CN111575285A

CN111575285A - Carrier containing oryza longistaminata promoter and capable of restoring male fertility of rice OsCYP704B2 mutant and application

Info

Publication number: CN111575285A
Application number: CN202010384451.5A
Authority: CN
Inventors: 李新鹏; 陈磊; 李燕群; 陈江淑; 陈思兰; 安保光; 龙湍; 吴永忠; 黄培劲
Original assignee: Hainan Bolian Rice Gene Technology Co ltd
Current assignee: Hainan Bolian Rice Gene Technology Co ltd
Priority date: 2020-05-07
Filing date: 2020-05-07
Publication date: 2020-08-25
Anticipated expiration: 2040-05-07
Also published as: CN111575285B

Abstract

The invention relates to a carrier containing a oryza longistaminata wild rice promoter and capable of restoring male fertility of a rice OsCYP704B2 mutant and application thereof. The OlCYP704B2 gene promoter of the oryza longistaminata is shown as SEQ ID NO.1, and the OsCYP704B2 gene expression cassette of rice driven by the promoter and optimized in sequence is shown as SEQ ID NO. 4. The promoter provided by the invention can drive the gene to be efficiently expressed in the plant anther; the rice OsCYP704B2 gene expression cassette provided by the invention can realize high-level expression of the OsCYP704B2 gene in anthers under the drive of the promoter, completely recovers rice male sterility caused by OsCYP704B2 function deletion, has high genetic stability, and can be used for recovering rice male fertility or preparing rice male genic male sterile maintainer lines and sterile lines.

Description

Carrier containing oryza longistaminata promoter and capable of restoring male fertility of rice OsCYP704B2 mutant and application

Technical Field

The invention relates to the field of plant molecular biology, in particular to an expression cassette containing a promoter of an OlCYP704B2 gene of oryza longistaminata and capable of restoring male fertility of a rice OsCYP704B2 mutant and application thereof.

Background

Rice is one of important grain crops, and hybrid rice plays an important role in increasing grain yield. The hybrid rice is produced by using two rice parents with different genetic backgrounds and complementary properties and through hybridization to produce the first generation hybrid with heterosis for rice production. The hybrid rice has obvious heterosis phenomenon, and the yield is increased by 30% compared with the conventional rice. At present, hybrid rice can be classified into three-line hybrid rice and two-line hybrid rice. Three-line hybrid rice (three-line hybrid rice) means that three rice lines are required to produce this hybrid rice: rice cytoplasmic male sterility maintainer line and rice cytoplasmic male sterility restoring line. Rice cytoplasmic-nuclear interactive male sterile line (sterile line, code A) has wild abortive type and red lotus type cytoplasm. The rice cytoplasmic male sterile maintainer line (maintainer line, code B) is used for breeding sterile line, and its nucleus genotype is identical to that of sterile line, but contains fertile cytoplasmic gene, can produce fertile pollen, and can self-copulate and fruit. Since the nuclear gene of the maintainer line does not contain a restorer gene, progeny produced by pollination of the sterile line are also sterile. The rice cytoplasmic male sterility restoring line (restoring line, code R) carries restoring gene, and can repair cytoplasmic male sterility, so that the hybrid produced by hybridization with sterile line (i.e. hybrid rice) can be normally bred. The three-line breeding method has the problems of susceptibility to diseases, poor quality, low breeding efficiency and the like, and 95 percent of rice resources cannot be used for cross breeding due to the restriction of the relationship between restoration and conservation. The two-line hybrid rice utilizes photo-thermo-sensitive genic male sterile mutant, which shows male sterility under the condition of high temperature in long day, can be used for hybrid seed production, shows male fertility under the condition of low temperature in short day, and can be self-bred. Compared with the three-series method, the two-series method has remarkable advantages: the sterile line and the restorer line are freely matched and have high probability of matching excellent combinations; the sterile line can be used for two purposes. However, the sterile line of the two-line method is easily influenced by the light-temperature environment, and the breeding risk is huge: for example, when the sterile line is changed into fertile one at low temperature during the seed production period, the selfing seeds are generated, and the purity of the hybrid seeds is influenced; some two-line hybrid species are sterile when encountering high temperature, which affects the maturing rate and results in reduced yield. Therefore, the search for new methods for male-sterile seed production remains an important research topic in crop breeding.

The SPT technology of the American Pioneer company successfully solves a series of problems in corn hybrid seed production by using a transgenic technology, and realizes commercial application in corn in 2012. The technique can also be applied to hybrid seed production of other crops (Wu et al, 2016, Development of a novel genetic male sterility system for hybrid seed production in mail and other cross-linking crops). The core of the SPT technology is to screen male sterile mutants and construct a vector containing a fertility restorer gene, a pollen abortion gene and a screening marker gene; through a large amount of screening and identification, the transformation events with single copy, stable heredity and stable and reliable work of each element are obtained and are used for producing new sterile lines and transgenic maintainer lines. The rice hybrid seed production can also realize the propagation of the common genic male sterile line and the maintainer line thereof by utilizing the technology, thereby overcoming the defects of the hybrid seed production by a three-line method and a two-line method and improving the rice hybrid seed production technology.

The rice CYP704B2 gene (i.e., OsMs26 gene) belongs to the P450 family, and its coding region contains 4 exons and encodes a protein of 544 amino acids (Li et al, 2010, Cytochrome P450 family member CYP704B2 genes the. omega. -hydroxylation of fatty acids and is required for antibiotic biosynthesis and polene expression in rice Cell, vol.22: 173-190). The CYP704B2 gene is specifically expressed in the mucoid layer and microspores before and after meiosis to form microspores. CYP704B2 protein is involved in fatty acid metabolism, and the fatty acid metabolites are precursors of cutin and sporopouenin. The deletion mutation of CYP704B2 hinders the synthesis and transportation of sporopouenin, so that the exine of microspore can not be normally formed, and the microspore is further inhibited from developing, thereby causing complete male sterility; in addition, the cutin on the surface of the mutant anther is lost. Li et al transferred a 3.8kb genomic fragment containing the CYP704B2 gene into the CYP704B2 mutant, restoring male fertility to the mutant.

