CN112251523B - Application and implementation method of rice coleoptile purple line gene OsMYB76 - Google Patents
Application and implementation method of rice coleoptile purple line gene OsMYB76 Download PDFInfo
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Abstract
The invention discloses an application and an implementation method of a rice coleoptile purple line gene OsMYB76, wherein the nucleotide sequence of the gene is shown as SEQ ID NO.2, the sequence of the gene after modification is shown as SEQ ID NO.1, and the sequence of an interference fragment of the gene is shown as SEQ ID NO. 3; the gene, the modified gene or the down-regulated expression of the gene can regulate and control the synthesis of anthocyanin at parts such as rice coleoptiles and the like, so the gene can be used as a report gene in transgenic identification, and the method for identifying transgenic positive offspring is simple and high in accuracy, and has important effects on simplifying gametophyte male sterility identification, identifying the transfer ratio of transgenic components along with pollen, removing transgenic pollution, improving the accuracy and the like.
Description
Technical Field
The invention belongs to the field of genetic engineering, and relates to a rice coleoptile purple line gene OsMYB76, a modified gene OsMYB76R thereof and an expression down-regulation element in transgenic positive strain identification, gametophyte male sterility identification and transgenic pollution identification and removal, and an application realization method thereof.
Background
The MYB transcription factor family is one of the most abundant transcription factor families in plants. Rice shares many MYB transcription factors 150 (Katiyar et al, 2012), of which OsMYB76 gene (Loc _ Os06g10350, C gene or OsC1, hereinafter collectively referred to as OsMYB76) is found to be essential for anthocyanin synthesis regulation of rice coleoptile purple line and the like (Saitoh et al, 2004; Zhang Yi 2009; Zhaosha 2014). At present, the research on the gene mainly focuses on evolutionary analysis, gene localization and function verification, and application research of the gene is reported to be used for identifying hybrid purity (Zhang Yi et al, 2005), but the gene is not used as a transgenic reporter gene, and the gene is not used as a research for identifying male gametophyte sterility and the like.
The reporter gene currently used in transgenosis is mainly exogenous GUS gene or some fluorescent protein genes, and the use of the genes as the reporter gene has the following defects which are difficult to overcome: the detection process is complex, a special detection reagent (such as GUS dye solution) needs to be prepared or a special detection device (such as a fluorescent emitter) needs to be used, false positives are easy to generate, and the like. The male gametophyte sterility and the sporophyte sterility are two important types of male sterility, the gametophyte sterility is controlled by the genotype of the pollen, and the sporophyte sterility is controlled by the genotype of the plant, so that the gametophyte sterility gene can only be transferred by female but not by pollen, and is only stored by heterozygote in nature. The sterile pollen of the sporophyte is completely abortive, and the pollen usually shows abnormal anther shape, color and size and is not fruitful in the field. The gametophyte sterile anther has half of pollen fertile, has no obvious difference compared with wild type, and is normal in fructification. Therefore, the discovery and identification of gametophyte sterility in the field is very difficult and requires a combination of indoor microscopic observation and molecular identification.
The anthocyanin character can be seen in plants with naked eyes and can be expressed in each organ. Therefore, the method for developing a simple and feasible transgene identification and gametophyte sterility identification method by utilizing the anthocyanin synthesis regulation essential gene OsMYB76 has important significance.
Disclosure of Invention
In view of the above, the invention aims to provide application and a realization method of an essential gene OsMYB76 for anthocyanin synthesis regulation such as rice coleoptile purple line, a modified gene OsMYB76R and a down-regulation element (preferably an interference fragment) thereof. The OsMYB76 gene has the function of regulating anthocyanin synthesis of organs such as bud sheaths and the like, and once functional deletion mutation or down-regulation expression occurs, all organs cannot express purple. The OsMYB76 gene with normal function or the OsMYB76R gene modified by the gene is linked with a target gene or fragment to be transferred and is transferred into a plant with the OsMYB76 gene or the homologous gene thereof being abnormal; or the down-regulation element (preferably an interference fragment) of the OsMYB76 gene is linked with a target gene or fragment to be transferred, and the down-regulation element and the fragment are transferred into a plant which has the OsMYB76 gene or homologous gene thereof normal and any organ of the plant shows purple; the existence of the positive event of the transgene can be directly prompted by the existence of purple in the plant organ. The nucleotide sequence of an anthocyanin synthesis regulation essential gene OsMYB76 such as rice coleoptile purple line is shown as SEQ ID No.1, the nucleotide sequence of a modified gene OsMYB76R of OsMYB76 is shown as SEQ ID No.2, and the nucleotide sequence of an interference fragment of OsMYB76 is shown as SEQ ID No. 3.
The invention provides a method for identifying a transgenic positive plant by using a rice coleoptile purple line gene, which comprises the following steps:
(1) according to the CDS nucleotide sequence and the coded amino acid sequence of the OsMYB76 gene, partial nucleotides are changed to remove common enzyme cutting sites, and the CDS sequence of the OsMYB76R gene after the OsMYB76 gene modification is obtained and artificially synthesized. On the premise of not influencing the function of regulating anthocyanin synthesis by the gene, at least one nucleotide can be substituted, deleted or added in the OsMYB76R gene sequence, so that the application of the OsMYB76 gene and the OsMYB76R gene and the protection of the implementation method thereof are not limited; or by PCR or artificially synthesizing an interference fragment of the OsMYB76 gene.
(2) The CDS sequence or interference fragment of the OsMYB76 gene or the OsMYB76R gene obtained after modification is placed at the downstream of a constitutive promoter such as 35S or the like, or a promoter of the OsMYB76 gene per se, or a specific promoter such as endosperm or the like, and at the upstream of a terminator such as Tnos or the like, and then the promoter + the OsMYB76 gene or the OsMYB76R gene or the interference fragment + the terminator region are loaded into a plant expression vector to obtain a vector p1(OsMYB76), a vector p2(OsMYB76R gene) or a vector p3 (interference fragment). The promoter and terminator described in this step can be flexibly selected.
(3) Any target gene X is connected into the vector p1, p2 or p3 with the rice anthocyanin synthesis regulatory gene OsMYB76 or OsMYB76R to obtain a vector pX1, a vector pX2 or a vector pX 3.
(4) The vector pX1 or pX2 is transferred into a plant with an OsMYB76 gene (rice) or homologous genes (other species) thereof abnormal and anthocyanin deletion by a transgenic technology, or the vector pX3 is transferred into a plant with an OsMYB76 gene (rice) or homologous genes (other species) thereof normal and any organ has anthocyanin. In rice, the OsMYB76 genes in the lines 9311, Minghui 63, ZhongjiuB and the like are subjected to function deletion mutation, all organs have no purple expression, and the genes can be used as host plants to be transferred into pX1 or pX2, but the application of other OsMYB76 genes (rice) or homologous genes (other species) of the genes is not excluded, and plant lines with anthocyanin deletion are used; the OsMYB76 genes in G46B, II-32B and other lines are normal, purple is expressed in organs such as coleoptiles, leaf sheaths, stigmas, glumes and the like, and all the genes can be used as host plants to be transferred into pX3, but the use of other plant lines with normal OsMYB76 genes (rice) or homologous genes (other species) thereof is not excluded.
