure domain of OsLFR.
Embodiment 2, quantitative PCR.
With reference to TaKaRa SYBR Premix Ex Taq reagents and 7500 real-time PCR instrument (Applied Biosystems) specification carries out.
PCR reaction systems are as follows（10 μl）：
SYBR Premix Ex Taq（2×） 5 μl；
Rox Reference Dye II（50×） 0.2 μl；
ddH2O 2.4 μl；
cDNA template 2 μl；
Primer mix（10 μM） 0.4 μl.
Using 25s as internal reference, same template and primer need to repeat to do three repetitions, need at least biology repetition three times Final result can just be obtained.7500 Real-time PCR instruments of ABI are selected to be expanded by three-step approach：First stage：95 DEG C 10 sec, second stage：95 DEG C of 5 sec, 60 DEG C of 34 sec are recycled 40 times.
Expression analysis：The position of left and right sets fluorescence threshold among the increased logarithmic phase of PCR reactions, reads and exports Ct values.It is analyzed by 2-△ △ Ct algorithms.Calculation formula：
。
By calculating 2 - △△CtIt obtains target gene and expresses times for raising or lowering relative to wild type in rice to be measured Number, it is considered that higher than 1.5 or less than 0.5 difference be more believable.Obtained data are repeated with biology three times to make Provide the column diagram of standard deviation.Different batches experiment can use 2-△Ct WTCalculate the standard deviation of wild type.
Embodiment 3 edits carrier based on CRISPR technologies structure OsLFR.
Using CRISPR technologies, rice is constructedOsLFREditor's carrier.Building flow is：Teacher Qu Lijia is real first It tests room and has asked for entry vector pOs-sgRNA, expression vector Ph-Ubi-cas9-7.DesignOsLFRSpecific target spot primer （sgOsLFR-22-FP：GGCAGCGAAGCGCGGGCGCCCCTT is shown in SEQ ID NO：2；sgOsLFR-22-RP： AAACAAGGGGCGCCCGCGCTTCGC is shown in SEQ ID NO：3）.Two single-stranded primers form double-strand through denaturation annealing, withBsa The entry vector pOs-sgRNA plasmids of I digestion are attached reaction, will successfully be connected into the entry vector and Ph- of gene order Ubi-cas9-7 carries out LR reactions, finally obtains OsLFR-cas9 and edits carrier.
The cultivation of embodiment 4, OsLFR-cas9 transgenic paddy rices.
The transgenosis that Rice Callus is carried out to building correct OsLFR-cas9, seedling to the transgenosis present age and Its progeny seed carries out DNA level sequencing and Phenotypic Observation experiment.In being tested for the Phenotypic Observation of seed, for there is phenotype Single mutant seeds carry out DNA extractions after removing kind of skin, using kit（TransDirect Plant tissue PCR Kit, TransGen Biotech）Extraction.Software used in the analyses and comparison of sequence is OMIGA.
The rataria callus transgenosis of agriculture bacillus mediated rice：The transgenic method of rice callus is with reference to (Wang et Al., 2012a), slightly change.Rice paddy seed removes glume, is sterilized 1-3 minutes with 75% ethyl alcohol of volume fraction, then use matter The sodium hypochlorite of amount score 2.5% is handled twice respectively, 15 minutes every time.Then 3-4 rear immersion of aseptic water washing 40 minutes is used Afterwards with it is sterile washing 10 times after be uniformly laid on calli induction media（MS+）The incubator of upper 28 DEG C of hairs seed is protected from light culture and lures Lead the generation of callus.Collection has induced 28 days or so callus, and smooth, yellow is selected from callus, and Harder, size is placed in the embryo callus subculture of 2-3mm on MS culture mediums, and 28 DEG C are protected from light squamous subculture and carry out agriculture bar after 7-8 days After bacterium dip dyeing, it is placed on the co-cultivation base for having added filter paper（NBCO）In 22 DEG C co-culture 3 days.When being cultivated from co-culturing to selecting, altogether Cultivate callus first with sterilizing washing three times, after with containing 500mg/l carboxylic Bian penicillin aqua sterilisa impregnate 10-15 minutes, with nothing Bacterium filter paper blots, and is put into Selective agar medium.It selects the callus newly grown and is put into pre- differential medium after 7 days to go to and break up culture It is cultivated on base.
Embodiment 6 is edited through CRISPROsLFRMutant and the seeds abortion of wild type are observed.
Referring to attached drawing 2, using stereomicroscope, we observe the seed morphology of different developmental phases, findOslfr-1Mutant will appear the apparent phenotype for being different from wild type in after fertilization the 5th day, be mainly manifested in seed morphology Irregularly, endosperm fraction is not full without filling or filling.
Referring to attached drawing 3, after rice paddy seed maturation, the spike of rice chosen on main tiller carries out Phenotypic Observation and statistics.As a result it sees below Table：
。
Based on the above embodiments as it can be seen that the normal expression of OsLFR genes for rice paddy seed normal development have it is important Physiological action, in basis and important regulation and control node in the molecular information network of seed development, when transgenic breedingOsLFRThe sequence variations and abnormal expression of gene are the major reasons for causing rice abortion.The present invention is based on the studies above achievements Develop withOsLFRGenetic test avoids transgenic paddy rice breeding abortion method for main implementation means.
Foregoing description is only proposed as the enforceable technical solution of the present invention, not as to the single of its technical solution itself Restrictive condition.