However, the breeding engineering of crops requires extremely high genetic stability and requires easy follow-up identification. The DNA fragment such as Li for restoration of fertility cannot satisfy both requirements. The main reasons are as follows: firstly, the longer the continuous homologous fragment is, the higher the probability that homologous recombination occurs to delete a gene is; secondly, in engineering application, the genetically transformed plant needs to be subjected to rechecking and backcross transformation, the genetic background of a mutation site needs to be identified in the two processes, the three genes of the original genome wild type, the mutant and the transformed restoring gene need to be distinguished simultaneously, and the sequence of the restoring gene in the transformed fragment is consistent with that of the wild type gene. The DNA fragment transformed by Li and the like is completely the same as the wild type, and the three fragments are difficult to be distinguished by designing corresponding molecular markers. Finally, the larger the transformation fragment, the lower the transformation efficiency. Therefore, Li and the like adopt the rice original genome sequence with the length of 3.8kb (not including flanking vector sequences) as a restoring gene segment, the possibility of genetic instability in engineering production application is higher, and the difficulty of vector construction and transformation is increased; it is also difficult to identify the genotype of the mutation site. In conclusion, the recovery DNA fragments and vectors developed by Li and the like are not suitable for engineering seed breeding technology, and the development of recovery DNA fragments and vectors with higher genetic stability, more convenient construction and transformation and convenient mutant genotype detection is urgently needed.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide an expression cassette containing a promoter of a long male wild rice (Oryzalongistamina) OlCYP704B2 gene and capable of restoring male fertility of a rice OsCYP704B2 mutant, a vector, a detection method and application thereof.

The rice genome is analyzed and screened to obtain the promoter from the OlCYP704B2 gene of the Oryza longistaminata (Oryza longistaminata) which can drive the specific expression of endogenous or exogenous genes in the rice anther, and the sequence of the promoter is shown as SEQ ID NO. 1.

First, the present invention provides a promoter of the OlCYP704B2 gene of oryza longistaminata, which has any one of the following nucleotide sequences:

(1) a nucleotide sequence shown as SEQ ID NO. 1;

(2) the nucleotide sequence shown as SEQ ID NO.1 is obtained by replacing, inserting or deleting one or more bases of the nucleotide sequence and has the function of driving the expression of the gene plant anther;

(3) a nucleotide sequence which can be hybridized with the nucleotide sequence shown as SEQ ID NO.1 under strict conditions.

In the present invention, the stringent conditions are those for nucleotide hybridization known in the art, such as: hybridization was carried out at 60 ℃ for 12 to 16 hours in a hybridization solution containing 400mM NaCl, 40mM PIPES (pH6.4) and l mM EDTA, followed by washing with a washing solution containing 0.1% SDS and 0.1 XSSC at 65 ℃ for 15 to 60 minutes.

On the basis, the invention provides the application of the OlCYP704B2 gene promoter of the oryza longistaminata in driving the expression of endogenous or exogenous genes in plant anthers.

The invention also provides application of the OlCYP704B2 gene promoter of the oryza longistaminata in preparing transgenic plants.

In the present invention, the plant is a monocotyledonous or dicotyledonous plant, including but not limited to rice, corn, sorghum, barley, oat, wheat, chestnut, sugarcane, soybean, cotton, tobacco, alfalfa, sunflower, and the like.

On the basis, the invention provides an expression cassette containing the OlCYP704B2 gene promoter of the oryza longistaminata.

The OlCYP704B2 gene promoter of the oryza longistaminata provided by the invention can drive any endogenous or exogenous gene to express in anthers, so that the expression cassette can comprise any endogenous or exogenous gene besides the OlCYP704B2 gene promoter of the oryza longistaminata, such as: genes encoding fluorescent proteins such as GUS and GFP, plant fertility control genes, and the like are all within the scope of the present invention as long as the expression cassette using the OlCYP704B2 gene promoter of oryza longistaminata as a gene expression element is used.

The OsCYP704B2 gene is an important regulation gene of rice male fertility, and the OlCYP704B2 gene promoter of the oryza longistaminata can drive the OsCYP704B2 gene to express efficiently in anthers, so that the OsCYP704B2 gene can be used for restoring the male fertility of rice.

As an embodiment of the invention, the expression cassette further comprises a rice OsCYP704B2 gene or a rice OsCYP704B2 gene mutant, and the CDS sequence of the rice OsCYP704B2 gene is shown in SEQ ID NO. 2.

The rice OsCYP704B2 gene mutant may comprise a non-synonymous mutation causing an alteration of the coding amino acid sequence or a synonymous mutation not causing an alteration of the coding amino acid sequence; however, neither the non-synonymous mutation nor the synonymous mutation should cause the OsCYP704B2 gene to be non-expressed.

In order to facilitate the identification of the rice OsCYP704B2 gene mutant and the rice genome in-situ OsCYP704B2 gene, a HaeIII enzyme cutting site is introduced into the rice OsCYP704B2 gene and/or eliminated under the condition of not changing the coding amino acid sequence.

In the invention, the mutant of the rice OsCYP704B2 gene has a nucleotide sequence with 660 th A mutation to C of a nucleotide sequence shown in SEQ ID NO.2, and/or a nucleotide sequence with 1032 th G mutation to A of the nucleotide sequence shown in SEQ ID NO. 2.