(5) Observing whether each organ has purple color or not for the transgenic progeny of pX1 or pX2, wherein the purple color of any organ is a transgenic positive strain; for the pX3 transgenic offspring, the purple change of each organ is observed, and any originally purple organ becomes a transgenic positive strain without purple.
The invention also provides application of OsMYB76 or modified gene OsMYB76R or down-regulation element (preferably interference fragment) thereof in identifying gametophyte male sterility, which comprises the following steps:
(1) according to the analysis of gene expression data (online expression database, gene expression data in literature, or self-sequencing anther or pollen transcriptome, gene chip or RT-qPCR, etc.), the candidate gene GMS with high and/or specific expression gene as gametophyte male sterility related gene in pollen is preliminarily determined.
(2) Constructing GMS gene down-regulation expression vector, preferably RNAi vector pGMSi, according to the candidate gene sequence.
(3) And (3) connecting the OsMYB76 gene and the interference fragment of the modified gene OsMYB76R or OsMYB76 into pGMSi to obtain a vector p1-GMSI, p2-GMSI or p 3-GMSI.
(4) Transferring the vector p1-GMSi or p2-GMSi into a plant with abnormal OsMYB76 gene (rice) or homologous gene (other species) thereof and anthocyanin deletion; or transferring p3-GMSI into a plant with normal OsMYB76 gene (rice) or homologous gene (other species) thereof and anthocyanin in any organ. In rice, the OsMYB76 genes in 9311, Minghui 63, ZhongjiuB and other lines have function deletion mutation, all organs have no purple expression, and the genes can be used as host plants to be transferred into p1-GMSi or p2-GMSi, but the use of other OsMYB76 genes (rice) or homologous genes thereof (other species) is abnormal, and plant lines with anthocyanin deletion are not excluded; the OsMYB76 genes in G46B, II-32B and other lines are normal, purple is expressed in organs such as coleoptiles, leaf sheaths, stigma, glumes and the like, and all the genes can be used as host plants to be transferred into p3-GMSi, but the use of other OsMYB76 genes (rice) or plant lines with normal homologous genes (other species) is not excluded.
(5) At T0And selecting transgenic positive strains in the generations.
(6) Crossing a female parent which is abnormal with an OsMYB76 gene (rice) or homologous genes thereof (other species) and lacks anthocyanin with a p1-GMSI or p2-GMSI positive strain serving as a male parent, and selfing a p1-GMSI or p2-GMSI positive strain; or using p3-GMSI positive strain as male parent, crossing with OsMYB76 gene (rice) or homologous gene (other species) thereof, and selfing the p3-GMSI positive strain. In rice, the OsMYB76 genes in the lines of 9311, Minghui 63, ZhongjiuB and the like are subjected to function deletion mutation, all organs have no purple expression, and the genes can be used as female parents for hybridizing with p1-GMSi or p2-GMSi positive strains, but the method does not exclude the use of other OsMYB76 genes (rice) or other homologous genes (other species) which are abnormal and plant lines with anthocyanin deletion; the OsMYB76 genes in the lines of G46B, II-32B and the like are normal, purple is expressed in organs such as coleoptiles, leaf sheaths, stigma, glume tips and the like, and all the genes can be used as female parents for hybridizing with p3-GMSI positive strains, but the use of other OsMYB76 genes (rice) or plant lines with normal homologous genes (other species) is not excluded.
(7) Hybridization F to p1-GMSI or p2-GMSI1Instead, the ratio of individuals with purple color to individuals without purple color was investigated. If all individuals have no purple color or the ratio of purple individuals is far less than 50 percent, the transgenic components can not or difficultly be transferred between generations along with pollen, and the candidate gene is a gametophytic sterility related gene; hybridization F to p3-GMSI1Next, the ratio of individuals with purple color to individuals without purple color was examined (observation site was positive for p 3-GMSI)T0Organs with purple loss in generations), if all individuals show purple color or the ratio of individuals without purple color is far less than 50%, the transgenic components can not or difficultly transmit with pollen among generations, and the candidate gene is a gametophytic sterility related gene. The principle is as follows: in general, down-regulation of gametophytic male sterility-associated genes results in complete or partial (depending on the suppression effect) lethality of transgenic pollen with reporter genes and down-regulating elements, and no or minimal progeny production, thus F1The probability of representing the now reported trait is theoretically 0 or much less than 50%, if the candidate gene is not a gametophytic male sterility-related gene, F1The probability of representing an individual with the now-reported trait is theoretically 50%.
(8) T for p1-GMSI or p2-GMSI1Investigating the separation ratio of purple in the generations, if the ratio of purple to colorless is in accordance with 1:1, the transgenic ingredients cannot or are difficult to transfer with pollen between generations, and only female gametes can transfer between generations, thereby further confirming that the candidate gene is a gametophyte male sterility related gene; or T of p3-GMSI1Purple color in the middle generation (observation site p3-GMSI positive T)0Organs with purple loss in generations) are investigated, if the purple-to-colorless ratio is in accordance with 1:1, transgenic components cannot or are difficult to transfer with pollen between generations, and can only be transferred between generations through female gametes, thereby further confirming that the candidate gene is a gametophyte male sterility related gene.
The invention also provides application of OsMYB76 or modified gene OsMYB76R thereof or down-regulation element (preferably interference fragment) thereof in identifying the transmission ratio of transgenic components along pollen and removing transgenic pollen pollution and a realization method, wherein the method comprises the following steps:
(1) and connecting the transgenic component Y to be identified into plant expression vectors p1, p2 or p3 with OsMYB76, OsMYB76R or interference fragments thereof to obtain vectors pY1, pY2 or pY 3.
(2) Transferring the vector pY1 or pY2 into a plant with OsMYB76 gene (rice) or homologous gene (other species) abnormality and anthocyanin deletion; or the vector pY3 is transferred into a plant with the OsMYB76 gene (rice) or homologous gene (other species) thereof being normal and any organ showing purple. In rice, the OsMYB76 genes in the lines 9311, Minghui 63, ZhongjiuB and the like are subjected to function deletion mutation, all organs have no purple expression, and can be used as host plants to be transferred into pY1 or pY2, but the application of other OsMYB76 genes (rice) or homologous genes (other species) thereof which are abnormal and plant lines with anthocyanin deletion are not excluded; the OsMYB76 genes in G46B, II-32B and other lines are normal, purple is expressed in organs such as coleoptile, leaf sheath, stigma, glume tip and the like, and all the genes can be used as host plants to be transferred into pY3, but the use of other plant lines with normal OsMYB76 genes (rice) or homologous genes (other species) thereof is not excluded.
(3) At T0And selecting transgenic positive strains in the generations.