Sequence table
<110>Hebei Normal University
<120>A method of avoiding transgenic paddy rice breeding abortion
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ccgctgctga accctcatca tgtcccacgt gaggtccgcc ccggcgggca agtccggcgg 180
gggcggcggc tccacgccgg cgaagcgcgg gcgccccttc ggaagcacca ccggcagcgg 240
ggcggcggcc gcggccgcgg cggctgcaat cggcgacgcg gcggcgccgg ccgcgctggt 300
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gtagtacact agtaatcata tgagtctatg acgaacattg gaaacaatgg ggacaaacaa 720
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acctagattt ttttccctgt tcatattggc tcattcaata gagatgctac ctcacatatg 840
tgtcatgagc ttgaaatgca caggcactcc ttttttctgc acacttaaca tgtactgtcg 900
acatggttac taagaaggag agtttatgtt tctttgtgga cttttcagat cagaacaata 960
aaagaattgt tcttgcattg caaagtgggt tgaagagtga gatactatgg gcattgaacg 1020
cccttacagt actctcattt aaggagaagg atgatctccg gcgggataca acacctcttg 1080
ctaaagttcc tgggctcttg gacgctctcc ttcaagttgt aggttcttaa tgttaacttc 1140
cccccttcca atttgatcta tatgttatgg aatgatgcat atcttctgaa gccaaaaatc 1200
atgtttccca attcctctag catgcattaa ttgatcaatt tgaatcagag ttcattttag 1260
aaaatgatta gtcagtcaat catcacccta ttcatttttt atggtggacc tccagtactg 1320
ttgatgccac cagtggccgc agccacctgt tccagaccag gaggatcagg aaatggattg 1380
tgagactgat ttatctttac tttttgttcg caaattattc tttcgtttaa tctgcagaag 1440
ttgttaggca atcctcctac ttaaggatgg gactatcgct acttgaaggc aaactagaat 1500
agttacccta ccccctgctt ctctgcgaac ttccgtgtac ctcctgtctt cctcctgcac 1560
aatggtgccc aagcgccatg gtccaggtct ccaggatctt ggcttcctgc ctctcgctcc 1620
tattcccttg ccaccagaat cttgcccaca atatccagac actgatgaaa atgtgatgat 1680
ctcatgcaat atccaggact ggcattgttg tccactgtag ccatagaaac gtaaataaag 1740
gttccgatat ttgcacttac acttaattgg ttggtggcca aagtatcgct ttatttaatt 1800
ggactaccgt gcttctttga tatgactaaa tttgctaaat aactaaattt tttaaacaat 1860
gcactgatca attttcatcc agaaaagaaa tgatatggtg atcatgtacg gatacgctta 1920
ttaggacttt atttctgcat tctgtttgat gcatttccat tttgtacaga tagatgactg 1980
gcgagatatt gcaatgccta aggatcatac aaaaccgcca agagtaagaa cattgggtgt 2040
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ggaccaagtt atgataacat gtaaactaac cttctattca tttttatcat gtaccagaag 2820
attatgagct cataacaaac taacttttta tgcattttta tcatgtaaca gaagatgatg 2880
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gtgccagcag tcgatgcaca ggcagctgct attagtgcgc tgtacaatgt agcagaagtg 3420
aatatggact tccggctgaa gcttgctagc gagcgatggt aatatctcct tcattctatt 3480
agatatagca cactgacatg agaacttaaa ttcaaactct taaatttcgt attatttgaa 3540
ccagggctgt cgatagactc cttaaggtcg tgaagacgcc acatcccgtg cctgaagttt 3600
gtaggaaagc atcaatgata gtggagagcc tagtctctga gcctcaaaac aggatgcatc 3660
tccttgtcca tgagaatact tttgctgaga tactcacgtc ggaagggaaa tactccgaca 3720
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Claims (4)

1. a kind of method avoiding transgenic paddy rice breeding abortion, it is characterised in that：With SEQ ID NO：Nucleotides sequence shown in 1 It is classified as target gene and carries out PCR respectively for transgenic paddy rice to be measured and wild rice, be then directed to pcr amplification product and carry out Gene sequencing, by sequencing result and SEQ ID NO：1 nucleotide alignments, if comparing result shows transgenosis to be measured There are genetic mutations for the correspondence gene of rice, then judging transgenic paddy rice to be measured, there are abortion possibility；If comparing result is shown The nucleotide sequence of the two is consistent, then passes through 2 - △△CtAlgorithm analyzed to obtain target gene expressed in mutant relative to The multiple that wild type is lowered, when target gene expresses the multiple value lowered relative to wild type in mutant is less than 0.5, then Judging transgenic paddy rice to be measured, there are abortion possibility.
2. the method according to claim 1 for avoiding transgenic paddy rice breeding abortion, it is characterised in that：The PCR reactions System is：
SYBR Premix Ex Taq 2× 5 μl；
Rox Reference Dye II 50× 0.2 μl；
ddH2O 2.4 μl；
cDNA template 2 μl；
Primer mix 10μM 0.4 μl。
3. the method according to claim 1 for avoiding transgenic paddy rice breeding abortion, it is characterised in that：The PCR amplification Process is：Using 25s as internal reference, same template and primer do three repetitions, and at least biology obtains finally after repeating three times As a result；7500 Real-time PCR instruments of ABI are selected to be expanded by three-step approach：First stage：95 DEG C of 10 sec, second Stage：95 DEG C of 5 sec, 60 DEG C of 34 sec are recycled 40 times.
4. the method according to claim 1 for avoiding transgenic paddy rice breeding abortion, it is characterised in that：Described 2 - △△CtIt calculates Method carry out calculation formula be：
。
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110229845A (en) * 2019-06-18 2019-09-13 湖南杂交水稻研究中心 A method of avoiding transgenic paddy rice breeding failure
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CN115873868B (en) * 2022-12-07 2024-04-12 扬州大学 Rice OsFIE1 mutant gene and encoding protein and application thereof

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