In order to enable the modified expression cassette to have a better expression effect so as to better realize fertility restoration of rice, the expression cassette comprises the anther promoter and the rice OsCYP704B2 gene mutant, and is also added with the 3' -UTR of the rice OsCYP704B2 gene.

Preferably, the added 3' -UTR is a 274bp fragment downstream of the stop codon of the OsCYP704B2 gene of the rice variety 93-11.

To facilitate excision, ligation, and fragment replacement of this expression cassette in a variety of plant expression vectors, several sites in the sequence of the expression cassette are optimized, including: the 348 th G of the rice OsCYP704B2 gene is mutated into C, 525 th A is mutated into G, 717 th G is mutated into C, 888C is mutated into G, 1206G is mutated into C, 1335C is mutated into G, 1422G is mutated into C, 1428G is mutated into C, and the first base of the initiation codon of the OsCYP704B2 gene is used as the 1 st base of each site.

The sequence of the expression cassette obtained by optimizing and modifying the whole sequence is shown as SEQ ID NO. 3. The nucleotide sequence shown as SEQ ID NO.3 comprises a promoter of the OlCYP704B2 gene of the oryza longistaminata shown as SEQ ID NO.1, the OsCYP704B2 gene shown as SEQ ID NO.2 is mutated into C from 660A, A from 1032G, C from 348G, G from 525A, G from 717G, G from 888C, G from 1206G, G from 1335C, C from 1422G, C from 1428G into C sequence, and the male fertility restoration mutant of the OsCYP704B2 gene obtained by optimizing and modifying the sequence and the 3' UTR of 274bp downstream of the OsCYP704B2 gene.

The expression cassette shown in SEQ ID NO.3 can be expressed efficiently in anthers to restore rice fertility.

Furthermore, in order to facilitate the construction and transformation of the vector of the expression cassette, the sequence shown as SEQ ID NO.3 is modified by any one or more of the following:

(1) adding a multiple cloning site at the 5' end of the sequence shown as SEQ ID NO. 3;

(2) adding a restriction enzyme cutting site before a 5' -UTR and an initiation codon of a sequence shown as SEQ ID NO. 3;

(3) adding a restriction enzyme cutting site between a stop codon of a sequence shown as SEQ ID NO.3 and a 3' UTR;

(4) adding a multiple cloning site at the 3' end of the sequence shown in SEQ ID NO. 3.

As a preferred embodiment of the invention, the nucleotide sequence of the expression cassette is shown as SEQ ID NO.4, and the expression cassette is obtained by transforming the nucleotide sequence shown as SEQ ID NO.3 into the nucleotide sequences in the above (1) to (4).

The invention also provides a biological material containing the OlCYP704B2 gene promoter or the expression cassette of the oryza longistaminata, wherein the biological material comprises a vector and a host cell.

The vector includes but is not limited to cloning vector, expression vector, non-replicative vector and other vectors, including but not limited to pCAMBIA1300 vector, pCAMBIA1301 vector, pCAMBIA3300 vector, pCAMBIA3301 vector and the like.

Preferably, the host cell comprises a microbial cell, a plant cell.

The microbial cells include, but are not limited to, Escherichia coli, Agrobacterium, and the like.

Such plant cells include, but are not limited to, plant anther cells and the like.

The OlCYP704B2 gene promoter of the oryza longistaminata provided by the invention can drive the expression of genes in anthers, so that the invention further provides application of the promoter or an expression cassette containing the promoter or the biological material in driving the expression of the genes in plant anthers or preparing transgenic plants.

The expression cassette containing the long-male wild rice OlCYP704B2 gene promoter and the OsCYP704B2 gene mutant provided by the invention can effectively restore the male fertility of male sterile rice, so that the invention further provides application of the promoter or the expression cassette containing the promoter or the biological material in plant fertility restoration, preparation of invisible male sterile plants, plant genetic breeding or plant germplasm improvement.

Preferably, the plant fertility restoration is male fertility restoration of male sterility caused by loss of function of ospcyp 704B 2.

The invisible male nuclear sterile plant includes but is not limited to a male sterility maintainer line, a male sterile line and the like.

When the expression cassette or the biological material containing the OlCYP704B2 gene promoter and the OsCYP704B2 gene mutant of the oryza longistaminata is used for preparing transgenic plants or the prepared transgenic plants are further cross-bred, an efficient detection method is needed for tracking and identifying. When the sequence of the OsCYP704B2 gene mutant is optimized, specific enzyme cutting sites are introduced and eliminated for subsequent identification.

Therefore, the invention also provides a method for detecting rice carrying an expression cassette containing the OlCYP704B2 gene promoter and the OsCYP704B2 gene mutant of the oryza longistaminata, which specifically comprises the steps of taking the genome of the rice to be detected as a template, carrying out PCR amplification by adopting the following primer combination (1) or (2), carrying out enzyme digestion on an amplification product by adopting HaeIII, and judging whether the rice to be detected carries the expression cassette containing the OlCYP704B2 gene promoter and the OsCYP704B2 gene mutant of the oryza longistaminata according to the fragment type of the enzyme digestion product:

(1) forward primer F1: AGGTCGGGTTTGGGGTT, respectively;

reverse primer R1: GATGTTGGCAGCGTCGAA, respectively;

(2) forward primer F2: AGCTTCGGGGACGACAAGA, respectively;

reverse primer R2: GCGCCGGAGCTTGTC are provided.

When the forward primer F1 and the reverse primer R1 are used for detection, if the enzyme digestion product contains a66 bp fragment, the rice to be detected carries the expression cassette;

when the forward primer F2 and the reverse primer R2 are used for detection, if the enzyme digestion product contains a 138bp fragment, the rice to be detected carries the expression cassette.