(4) Using a pY1 or pY2 positive strain as a male parent, and hybridizing the male parent with a female parent which is abnormal in an OsMYB76 gene (rice) or homologous genes (other species) and is deficient in anthocyanin; or using pY3 positive strain as male parent, crossing with OsMYB76 gene (rice) or homologous gene (other species) normal, female parent with anthocyanin in any organ. In rice, the OsMYB76 genes in the lines 9311, Minghui 63 and ZhongjiuB are subjected to function deletion mutation, all organs have no purple expression, and can be used as female parents for hybridization with pY1 or pY2 positive strains, but the application of other OsMYB76 genes (rice) or other homologous genes (other species) are abnormal, and plant lines with anthocyanin deletion are not excluded; the OsMYB76 genes in the lines of G46B, II-32B and the like are normal, purple is expressed in organs such as coleoptiles, leaf sheaths, stigmas, glume tips and the like, and all the genes can be used as female parents for hybridizing with the pY3 positive strain, but the use of other OsMYB76 genes (rice) or plant lines with normal homologous genes (other species) is not excluded.
(5) Hybridization F to pY1 or pY21Second generation, survey F1The total number of individuals N, the number of purple individuals N, and the transfer ratio (%) of the transgenic component to be detected with pollen according to the following formula: 100 x 2N/N; hybridization F to pY31Second generation, survey F1The total number of individuals M, the number of individuals M without purple color, and the transfer ratio (%) of the transgenic component to be detected with pollen were calculated according to the following formula: 100X 2M/M.
(6) For pY1 or pY2 and OsMYB76 gene (rice) or homologous gene (other species) thereof is abnormal, and anthocyanin is deleted from filial generation of parents, and individuals with purple color in any organ can be directly removed, so that transgenic pollution generated along with pollen transmission can be removed; progeny of plants which are normal to the pY3 and OsMYB76 genes (rice) or homologous genes (other species) and have purple color in any organ are removed (the observed part is pY3 positive T)0Organs with purple loss in the generations) can remove transgenic contamination that occurs with pollen transmission.
As a general technical concept, the promoter, the gene to be identified and the species to be transformed can be flexibly selected; the same gene can use a plurality of target spots, the interference target spots can be different positions of the same gene, and the same target spot can be used in a plurality of copies; in addition, the carrier names used in the method statement are used only for convenience of statement and do not limit the use of other symbols to represent the relevant carrier in practical application. Thus, the practical use of the alternatives described above does not indicate a substantial difference from the invention, and thus does not affect the protection of the invention.
The invention has the beneficial effects that: the invention discloses OsMYB76, a modified gene OsMYB76R thereof and an expression down-regulation element (preferably an interference fragment) thereof, which are mainly used for regulating and controlling synthesis of anthocyanin such as coleoptile purple line and the like. The OsMYB76 gene or the OsMYB76R gene modified from the OsMYB76 gene or expression down-regulation element (preferably an interference fragment) of the OsMYB 8932 gene relates to nucleic acid or protein which is endogenous to rice, and the application of the OsMYB76 gene or expression down-regulation element (preferably an interference fragment) as a reporter gene can reduce the risk of transgenosis and reduce the worry of people about transgenic products.
The invention also has the following beneficial effects: the purple character controlled by the OsMYB76 gene or the OsMYB76R gene modified from the OsMYB76 gene or an expression down-regulation element (preferably an interference fragment) is obvious and visible to the naked eye. In the positive identification of the transgene, compared with the reporter genes such as GUS, GFP and the like: the detection procedure of the transgenic positive offspring can be simplified, the detection object is not damaged, medicines and instruments are not needed, and the judgment can be directly carried out according to the purple color and the useless naked eyes; the detection result can be accurate, and false positive can be reduced; the detection time can be saved, the culture and planting space can be saved, and the cost can be reduced. In the gametophyte male sterility identification, generally, molecular markers are utilized, the transmission condition of possible gametophyte male sterility linkage markers in offspring is analyzed through PCR amplification and electrophoresis or sequencing, the method has the problems of high cost, long time, false positive and the like.
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In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows purple color expression at coleoptiles, leaf sheaths and stem bases, A and B are the expression of the purple lines of the normal and abnormal parental coleoptiles of the OsMYB76 gene of the invention, respectively; c, judging whether the stem base of the tissue culture seedling is a positive plant by using the purple color; d is a purple leaf sheath material with normal OsMYB76 gene, and the color of the leaf sheath shows before and after the OsMYB76 gene interference; E. f and G are cross F for investigating transgenic component by coleoptile purple line1The transfer case of (1), wherein E is full transfer, F is partial transfer, and G is no transfer;
FIG. 2 shows the fine mapping of the OsMYB76 gene;
FIG. 3 is a schematic representation of the multiple cloning site engineered cleavage site of vector pOsMYB 76R;
FIG. 4 shows the restriction sites of pCAMBIA1300 after the modification of the multiple cloning site.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (fourth edition, Cold spring harbor laboratory Press) or in the compendium for molecular biology protocols (fifth edition, scientific Press), or according to the conditions recommended by the manufacturer.
The term "essential gene for regulating anthocyanin synthesis such as coleoptile purple line" refers to a nucleotide sequence and a degenerate sequence thereof, wherein the nucleotide sequence is shown as 1 st to 819 th positions in SEQ ID NO.2 or SEQ ID NO.1, and the gene encodes a MYB transcription factor gene OsMYB76 for regulating anthocyanin synthesis such as coleoptile purple line or an OsMYB76R gene modified from the gene. Degenerate sequences are sequences which are the result of one or more codons in the nucleotides of the sequence SEQ ID No.2 or SEQ ID No.1 being replaced by degenerate codons which code for the same amino acid. The term also includes a variant of OsMYB76 or OsMYB76R gene that is engineered therefrom. These variants include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 90, preferably 1 to 60, more preferably 1 to 20, most preferably 1 to 10) nucleotides, and addition of several (usually less than 60, preferably less than 30, more preferably less than 10, most preferably less than 5) nucleotides at the 5 'and/or 3' end.
In the examples, various vectors known in the art, such as commercially available vectors including plasmids, cosmids, and the like, may be used.
The full-length sequence of related nucleotides or fragments thereof can be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed based on the nucleotide sequences disclosed in this example, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art, or genomic DNA as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order. Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
Example 1 obtaining and morphological Observation of OsMYB76 Gene mutant plants
The OsMYB76 gene mutant is derived from mutation of indica rice R25 (purple line of coleoptile, abbreviated as coleoptile purple line). OsMYB76 mutant hybridized with R25, FlAll the generations are bud sheaths with purple lines and are selfedF2Segregation occurs in generations, and the ratio of purple line to non-purple line is 3:1, which indicates that the non-purple line mutant phenotype is caused by one nuclear gene mutation. Morphological observations of the OsMYB76 mutant plants: the main difference between the mutant and the wild type is that the wild type coleoptile has a distinct purple line (FIG. 1, A), whereas the OsMYB76 mutant coleoptile has no purple line (FIG. 1, B).
Example 2 mapping and cloning of the OsMYB76 Gene
1. Locating a population
Hybridizing the OsMYB76 mutant with purple-line II-32B, hybridizing non-purple-line medium-nine B with wild-type R25, and obtaining F1Selfing to obtain F2Generation and planting F2And obtaining a positioning population.
2. Extraction of Rice DNA
The parent is extracted by adopting an improved CTAB method, and the method comprises the following steps: taking 0.1-0.2 g (about half piece) of leaf blades, putting the leaf blades into a small mortar, adding a proper amount of liquid nitrogen, immediately grinding the leaf blades into powder, putting the powder into a 2m1 centrifuge tube, adding 700 mu L of a 1.5 XCTAB solution preheated at 100 ℃ into the centrifuge tube, carefully and uniformly mixing the solution and the powder, putting the mixture into a 65 ℃ water bath, taking out the centrifuge tube after 20 minutes, adding equal volume of chloroform/isoamyl alcohol into the centrifuge tube, violently and uniformly mixing the mixture, centrifuging the mixture at 13000rpm for 10 minutes, taking supernatant into a new tube, adding 900 mu L of absolute ethyl alcohol into the new tube, uniformly mixing the supernatant and putting the mixture at-20 ℃ for more than half an hour. The precipitated DNA was centrifuged at 14000rpm for 10 minutes. The supernatant was removed, and the precipitate was washed once with 1mL volume fraction of 70% ethanol, dried by centrifugation, dissolved in 200. mu.L of TE solution and stored in a refrigerator at 4 ℃. The individual plant DNA of the positioning population is extracted by adopting an improved alkaline cooking method, and the method comprises the following steps: cutting tender leaf into pieces of 1-2cm2The mixture was put into a 0.5M1 centrifuge tube, 100. mu.L of 0.125M NaOH solution was added, and the mixture was boiled in a water bath for 30 seconds, then 50. mu.L of 1.0M Tris-HCl (pH8.0) was added, and finally 100. mu.L of 0.125M HCl was added, and the mixture was kept in a refrigerator at 4 ℃ after 2 minutes in a boiling water bath for further use.
3. Initial localization of population segregation analysis
Take F2Respectively mixing 15 plants of each single plant with purple line and 15 plants without purple line, and extracting DNA to obtain a wired gene pool and a wireless gene pool. SSR primers were synthesized based on published sequences (see in particular httpHtml) was used to amplify the gene pool with the synthesized 302 pairs of primers, and the PCR amplification procedure was performed in 10. mu.L system, L. mu.L template, L. mu.L 10 pmol/. mu.L upstream primer, L. mu.L 10 pmol/. mu.L downstream primer, L. mu.L 10 Xbuffer (Mg. mu.L 10 Xbuffer)2 Ten) mu.L of 2mM dNTP, 0. mu.L of Taq, and 3.9. mu.L of water; and detecting the PCR product by PAGE gel electrophoresis with the mass volume fraction of 10% and a silver staining method. Through two F2And (3) carrying out primary positioning on the formed positioning group to obtain the same primary positioning result: the primers RM587 and RM5531 on the 6 th chromosome have differences among gene pools, and are proved to be linked with the purple line after being analyzed by recessive individuals, recombinants of the two primers are not included, so that the two primers are respectively positioned at two sides of the target gene, and the genetic distances between the two primers and the target gene are respectively 22.9cM and 20.3cM (figure 2). The primary targeting primer sequences are shown in Table 1.
4. Fine positioning
To further narrow the localization of candidate genes, we used F of nine B × R252The generation was used as a fine positioning group, and a total of 1020 wireless individuals were obtained. 24 pairs of SSR primers were newly synthesized between the preliminarily mapped primers RM587 and RM5531, with clear differences between the parents RM8258, RM5754 and RM 19665. Further analysis shows that they are all linked with the target gene. 20, 14 and 186 recombinants were found in 1020 recessive individuals, and the OsMYB76 gene was located between RM5754 and RM19665 according to the number of recombinants and inclusion relation of each primer (FIG. 2). According to the annotation of fine localization intervals at http:// www.gramene.org/website, only one MYB transcription factor, LOC _ Os06g10350, involved in anthocyanin formation was found, and homology analysis indicated that its homologous gene C1 in maize regulated transcription of structural genes associated with pigment synthesis. LOC _ Os06g10350 is most likely our candidate gene OsMYB 76. Sequencing alignment of R25 and Loc _ Os06g10350 gene in the mutant revealed: deletion of 10 bases (ACTGGAACAG) after 319 bases in CDS of Loc _ Os06g10350 gene in OsMYB76 mutant leads to frame shift of protein translation of the gene in the mutant from 107 amino acid and termination at 206 amino acid in advance; a T → C substitution at base 369 and a T → C substitution at base 716One T → C substitution. Therefore, it was preliminarily determined that Loc _ Os06g10350 is our target gene OsMYB 76. The nucleotide sequence of CDS of the gene is shown in SEQ ID NO. 2.
TABLE 1 Gene mapping and functional verification primer sequences
Example 3 functional analysis of OsMYB76 Gene and application in identification of transgenic Positive offspring
In order to further determine that the OsMYB76 gene is a gene causing a purple-line-free phenotype and detect whether the gene can be used as a reporter gene for transgenic positive progeny detection, CDS of the OsMYB76 gene and a modified OsMYB76 gene (SEQ ID NO.1) are used for replacing GUS genes in pCAMBIA1301 by means of enzyme digestion and homologous recombination respectively to obtain vectors p1(pOsMYB76) and p2(pOsMYB76R), and complementary fragments of the genes OsABCG15(Wu et al, CG 2014) related to rice pollen fertility are loaded into multiple cloning sites in MYp 1 (pOsB 76) and p2(pOsMYB76R) vectors to obtain vectors pX1(pOsABC 15-OsMYB76) and pAB 2 (pOsCG 15-OsB 76 MYB76R), and the purple vector fertility-related materials are subjected to specific operation and survey, wherein the following specific operations are carried out:
1. using R25 coleoptile cDNA as a template, using a primer OsMYB76CF/R (with BglII and BstEII, table 1) to amplify a target fragment, and using BglII and BstEII to connect the target fragment into pCAMBIA1301 to obtain a vector p1(pOsMYB 76); using the synthesized gene OsMYB76R as a template, a target fragment was amplified using OsMYB76RF/R (carrying BglII and BstEII homologous fragments of pCAMBIA1301, respectively, Table 1), and the target fragment was ligated to pCAMBIA1301 by homologous recombination, resulting in vector p2(pOsMYB 76R).
2. A complementary fragment of OsABCG15 gene was excised from the OsABCG15 gene complementation vector (Wu et al, 2014) using EcoRI and SbfI, and simultaneously loaded with p1(pOsMYB76) and p2(pOsMYB76R), to give vectors pX1(pOsABCG15-OsMYB76) and pX2(pOsABCG15-OsMYB 76R). After the sequences of the vectors are ensured to be correct, the agrobacterium tumefaciens LBA4404 is transformed by a freeze-thaw method, and the agrobacterium tumefaciens respectively containing recombinant expression vectors pX1(pOsABCG15-OsMYB76) and pX2(pOsABCG15-OsMYB76R) is obtained.