The detection method can also be used for efficiently identifying the transgenic events of OsCYP704B2 wild type, OsCYP704B2 mutant and OsCYP704B2 fertility restoration;

therefore, the invention also provides a method for identifying the OsCYP704B2 wild type, the OsCYP704B2 mutant and the OsCYP704B2 fertility restoring transgenic event, specifically, taking the genome of rice to be detected as a template, adopting the following primer combination (1) or (2) to perform PCR amplification, adopting HaeIII to enzyme-cut an amplification product, and judging the rice OsCYP704B2 genotype according to the band type of the enzyme-cut product:

(1) forward primer F1: AGGTCGGGTTTGGGGTT, respectively;

reverse primer R1: GATGTTGGCAGCGTCGAA, respectively;

(2) forward primer F2: AGCTTCGGGGACGACAAGA, respectively;

reverse primer R2: GCGCCGGAGCTTGTC, respectively;

the OsCYP704B2 fertility restorer transgenic event is the transformation of the expression cassette or a vector or a host cell containing the expression cassette.

Preferably, the OsCYP704B2 mutant is cyp704B2-2 and OsCYP704B2-3 mutants, which are disclosed in Chinese patents CN104894144A and CN105002191A, respectively.

As an embodiment of the invention, when the OsCYP704B2 mutant is cyp704B2-2, the forward primer F2 and the reverse primer R2 are adopted for amplification, if the enzyme digestion product contains a 115bp fragment, the wild-type genotype of the OsCYP704B2 in situ of the rice genome is contained, if the enzyme digestion product contains a 113bp fragment, the wild-type genotype of the OsCYP704B2 in situ of the rice genome is contained, if the enzyme digestion product contains a cyp704B2-2 mutant genotype, and if the enzyme digestion product contains a 138bp fragment, the fertility restoring transgenic event genotype of the OsCYP704B2 is contained.

As another embodiment of the invention, when the OsCYP704B2 mutant is cyp704B2-3, the forward primer F1 and the reverse primer R1 are adopted for amplification, if the enzyme digestion product contains 86bp fragments, the wild-type genotype of the OsCYP704B2 in situ of the rice genome is contained, if the enzyme digestion product contains 84bp fragments, the wild-type genotype is cyp704B2-3 mutant genotype is contained, and if the enzyme digestion product contains 66bp fragments, the fertility restoring transgenic event genotype of the OsCYP704B2 is contained.

The invention has the beneficial effects that:

(1) the OlCYP704B2 gene promoter of the oryza longistaminata provided by the invention can drive endogenous or exogenous genes to be efficiently expressed in plant anthers, and has good application potential in driving the genes to be efficiently expressed in the anthers and preparing transgenic plants.

(2) The expression cassette containing the rice OsCYP704B2 gene optimized by the sequence and the upstream and downstream expression regulatory sequences can realize high-level expression of the OsCYP704B2 gene in anthers under the drive of the long-male wild rice OlCYP704B2 gene promoter under the condition of not changing the amino acid sequence coded by the OsCYP704B2 gene, efficiently and completely recover the rice male sterile phenotype caused by the loss of the OsCYP704B2 function, and can be used for recovering the rice male fertility or preparing a rice male nuclear sterile maintainer line and a rice male sterile line in practice.

(3) Compared with the prior art, the OsCYP704B2 gene fertility restoration expression cassette provided by the invention has the advantages that the fragment length is obviously reduced, the fertility restoration effect is ensured, and meanwhile, the genetic stability, the construction of a vector and the genetic transformation efficiency are improved.

(4) According to the invention, enzyme cutting sites are introduced or eliminated inside the OsCYP704B2 gene of the expression cassette, so that a rapid and convenient detection method capable of simultaneously identifying the OsCYP704B2 mutant, the wild type and the fertility restoration expression cassette through PCR amplification and enzyme cutting is provided, and the genotype of the transgenic plant of the OsCYP704B2 fertility restoration expression cassette or the filial generation thereof can be efficiently identified by using molecular markers.

(5) The OsCYP704B2 fertility restoration expression cassette provided by the invention is added with common enzyme cutting sites at the 5 ' end of the promoter, between the promoter and the coding region, between the coding region and the 3 ' -UTR region and at the 3 ' end of the 3 ' -UTR, so that the promoter, the gene or the 3 ' -UTR segment can be conveniently replaced for the expression cassette according to the requirement in the subsequent application.

Drawings

FIG. 1 is a schematic structural diagram of the pOlMs26 expression cassette OsMs26m constructed in example 2 of the present invention.

FIG. 2 is the genotype identification electrophoresis of cyp704b2-3 homozygous seeds of middle flower 11 background in example 4 of the present invention, wherein lane 1 is cyp704b2-3 homozygous mutant seeds, lane 2 is middle flower 11 seeds, and lanes 3-14 are seeds obtained by selfing backcross heterozygous individuals.

FIG. 3 is the electrophoretic image of the background genotype and the genotype of the expression cassette of T0 transformed plant in example 6, wherein lane 1 is wild type middle flower 11, lane 2 is the cyp704b2-3 homozygous mutant on the background of transformed recipient middle flower 11, and lanes 3-8 are the transformed plant with mutant background and containing the expression cassette.

FIG. 4 is a drawing showing the anther morphology and pink iodine-potassium iodide staining of the wild type and T0 generation transformed plants in example 7, in which A is the anther of the sterile mutant, B is the anther of the transformation positive strain, and C is I₂-KI-stained mutant anthers D being I₂KI-stained anthers of transformation positive strains.