3. The obtained positive agrobacterium is transformed into rice osmyb76 mutant, the purple performance condition is observed, and the result shows that the T parts of the two plasmids are transferred0The base of the stem of the plant appeared purple (FIG. 1, C), the sheath, stem, stigma and tip at maturity also appeared purple, its T1The generation coleoptile purple line shows the separation of 3:1, the leaf sheath, stem and stigma in flowering and the rice tip in maturation all show the purple color, and are separated from the coleoptile purple line; t with colorless stem base0The plants have no purple color at any time and organs, and the T of the plants is1Generation is also when all organs are purple-free at any time. The results show that the cloned OsMYB76 is a target gene for controlling anthocyanin synthesis, and the modified OsMYB76R gene has the same function as the OsMYB76 gene and can regulate anthocyanin synthesis.
4. Nine B-OsABCG15 in colorless genic sterile materials with abnormal OsMYB76 gene and all organs without purple are bred by taking the nine B with abnormal OsMYB76 gene as a recurrent male parent and the genic sterile mutant of OsABCG15 gene as a non-recurrent female parent. The above-mentioned positive Agrobacterium was transformed into colorless nonaB-osabcg 15, and as a result, it was found that all of the T's were purple-colored at the base0Pollen of the plants is restored to be fertile, and T of the plants1Purple of the generation is separated from pollen fertility, and colorless is separated from pollen sterility. The colorless nuclear sterile material OsABCG15 becomes fertile after being transgenic, and a transgenic positive event occurs for OsABCG15 gene; fertile and purple color at T0Concomitant in plants, at T1Co-segregation occurs in the generations, indicating that purple color can be used to indicate a positive event for the transgene. Therefore, the gene OsMYB76 and the OsMYB76R gene transformed from the gene OsMYB76 can be used as a plant endogenous reporter gene for identifying transgenic positive plants.
Example 4 use of the OsMYB76R Gene in identifying gametophytic male sterility, identifying pollen-transmitted transgene Components, and screening for transgene contamination by pollen
The male gametophyte development-associated gene is often expressed in flowers/pollen in the middle and late stages. In order to find some gametophyte development related genes, a BAR rice gene expression database (http:// BAR. utontono. ca/efprice/cgi-bin/efpWeb. cgi) is analyzed, a plurality of genes expressed in flowers at the middle and later stages are found, and a batch of genes possibly related to the development of male gametophytes are selected through qPCR verification. The rice Loc _ Os05g40740 gene is specifically and highly expressed in late flowers, the arabidopsis thaliana homologous gene is specifically expressed in pollen, the Loc _ Os05g40740 gene is listed as a male gametophyte development related gene preliminarily, and the promoter thereof is listed as a pollen specific promoter. In order to prepare and identify the gametophytic male sterile material, the creation and identification of gametophytic male sterility was carried out with Loc _ Os05g40740 as the target:
1. the kind and sequence of the enzyme cutting sites of the multiple cloning sites of the vector p2(pOsMYB76R) are modified. Firstly, a new multiple cloning site sequence is artificially synthesized, and the nucleotide sequence is shown as SEQ ID NO. 18. The synthetic sequence was then replaced by homologous recombination with the original sequence of the multiple cloning site, and the new multiple cloning site was shown in FIG. 3. The vector after the multi-cloning site modification is named as pOsMYB76R 1.
2. Artificially synthesizing a Nos terminator sequence A (with a restriction enzyme site Sbf I + Asc I), wherein the nucleotide sequence is shown as SEQ ID NO. 19; loading it into pOsMYB76R1 to obtain vector pOsMYB76R 2.
3. Artificially synthesizing an interference fragment (with enzyme cutting sites EcoRI + Sbf I) of Loc _ Os05g40740, wherein the nucleotide sequence of the interference fragment is shown as SEQ ID NO. 20; loading it into pOsMYB76R2 to obtain vector pOsMYB76R 3.
4. The promoter of Loc _ Os05g40740, the nucleotide sequence of which is shown in SEQ ID NO.23, was amplified from Zhonghua 11 using primers P5F (5'-GCGTCGACgtcccatgtcaccgacagtact-3', SEQ ID NO.21, with restriction site SalI) and P5R (5'-CGGAATTCcgtggaaatgtgatcgctaggct-3', SEQ ID NO.22, with restriction site EcoRI). Loading the vector pOsMYB76R3 to obtain a gametophyte male sterility vector pOsMYB 76R-GM.
5. pOsMYB76R-GM was transferred into Agrobacterium LBA4404, and positive Agrobacterium was transfected into colorless ZhongjiuB in all organs.
6. At T0Selecting positive plants according to the existence of purple at the stem base, selfing, using the positive plants as male parent, and using the positive plants as the nine B-os in the colorless nuclear sterile materialabcg 15. The purple line investigation is carried out on the inbred and hybrid seeds, and the inbred F is found2The separation ratio of the purple line of the seeds is 1:1, and the separation ratio of the purple line of the seeds is not 3:1, 97.1 percent1The seeds show no purple line, which indicates that the transgenic components are difficult to transfer with pollen between generations, but with female gametes, which is characteristic of gametophytic male sterility. Thus, Loc _ Os05g40740 was associated with male gametophyte pollen development, and by interfering with this, we obtained gametophytic male sterile material and successfully performed genetic identification by genetic investigation of linked purple line traits.
Genetic analysis of gametophyte male sterile material using F used in the above creation1The seed can also realize the identification of the probability of the transgenic components transmitted by the pollen by investigating the proportion of purple line individuals, and can screen out the transgenic pollution transmitted by the pollen by eliminating individuals with purple lines.
Example 5 application of OsMYB76 gene interference in identification of transgenic positive strains
In rice, the OsMYB76 gene of many materials is normal, the materials usually show purple color in one or more organs, such as II-32B organs such as coleoptile, leaf sheath, leaf margin and stigma, and the materials cannot be used as transgenic objects to identify transgenic positive strains by using the OsMYB76 gene or the modified OsMYB76R gene as a reporter gene. Therefore, a method for identifying the transgenic positive strains of the material by using the interference of OsMYB76 is designed, and specific operations and results are as follows:
1. the kind and sequence of the restriction enzyme sites of the multiple cloning site of the vector pCAMBIA1300 are modified. Firstly, a new multiple cloning site sequence is artificially synthesized, and the nucleotide sequence is shown as SEQ ID NO. 24. The synthetic sequence was then replaced by homologous recombination with the original sequence of the multiple cloning site, and the new multiple cloning site was shown in FIG. 4. The vector after the multi-cloning site modification is named pRSVM-1300.
2. Interference elements of the OsMYB76 gene were loaded sequentially with isocaudarner BamHI and BglII in pRSVM-1300:
(1) artificially synthesizing a Nos terminator sequence B (with enzyme cutting sites SpeI + BamHI and BglII), wherein the nucleotide sequence is shown as SEQ ID NO. 25; it was loaded into pUC57 to obtain vector pTnosB.
(2) The target fragment TnosB was excised from pTnosB with SpeI + BglII, and pRSVM-1300 was digested with SpeI + BamHI to recover the scaffold, and the target fragment and the scaffold were ligated to obtain vector pRTnosB.