FIG. 5 is the electrophoresis chart of the wild type OsCYB704B2 gene, cyp704B2-2 mutant and pOlMs26 of example 8. OsMs26m expression cassette identification, wherein lane 1 is cyp704B2-2 mutant, lane 2 is wild type plant, and lanes 3-10 are transformed plant. Wherein lanes 3-5, 7, 9 are positive transformants of homozygous mutant background;

lanes

6, 8 are false positive plants with homozygous mutant background but no expression cassette.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples, unless otherwise specified, the nucleotide positions are calculated with the first nucleotide of the start codon of the gene being at position +1 and the first nucleotide upstream of the start codon being at position-1.

Example 1 development and functional characterization of the promoter of the OlCYP704B2 Gene from Oryza longistaminata

The upstream sequence of an OlCYP704B2 (namely OlMs26) gene in a genome of oryza longistaminata is analyzed by a promoter element prediction website New PLACE (https:// www.dna.affrc.go.jp/PLACE/. The DNA fragments with different lengths and containing different numbers of POLLEN1LELAT52 elements are respectively intercepted for carrying out promoter activity identification, the result shows that some of the intercepted fragments do not have promoter activity or have lower promoter activity although containing an anther-specific promoter element POLLENT 52 sequence, the fragments are screened layer by layer and are compared in promoter activity, finally, the 1110bp fragment (SEQ ID NO.1) containing 5 POLLENT 52 elements is determined to be used as a promoter sequence, the fragment has excellent function of driving the expression of genes in anthers, and can be used as a promoter for driving the high-efficiency expression of exogenous genes or endogenous genes of rice in anthers, and the promoter is named as pOLMs 26.

Example 2 construction of expression cassette and vector containing promoter of Oryza longata (Oryza longistaminata) OlCYP704B2 gene and OsCYP704B2 gene

According to the coding region and the upstream and downstream sequences of the OsCYP704B2(OsMs26) gene of indica rice variety 93-11 disclosed by the Gramene website, the sequence of an expression cassette OsMs26m containing the OlCYP704B2 gene promoter and the OsCYP704B2 gene of oryza longistaminata is designed, and the sequence is optimized to add or eliminate recognition sites of certain endonucleases, so that subsequent operations of enzyme digestion, transfer and the like of the expression cassette are facilitated, and the method specifically comprises the following steps:

(1) the OlCYP704B2 gene promoter pOlMs26 of the oryza longistaminata obtained in example 1 is linked with the CDS sequence (shown as SEQ ID NO. 2) of the OsCYP704B2 gene of rice to drive the expression of the OsCYP704B2 gene.

(2) On the premise of not changing the coded amino acid sequence, the 660 th site of the CDS sequence of the OsCYP704B2 is mutated from A to C so as to introduce a HaeIII enzyme cutting site; the 1032 th G is mutated into A, and a HaeIII site and an MspI site are eliminated, so that the molecular marker identification after the expression cassette is transformed into rice is facilitated.

(3) The 3 'UTR sequence 274bp downstream of the OsCYP704B2 gene was added to the 3' end of the above-obtained sequence.

(4) And (3) carrying out expression optimization of the whole sequence on the sequence obtained in the step (3) under the premise of not changing the coded amino acid sequence, and obtaining the following modified combination by screening: g348 → C348; a525 → G525; g717 → C717; c888 → G888; g1206 → C1206; c1335 → G1335; g1422 → C1422; g1428 → C1428, the resulting sequence is shown in SEQ ID NO. 3.

(5) Adding a multiple cloning site with the total length of 58bp to the 5 ' end of the sequence obtained in the step (4), wherein the 5 ' end is a HindIII enzyme cutting site, and the 3 ' end is a SacI enzyme cutting site.

(6) Modifying the base A at position-8 of the sequence obtained in the above (5) to GT to form a KpnI cleavage site with the upstream and downstream bases.

(7) A BamHI cleavage site sequence was inserted between the stop codon of the OsCYP704B2 gene and the 3' UTR of the sequence obtained in (6) above.

(8) A93 bp multiple cloning site was added to the 3 ' end of the sequence obtained in (7), the 5 ' end was XbaI cleavage site, and the 3 ' end was EcoRI cleavage site.

After the transformation of the (1) to (8), a pOlMs26 is obtained, wherein an OsMs26m expression cassette (the base-transformed OsCYP704B2 gene CDS and the 3' UTR are called OsMs26m for short) has a sequence shown in SEQ ID NO.4, and a structural schematic diagram of the expression cassette is shown in FIG. 1. This sequence was synthesized by Nanjing Kinshire and cloned into the pUC57 vector. The synthesized pOlMs26:: OsMs26m expression cassette sequence is connected to pCAMBIA1300 by double digestion and connection of HindIII and XbaI to obtain vector pCAMBIA-pOlMs26:: OsMs26 m.

Example 3 backcrossing of cyp704b2-3 to mid-flower 11 Rice plants

In previous studies, the inventors obtained two indica rice 93-11 background OsCYP704B2 mutants, cyp704B2-2 and cyp704B2-3, respectively (see patents CN 104894144B and CN105002191B, respectively), and both mutants exhibited pollen-free male sterility. In this example, the method for breeding by backcross of mutant genes described in example 9 of patent CN105002191B was adopted, and 1 of individuals BC2 was selected 1 of individuals with middle flower 11 background over 90% of cyp704b 2-3-wild type hybrid strains by backcrossing two generations with the cyp704b2-3 mutant as the mutant gene donor and the middle flower 11 as the backcross recipient parent, and the selfed seeds were used for identification of homozygous mutant and mutant-wild type hybrid.

Example 4 obtaining of seed homozygous for cyp704b2-3 on flower 11 background

Taking the heterozygous selfed seed harvested in the example 3 as a sample to be detected, cutting the selfed seed into two halves, wherein the half of the seed containing the embryo is reserved as a transformation receptor, and the other half of the seed without the embryo is used for rapidly extracting DNA by adopting the following method:

(1) taking half seeds without embryos and placing the half seeds into a 96-hole PCR plate hole of 200 mu L;

(2) buffer a was prepared before use: 800 μ L of 20% Tween20, 160 μ L of 5mol/L NaOH, 7.04mL ddH₂O (in one piece 96 well PCR plate); and (3) buffer solution B: 1mL of 1mol/L Tris-HCl (pH8.0), 40. mu.L of 0.5mol/L EDTA (pH8.0), 8.96mL of ddH₂O。

(3) Adding 50 μ L buffer solution A into each well, sealing with a rubber cover, heating to 95 deg.C in a PCR instrument, and maintaining for 10 min;

(4) taking out the plate, quickly adding buffer solution B with the same volume, centrifuging at 1000 Xg, and mixing uniformly for 1min to obtain an extracting solution which can be used as template DNA for PCR detection;

taking 1 μ L of the above extractive solution as template, and identifying the seed carrying homozygous recessive male sterile gene by using molecular marker amplification primer and identification method used in example 9 of patent CN105002191B, the result is shown in FIG. 2. Using the half of the seeds with embryos corresponding to the identified seeds carrying the homozygous recessive male sterile cyp704b2-3 mutant gene for the subsequent agrobacterium transformation to construct transgenic plants; the sterile mutant-half of the seeds with embryos corresponding to the wild type heterozygous seeds were continuously planted for selfing and expanding the mutant and the heterozygous seeds, and the homozygous mutant was continuously identified and screened for genetic transformation according to the method of this example, or the heterozygous seeds were used for expanding propagation.

Example 5 transformation of pOlMs26 construction of transgenic Rice plants with OsMs26m expression cassettes

The pCAMBIA-pOlMs26 constructed in example 2 is that OsMs26m recombinant plasmid is introduced into agrobacterium EHA105 strain, transformed into the callus of the seeds which have the background of japonica rice middle flower 11 and carry the homozygous recessive male sterile gene cyp704b2-3 and obtained in example 4, and then the callus is screened by hygromycin resistance, differentiated and rooted. Finally, the regenerated pCAMBIA-pOlMs26 is obtained, wherein OsMs26m transform strains 72, hygromycin resistance genes transferred into the transgenic plants are identified through PCR to obtain 69 positive plants identified through PCR, and the positive plants are transplanted into soil to survive 64 plants.

Example 6 genetic background of Positive plants and pOlMs26 identification of the OsMs26m expression cassette

The leaves of the pCAMBIA-pOlMs26 positive OsMs26m transgenic line were cut, and the DNA of the leaves was extracted by the CTAB method.

Specific primers are adopted: forward primer F1: AGGTCGGGTTTGGGGTT; reverse primer R1: GATGTTGGCAGCGTCGAA; PCR amplification and HaeIII enzyme digestion identification of PCR products were performed on the transgenic positive individuals obtained in example 5. The primer amplification product contains a cyp704b2-3 mutant mutation site, and 2bp deletion mutation existing in a background plant of the cyp704b2-3 mutant can be observed in polyacrylamide gel electrophoresis; the primer amplification product also comprises pOpMs26:: OsCYP704B2 gene segment carrying A660C mutation in OsMs26m expression cassette (the mutation introduces HaeIII restriction site), therefore, transgenic plants successfully integrating pOlMs26:: OsMs26m expression cassette into rice genome can be restricted by HaeIII, while original OsCYP704B2 gene segment of rice genome can not be restricted by HaeIII. Using the above-described test method, a homozygous mutant with the genetic background of cyp704b2-3, which contained pOlMs26:: 50 transgenic plants of the OsMs26m expression cassette, was identified from 64 transformed plants obtained in example 5 that were transplanted to survive. The enzyme digestion identification result of the PCR product of the partial plant is shown in figure 3, wherein an enzyme digestion strip of 86bp is the original wild type OsCYP704B2 genotype of the rice genome, an enzyme digestion strip of 84bp is the cyp704B2-3 mutant genotype, and an enzyme digestion strip of 66bp is pOlMs26, wherein the OsMs26m expression cassette transforms the OsMs26m genotype of the segment; the wild type plant only generates 86bp enzyme cutting bands, the transgenic plant with the wild type background generates 86bp and 66bp enzyme cutting bands, and the transgenic plant with the cyp704b2-3 homozygous mutant background generates 84bp and 66bp enzyme cutting bands.

Example 7 Male fertility identification of transformation Positive plants

After the T0 generation plants of the transformed positive plants obtained in example 6 are spilt, before flowering, the anthers are tabletted, the pollen viability of the transgenic plants is identified by using a potassium iodide staining microscope, and the pollen morphology of the transgenic plants is observed by a stereomicroscope. As shown in FIG. 4, the cyp704b2-3 mutant plants showed no visible pollen, while the transformed positive plants had a large amount of fertile pollen, indicating restoration of fertility. In 50 cyp704b2-3 homozygous mutant backgrounds and carrying T0 generation plants containing pOlMs26: OsMs26m expression cassette transformation gene, 33 of the plants were fertile by microscopic examination; in 33 plants which can be bred by microscopic pollen, 26 plants can be bagged for selfing to fruit, the fruit setting rate of 16 plants is more than 50 percent, 11 plants are more than 80 percent, and the fruit setting rate of wild type is achieved.

Selecting a transgenic fertile T0 single plant, harvesting seeds, taking 30 grains for planting, emerging seedlings and surviving 22 plants, performing microscopic examination on the activity of the pollen according to the method of the embodiment, and counting the self-bred fructification condition of the T1 generation. The results show that all 22 original genetic backgrounds are cyp704b2-3 mutants, wherein 15 of 18 individuals containing the transgenic expression cassette can normally selfe and fruit, and 4 individuals without the transgenic expression cassette can not selfe and fruit. The expression cassette is proved to be capable of stably inheriting the fertility restoring capability.