(3) Artificially synthesizing an OsMYB76 interference fragment IC1 (with enzyme cutting sites SpeI + BamHI and BglII), wherein the nucleotide sequence of the OsMYB76 interference fragment IC1 is shown as SEQ ID NO. 3; it was loaded into pUC57 to obtain vector pIC 1.
(4) The target fragment IC1 was excised from pIC1 using SpeI + BglII, and the backbone was recovered by digesting pRTnosB with SpeI + BamHI, and the target fragment and backbone were ligated to give vector pIC 2.
(5) Primer PCF5(Ggactagtggatccctccctccgtcccaaaatataag, SEQ ID NO.26 with SpeI _ BamHI)/PCR5(GAagatctgcagcaagctctcctccccat, SEQ ID NO.27 with BglII) is designed, and OsMYB76 gene promoter is amplified from R25 genome and is recovered for standby, and the nucleotide sequence of the promoter is shown as SEQ ID NO. 28.
(6) And (3) digesting the OsMYB76 gene promoter by using SpeI + BglII for recycling and standby, digesting the pIC2 by using SpeI + BamHI for recycling and standby, and connecting the target fragment and the skeleton to obtain a vector pIC.
(7) The complementary fragment of OsABCG15 gene was excised from the complementary vector of OsABCG15 gene using EcoRI and SbfI, and loaded into pIC to obtain vector pOsABCG 15-IC.
(8) Purple II-32B-OsABCG15 nuclear sterile materials such as OsMYB76 gene normal and leaf sheath are bred by taking purple II-32B of leaf sheath as a recurrent male parent and taking a nuclear sterile mutant OsABCG15 of OsABCG15 gene as a non-recurrent female parent. Agrobacterium containing pOsABCG15-IC was transformed into II-32B-osabcg15 to obtain 15T cells0Individuals with purple 6 sheaths, colorless 9 sheaths (FIG. 1, D), and purple sheath T0The pollen of the plant is still sterile and colorless T0Pollen of plants is restored to fertility, and T of these fertile plants1The colorless leaf sheaths of the generations are separated from the pollen in a fertile way, and the purple leaves are separated from the pollen in a sterile way. The nuclear sterile material II-32B-OsABCG15 becomes fertile after being transgenic, and a transgenic positive event occurs for OsABCG15 gene; fertile and colorless leaf sheathAt T0Concomitant in plants, at T1Co-segregation occurs in the generations, indicating that colorless leaf sheaths generated by the interference can be used to indicate a positive event for the transgene. Therefore, when the host is transformed to have a purple character (such as purple leaf sheath), a colorless character report transgene generated by the interference of the gene OsMYB76 can be used for positive plant identification (FIG. 1, E-F).
Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that, while the invention has been described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> university of Yunnan
<120>Rice coleoptile purple line geneOsMYB76Application and implementation method of
<160> 28
<170> SIPOSequenceListing 1.0
<210> 1
<211> 819
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgggacgca gggcttgctg tgcaaaggaa ggaatgaagc ggggtgcctg gacgtccaag 60
gaggacgatg tcctggcgag ctacattaag tctcacggag agggtaagtg gcgggaggtt 120
ccgcagagag ccggacttcg gagatgcggc aagtcctgtc gccttagatg gttgaactat 180
ctccgcccaa atatcaagag gggcaacatt gacgatgacg aggaagagct gatcgtgcgg 240
cttcatacgc tcctggggaa tagatggtcc ctgatcgcag gccgccttcc gggaaggacc 300
gataacgaga ttaagaacta ctggaatagt acactctcca ggaagatcgg aacggccgcg 360
accgccgccg ccggctcacg cggcggctcc acccctgata cagccagggc gactgacgcg 420
gcttccagct cttcagtggt cccaccagga cagcaacagc aaccagcaag ccgggcggat 480
actgacaccg caaccgccgc cgccgccgcc gccgccacta caacgactgt ttgggctcca 540
aaggcagtgc ggtgcacgag aggcttcttt ttccacgata gagaaactgc accattggct 600
gctgcggcgc ctgctcccgc aggagagctg ggagacggag atgacgtgga ttgcgactac 660
tattgttctg gttcgagttc cgctgcaacc acaacctcct cctcctccct cccggttgtg 720
gtcgagccat gctttagtgc cggcgatgac tggatggatg acgtccgcgc cttggcgtca 780
ttcctcgaca cagatgacgc ttggaacctg tgtgcgtga 819
<210> 2
<211> 819
<212> DNA
<213> Rice (oryza. sativa L.)
<400> 2
atggggagga gagcttgctg cgcaaaggaa gggatgaaga gaggggcatg gacgagcaag 60
gaggacgacg tgcttgcctc ctacatcaag tcccatggcg aaggcaagtg gcgcgaggtc 120
ccccaacgag ctggtttgag gcggtgcggc aagagctgca ggctccggtg gctcaactat 180
ctccggccta acatcaagcg cggcaacatc gacgacgacg aggaggagct catcgtcagg 240
ctccacaccc tcctcggcaa caggtggtct ctcattgcag gcaggctgcc gggccgaaca 300
gacaatgaaa tcaagaacta ctggaacagc acgctcagcc gcaagatcgg caccgccgcc 360
accgccgctg ccggcagccg cggtggcagc acgccggaca ccgccagagc gacggacgcg 420
gcgtcgtcca gctccgtcgt gccgccgggc cagcagcagc agccagcctc ccgcgccgac 480
accgacacag caacggcagc ggcggcggcg gcggcgacga cgaccaccgt gtgggcgccc 540
aaggccgtgc ggtgcacgcg cgggttcttc ttccacgacc gtgaaacggc gccgctcgcc 600
gcggcggcgc cggcgccggc aggggaatta ggagacggcg atgacgtcga ctgcgactac 660
tactgcagcg gcagcagctc ggcggcgacg acgacgtcgt cgagctcatt accggtggtc 720
gtcgagccgt gcttctccgc cggcgacgac tggatggacg acgtgagagc cttggcgtcg 780
tttcttgaca ccgacgacgc ctggaacttg tgtgcgtga 819
<210> 3
<211> 712
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ttacactagt actagcagac cattaggatc caacaaagca ccagtggtct agtggtagaa 60
tagtaccctg ccacggtaca gacccgggtt cgattcccgg ctggtgcatg gggaggagag 120
cttgctgcgc aaaggaaggg atgaagagag gggcatggac gagcaaggag gacgacgtgc 180
ttgcctccta catcaagtcc cttggcgaag gcaagtggcg cgaggtcccc gaacgagctg 240
gtttgaggcg gtgcggcaag agcagcaggc tccggtggct caactatctc cggcctaaca 300
tcaagcgcgg caacatcgac gacgacgagg aggagctggt gagctagcta ttacctaatc 360
gatcgatggt catcgatcat gagatgatga tgatgagatt tgtacttaat tgtgatctgt 420
atggatgctg ttgttgatca agttcttgcg atcgatcgat ctgaattttc aggtttgact 480
cctcgtcgtc gtcgatgttg ccgcgcttga tgttaggccg gagatagttg agccaccgga 540
gcctgctgct cttgccgcac cgcctcaaac cagctcgttc ggggacctcg cgccacttgc 600
cttcgccaag ggacttgatg taggaggcaa gcacgtcgtc ctccttgctc gtccatgccc 660
ctctcttcat cccttccttt gcgcagcaag ctctcctccc caagatctgg ca 712
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ctttgctacc agtagatcca gc 22
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
accctctatc tgtgcatcaa 20
<210> 7
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ctcgactaaa ctcgaaatcc t 21