As can be seen from the phenotypes of the plants of the above T0 and T1 generations, the anther-specific promoter of the oryza longistaminata pOlMs26 with the length of 1110bp, which is developed in example 1, can drive the subsequent genes to be efficiently expressed in the anther of the oryza sativa, and the strength of the promoter is enough to meet the requirement of the wild-type OsCYP704B2 gene for restoring the male sterile phenotype of the corresponding mutant; transgenic rice transformed with the OsMs26m expression cassette can restore the male sterile phenotype caused by the OsCYB704B2 gene mutation of rice and can be stably inherited.

Example 8 application of molecular marker detection method in identifying wild type OsCYB704B2 gene, cyp704B2-2 mutant and pOlMs26:: OsMs26m expression cassette genotype

The purpose of this example is to verify the application of the molecular marker detection method provided by the present invention in identifying wild type, cyp704b2-2 mutant and pOlMs26:: OsMs26m expression cassette genotype.

Shearing leaves of wild rice 93-11 and cyp704b2-2 mutant, extracting DNA of the leaves by using a CTAB method, and diluting to 100 ng/. mu.L; pCAMBIA-pOlMs26:: OsMs26m recombinant plasmid was diluted to 10 ng/. mu.L. The genomic DNA of the leaves of the wild rice 93-11 and the cyp704b2-2 mutant and pCAMBIA-pOlMs26: OsMs26m recombinant plasmids are respectively used as templates, and a forward primer F2: AGCTTCGGGGACGACAAGA and reverse primer R2: GCGCCGGAGCTTGTC PCR amplification and HaeIII enzyme digestion verification: the primer amplification region comprises pOlMs26, the G1032 → A1032 base modification carried by the OsMs26m gene of the OsMs26m expression box eliminates a HaeIII, therefore, the pOlMs26 shows that the vector of the OsMs26m expression box still keeps 138bp after the amplification enzyme digestion; the OsCYB704B2 gene amplified fragments of the wild 93-11 and cyp704B2-2 mutants still have the enzyme cutting site, the fragments after enzyme cutting are respectively 115bp and 113bp, and the detection result is shown in FIG. 5.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Hainan Borax Rice Gene science and technology Co., Ltd

<120> vector containing oryza longistaminata wild rice promoter and capable of restoring male fertility of rice OsCYP704B2 mutant and application