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
<210> 10
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cgatgtcttc gagtccctta acagg 25
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
acggttggtg atgctcttag gc 22
<210> 12
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tttgtgttgg taagttgctt c 21
<210> 13
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ttaaggagag tgttttcttt tctc 24
<210> 14
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gaagatctat ggggaggaga gcttgctg 28
<210> 15
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
ctggtgacct cacgcacaca agttccagg 29
<210> 16
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ggactcttga ccatgatggg acgcagggct tgct 34
<210> 17
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
attcgagctg gtcactcacg cacacaggtt ccaag 35
<210> 18
<211> 227
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
acagctatga ccatgattac aagcttcata cctctagaca cccatggagt gtatgagtaa 60
ggtacacgtg cacgagctct ttttagcggg gtcgacacaa ccgaattcca cagatctatg 120
gaatgagtat gtgaggtgac ccaccctgca ggtttttgaa gggggcgcgc cacaaccact 180
agtcaccacg gtaccagcgg gggatccggc actggccgtc gttttac 227
<210> 19
<211> 286
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gccctgcagg gaatttcccc gatcgttcaa acatttggca ataaagtttc ttaagattga 60
atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac gttaagcatg 120
taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg attagagtcc 180
cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac taggataaat 240
tatcgcgcgc ggtgtcatct atgttactag atcgggggcg cgccac 286
<210> 20
<211> 803
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cggaattcgc agaaggtgat cctgatcaac ggcgagttcc cgggcccgcg gatcaactgc 60
tcgtccaaca acaacatcgt ggtgaacgtg ttcaaccagc tggacgagcc gctgctcttc 120
acctggaacg ggatgcagca ccgcaagaac tcgtggcagg acggcctcgc cgggacgcag 180
tgccccatcg cgccgggcac caactacacg tacaagtggc agcccaagga ccagatcggc 240
agcttcttct acttcccgtc gctggggatg caccgcgccg ccggcggcta cggcgggatc 300
agcgtcgtca gccgcctgca gatctacgag ctggtgagct agctattacc taatcgatcg 360
atggtcatcg atcatgagat gatgatgatg agatttgtac ttaattgtga tctgtatgga 420
tgctgttgtt gatcaagttc ttgcgatcga tcgatctgaa ttttcaggtt tgaggggtga 480
ccgcaggcgg ctgacgacgc tgatcccgcc gtagccgccg gcggcgcggt gcatccccag 540
cgacgggaag tagaagaagc tgccgatctg gtccttgggc tgccacttgt acgtgtagtt 600
ggtgcccggc gcgatggggc actgcgtccc ggcgaggccg tcctgccacg agttcttgcg 660
gtgctgcatc ccgttccagg tgaagagcag cggctcgtcc agctggttga acacgttcac 720
cacgatgttg ttgttggacg agcagttgat ccgcgggccc gggaactcgc cgttgatcag 780
gatcaccttc tgccctgcag gga 803
<210> 21
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gcgtcgacgt cccatgtcac cgacagtact 30
<210> 22
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
cggaattccg tggaaatgtg atcgctaggc t 31
<210> 23
<211> 1887
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gtcccatgtc accgacagta ctaaatgggt aaagattgga taaagtatat ggggtatttg 60
tgaggtatta ttagaaaact tcgtgtggtt ttgatggacc tgttttatgt gttgaaaata 120
tgaatggtta tagggtgtgt ttgcaagtgc aggatgggaa ctcatccctc ctgcacgcaa 180
aacggagcgg ctttttaaca catgattaat taaatattag ctaatttttt taaaaaaaat 240
ggattaattt gattttttta agcaactttc atatagaaat tttttgcaaa aaacacaccg 300
tttaatagtt taaaaacgtg cgcgcgaaaa acgagggaga ggggttggga acatgggttt 360
gcaaacacaa ccatagtatt ggcgattcct tttcgtttga gtaaatttta caaaactaca 420
ggtattttga ccaaattatc acaaaactac agatttaagg agttgtatca taaaactaca 480
catttagcat caaatttatc acaaaactgc agattttagg ttaagtatca caaaaataca 540
tatttaatat tgaacttatc acaaaactat aacttttgga gtttaaatcc ctagcaccat 600
tgttatggtg gagctataaa cattattact ttgtgattaa attggttcta aacctttagt 660
tttatgataa tttagtaact aaacgtgtag ttttgtaaca cttcatcttt aatatgtagt 720
tttgtgctaa atttggtgct aaatgtgtaa ttttgtgata taattcctta aatatgtagt 780
tttgtgatag tttggttata atatctgtag ttttatgaaa tttactcttt tcgttttcac 840
tgcaatttgg aatgatggaa ttgactagat ccggcattac cgatgggctg ccgaacgctg 900
tgatgcggtt gatcttgagc gatccgggac gccacaagca ccgatgggtt ctgggagttc 960
atacggctgg tgcagcagtg tgtcaatagc agccgggatg tgcgcccaac catggtcgcc 1020
gtcgagagga ggatcgaaga catcctgaac tcggttgtca ggtcatccac caccgggttc 1080
atgactgccg gaggcgacac acccagcaac gagccaaatc gtgaagataa cggaaacgag 1140
ccaaatccca gcaacgagat cgccagggac tagtagtacg tacagcagtg gtgatttgtc 1200
atataggtgt atatcggctg ttttcgcatc tcaaggcctc aagcagtgtg tgcaatctgg 1260
agtagtatat aaatatgtaa aatgttcatt tcgatatact gtcaaatgcg tgtaaattaa 1320
ccaatgctaa aacaacacac tgtgactaaa tttactgagt tggatgatga ggatgattat 1380
gttgcgtgca cacctgatca ggaggacata taatataggc catttgggcc gtcttggaca 1440
ccaccgtttg atttgtatga agttgggccg aactatgcaa gcccagaggc gctgcctctg 1500
tgccacggcc cacgggcatc gctggatggt caagcaggtg atcggtggag cgccaatggc 1560
ggcggcgaga cacacagcgc ggcgcgcgcg cgaacgtgcg gacgcgcgcg ccccggccac 1620
ggccgccgcg ctcgtctcct ggcctcccgc gcccgctaca aatggcggcc ccggcgtccc 1680
ctcctcactc cgaagcttcc cggttgacga cctctccggt ctcccccctc accccaccgc 1740
aacccgggac gtcttccatg gccgccgccg ccgccgcccc cgcctactaa accaccctac 1800
ccaccccctc caaactccca cacattacat ccttcaaaga gagcatcaca cacacacaca 1860
caccagccta gcgatcacat ttccacg 1887
<210> 24
<211> 446
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ttacaagctt cgtgcaggag aggacggtac ccacacaatc tgccctgtta accaaatttt 60
ctgtcagcct caggggagag ggtgaaggtg ggccccatac ggaaaactta ccatggcctt 120
aaatttattt gggcgcgccg gaaaactacc tgttcacgtg cacttgtcac tacttggtga 180
cctgttcaat gcttttcgag ctcacccaga tcatatgatc tagacacgac ttcttcaaga 240
ggcctcatgc ctgagggata cctgcaggag tgtatgagta aggtactgtc gacggagaca 300
ccctcgtccc tagggatcga gcttaaggga tttaaatatt tcaaggagga cggaattccg 360
gccacaagtt ggaactagta ctacaactcc cacaagcgct actagcagac cattaggatc 420
ccattgaaga tggaagagat ctggca 446
<210> 25
<211> 311
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ttacactagt actagcagac cattaggatc cgctcgaatt tccccgatcg ttcaaacatt 60
tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa 120
tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg 180
agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa 240
atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg 300
gagatctggc a 311
<210> 26
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
ggactagtgg atccctccct ccgtcccaaa atataag 37
<210> 27
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gaagatctgc agcaagctct cctccccat 29
<210> 28
<211> 525
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ctccctccgt cccaaaatat aagcattttt agaatagtgt caagtcaaac tttttaaatt 60
ttgagtatta gtagcaaaaa ttttagaaag attaatcatg taaatttgat cttactatat 120
ttatcattaa acgaactatc ataacatgca actcttttta tttaaaacat tctactttta 180
tagatattgt tggtcaaaat agtatctcga aaaccgtgtc aagaccaaaa atacttatat 240
tttgggacgg aggggtagta ttcattgatt ctctccatcc gtagcagcca agtcccatgg 300
atggttgcat gtgaccacat cgtacggggc tacaaggtgg tgtgtattca tgcccacacc 360
gttgcatgta taagtacagc gcaaaagtgg tagaacgaga ggctctgatg cgcaccagat 420
cgctcagtct cacaccgcac agagacagag aagagctcta gagagaacga gagagagaga 480
cagagagaga gagagagagg gagaatgggg aggagagctt gctgc 525
Claims (8)
1. The application of the rice coleoptile purple line gene OsMYB76 in identification of transgenic positive offspring, identification of gametophyte male sterility related gene, identification of transgenic component transfer rate along pollen and removal of transgenic pollution is characterized in that: the nucleotide sequence of the rice anthocyanin synthesis regulatory gene OsMYB76 is shown in SEQ ID No. 2.
2. Modified gene OsMYB76R of rice coleoptile purple line gene OsMYB76, which is characterized in that: the nucleotide sequence of the modified gene OsMYB76R is shown in SEQ ID NO. 1.
3. The use of the modified gene OsMYB76R in claim 2 for identifying transgenic positive progeny, identifying gametophytic male sterility associated genes, identifying the rate of transmission of transgenic components with pollen, or removing transgenic contamination.
4. The expression down-regulation element of the rice coleoptile purple line gene OsMYB76 is characterized in that: the expression down-regulation element is an interference fragment, and the nucleotide sequence of the expression down-regulation element is shown as SEQ ID NO. 3.
5. Use of the expression down-regulation element of the gene OsMYB76 in claim 4 for identification of transgenic positive progeny, identification of gametophytic male sterility related gene, identification of the rate of transmission of transgenic components with pollen, or removal of transgenic contamination.
6. The method for identifying the transgenic positive plant by using the rice coleoptile purple line gene is characterized by comprising the following steps: the OsMYB76 gene or the modified gene OsMYB76R and a certain target gene X to be identified are linked and transferred into a plant with anthocyanin deficiency, whether each organ is purple or not is observed, and the purple organ in any organ is a transgenic positive strain; the nucleotide sequence of the OsMYB76 gene is shown as SEQ ID NO.2, and the nucleotide sequence of the modified gene OsMYB76R is shown as SEQ ID NO. 1; or the expression down-regulation element of the OsMYB76 is linked with a certain target gene X to be identified and transferred into a plant with normal anthocyanin, whether each organ has purple color or not is observed, and the transgenic positive strain without purple color in any organ is observed; the interference fragment of the OsMYB76 is shown in SEQ ID NO. 3.
7. An application method for identifying male gametophyte sterility by using a rice coleoptile purple line gene is characterized by comprising the following steps: transferring OsMYB76 gene or modified gene OsMYB76R and a down-regulation element of a possible gametophyte male sterility related gene GMS to be identified into anthocyanin-deficient plants in a linkage manner, pollinating a colorless female parent by taking a purple positive strain as a male parent, and observing a hybrid F1Judging whether organs of the generation have purple color and proportion, if all the individuals have no purple color or the proportion of colored individuals is far less than 50%, judging that the GMS is a male gametophyte sterility related gene; the nucleotide sequence of the OsMYB76 gene is shown in SEQ ID NO.2, and the nucleotide sequence of the modified gene OsMYB76R is shown in SEQ ID NO. 1.
8. The application method for identifying the transmission ratio of the transgenic components along pollen and removing transgenic pollution by using the rice coleoptile purple line gene is characterized by comprising the following steps of: the OsMYB76 gene or the modified gene OsMYB76R and a transgenic component Y to be identified are linked and transferred into a plant with anthocyanin deficiency, a colorless female parent is pollinated by a purple positive strain, and F is investigated1Calculating the number of purple individuals N as the total number of individuals N, and calculating the number of individuals to be detectedTransfer rate (%) of transgene components with pollen: 100 x 2N/N; directly removing purple individuals in any organ, namely removing transgenic pollution generated along with pollen transmission; the nucleotide sequence of the OsMYB76 gene is shown in SEQ ID NO.2, and the nucleotide sequence of the modified gene OsMYB76R is shown in SEQ ID NO. 1.
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| CN107142276A (en) * | 2017-05-23 | 2017-09-08 | 华南农业大学 | A kind of transgenic breeding method that anthocyanidin is synthesized in Crop Seed Endosperm |
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| CN107142276A (en) * | 2017-05-23 | 2017-09-08 | 华南农业大学 | A kind of transgenic breeding method that anthocyanidin is synthesized in Crop Seed Endosperm |
Non-Patent Citations (4)
| Title |
|---|
| 1个水稻紫叶基因pl41的遗传分析与定位;刘立龙等;《华南农业大学学报》;20180416(第03期);31-36页 * |
| Dominant complementary interaction between OsC1 and two tightly linked genes, Rb1 and Rb2, controls the purple leaf sheath in rice;HU,W.等;《Theor Appl Genet.》;20200526;2555-2566页 * |
| MYB类转录因子对花药和花粉发育的调控途径;邓志刚等;《西北植物学报》;20130415(第04期);214-220页 * |
| 植物细胞核雄性不育基因研究进展;杨莉芳等;《植物遗传资源学报》;20131022(第06期);128-137页 * |
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