<130>KHP181118259.6

<160>8

<170>SIPOSequenceListing 1.0

<210>1

<211>1110

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

attttgatac tcttgattga ttgtttattt agttactcgg tatctcagta acagatgaga 60

gatttattca gcaaatgctc cggtttgctc gaagattgta ataagtgtgg gcaagaatca 120

ggatcaatcc ataagagcag tattttcatg ctcttttgat cttggtttca gacttatttc 180

agtgttgaca ttggttattt ctcaattcat tcgagtattt gttgttacat cacaaaggat 240

aagttctata gaaaaaatct tccttttcaa gtgatgttct ttaaatttct gtagaattgt 300

gccctgcaat ttctcaaatc tttgatagcc ggcttatttg tattgactgg aaaataaatt 360

agttgtcaat aactagaaac attagagatg caaagtattg aatatatctt ggcaattgta 420

tttcatattg tctgtttctg tgagaatgtt ttaactagat ggcaactgat ttttgggaca 480

aaatcgcttc tacaatggct tatgtaactc gtactcgtcg atggcatttc tcatatttgg 540

atgtttgtca atggtatttc ttggattttc tcttcattga aatagcctat tcagatgaaa 600

tagaaaccaa ctactttggg ttcacaattt atatttcttt cgaggatgcc ccatttcatt 660

ttagttgtca tcaaagactc tgtctcttta cattaataat acaatatcga cagaaaatcg 720

taaccctggt ttcagttggt gacaatttaa cagaattcag atggatatgg ctctgatatt 780

agaaggtggc atacctttag tcgctgcaaa tgcttcagtt atctgaacaa aacaacgaac 840

ttctggctga gcaggggaaa aaaaatactg tggcattcat tttgtgttta cgtaacgatt 900

cttttctagg tggacagatc acaaaaagaa aactaaagct aagatccaac tcctaagggt 960

gttaggttag ggacaccata tgaatgagac aatcttaatt cttggtcaca caaagattgt 1020

ctcaaggttg gtagcatcag tgcccaatat atcacctaac tatgccatcc aaaatgctac 1080

atagcatctc ttgtagacac tgaacccttc 1110

<210>2

<211>1635

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

atgaagagcc ccatggagga agctcatgca atgccagtga catcattctt cccagtagca 60

ggaatccaca agctcatagc tatcttcctt gttgtcctct catggatctt ggtccacaag 120

tggagcctga ggaaccagaa agggccaaga tcatggccaa tcatcggcgc gacagtggag 180

caactgaaga actaccacag gatgcatgac tggcttgtcg agtacttgtc gaaggacagg 240

acggtgaccg tcgacatgcc tttcacctcc tacacctaca ttgccgaccc ggtgaacgtc 300

gagcatgtcc tgaagaccaa cttcaccaat taccccaagg gtgaagtgta caggtcttac 360

atggatgtgc tgctcggtga tggcatattc aatgccgacg gcgagatgtg gaggaagcaa 420

aggaagacgg cgagcttcga gtttgcctcc aagaacttga gagacttcag cactgtggtg 480

ttcagggagt actccctgaa gctatcaagc attctgagcc aagcatgcaa ggccggcaga 540

gttgtagaca tgcaggaatt gttcatgagg atgacactgg actcgatctg caaggtcggg 600

tttggggttg agatcgggac gctgtcacct gatctcccgg agaacagctt tgcccaggca 660

ttcgacgctg ccaacatcat cgtcacgctg cggttcatcg atcctctgtg gcgtctgaag 720

aagttcttgc acgtcggatc agaggctctc ctcgagcaga gcatgaagct ggttgatgac 780

ttcacctaca gcgtgatccg ccgccgcaag gctgagatct tgcaggctcg agccagcggc 840

aagcaagaga agatcaagca cgacatactg tcgcggttca tcgagctcgg ggaggccggc 900

ggcgacgagg ggggcggcag cttcggggac gacaagagcc tccgcgacgt ggtgctcaac 960

ttcgtgatcg ccgggcgtga cacgacggcg acgacgctgt cgtggttcac gtacatggcg 1020

atgacgcacc cggccgtcgc cgacaagctc cggcgcgagc tggccgcgtt cgaggctgag 1080

cgcgcgcgcg aggagggcgt cgcgctcgcc gacgccgccg gcgaggcgtc attcgcggcg 1140

cgcgtggcgc agttcgcgtc gctgctgagc tacgacgcgg tggggaagct ggtgtacctg 1200

cacgcgtgcg tgacggagac gctccgcctc tacccggcgg tgccgcagga ccccaagggg 1260

atcgtggagg acgacgtgct ccccgacggc accaaggtgc gcgccggcgg gatggtgacg 1320

tacgtgccct actccatggg gaggatggag tacaactggg gccccgacgc ggcgagcttc 1380

cggccggagc ggtggctcag cggcgacggc ggcgcgttcc ggaacgcgtc gccgttcaag 1440

ttcaccgcgt tccaggccgg gccgcggatc tgcctcggca aggactccgc ctacctccag 1500

atgaagatgg cgctcgccat cctcttccgc ttctacacct tcgacctcgt cgaggaccac 1560

cccgtcaagt accggatgat gaccatcctc tccatggctc acggcctcaa ggtccgcgtc 1620

tccacctccg tctga 1635

<210>3

<211>3019

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

attttgatac tcttgattga ttgtttattt agttactcgg tatctcagta acagatgaga 60

gatttattca gcaaatgctc cggtttgctc gaagattgta ataagtgtgg gcaagaatca 120

ggatcaatcc ataagagcag tattttcatg ctcttttgat cttggtttca gacttatttc 180

agtgttgaca ttggttattt ctcaattcat tcgagtattt gttgttacat cacaaaggat 240

aagttctata gaaaaaatct tccttttcaa gtgatgttct ttaaatttct gtagaattgt 300

gccctgcaat ttctcaaatc tttgatagcc ggcttatttg tattgactgg aaaataaatt 360

agttgtcaat aactagaaac attagagatg caaagtattg aatatatctt ggcaattgta 420

tttcatattg tctgtttctg tgagaatgtt ttaactagat ggcaactgat ttttgggaca 480

aaatcgcttc tacaatggct tatgtaactc gtactcgtcg atggcatttc tcatatttgg 540

atgtttgtca atggtatttc ttggattttc tcttcattga aatagcctat tcagatgaaa 600

tagaaaccaa ctactttggg ttcacaattt atatttcttt cgaggatgcc ccatttcatt 660

ttagttgtca tcaaagactc tgtctcttta cattaataat acaatatcga cagaaaatcg 720

taaccctggt ttcagttggt gacaatttaa cagaattcag atggatatgg ctctgatatt 780

agaaggtggc atacctttag tcgctgcaaa tgcttcagtt atctgaacaa aacaacgaac 840

ttctggctga gcaggggaaa aaaaatactg tggcattcat tttgtgttta cgtaacgatt 900

cttttctagg tggacagatc acaaaaagaa aactaaagct aagatccaac tcctaagggt 960

gttaggttag ggacaccata tgaatgagac aatcttaatt cttggtcaca caaagattgt 1020

ctcaaggttg gtagcatcag tgcccaatat atcacctaac tatgccatcc aaaatgctac 1080

atagcatctc ttgtagacac tgaacccttc atgaagagcc ccatggagga agctcatgca 1140

atgccagtga catcattctt cccagtagca ggaatccaca agctcatagc tatcttcctt 1200

gttgtcctct catggatctt ggtccacaag tggagcctga ggaaccagaa agggccaaga 1260

tcatggccaa tcatcggcgc gacagtggag caactgaaga actaccacag gatgcatgac 1320

tggcttgtcg agtacttgtc gaaggacagg acggtgaccg tcgacatgcc tttcacctcc 1380

tacacctaca ttgccgaccc ggtgaacgtc gagcatgtcc tgaagaccaa cttcaccaat 1440

taccccaagg gtgaagtcta caggtcttac atggatgtgc tgctcggtga tggcatattc 1500

aatgccgacg gcgagatgtg gaggaagcaa aggaagacgg cgagcttcga gtttgcctcc 1560

aagaacttga gagacttcag cactgtggtg ttcagggagt actccctgaa gctatcaagc 1620

attctgagcc aagcgtgcaa ggccggcaga gttgtagaca tgcaggaatt gttcatgagg 1680

atgacactgg actcgatctg caaggtcggg tttggggttg agatcgggac gctgtcacct 1740

gatctcccgg agaacagctt tgcccaggcc ttcgacgctg ccaacatcat cgtcacgctg 1800

cggttcatcg atcctctgtg gcgtctcaag aagttcttgc acgtcggatc agaggctctc 1860

ctcgagcaga gcatgaagct ggttgatgac ttcacctaca gcgtgatccg ccgccgcaag 1920

gctgagatct tgcaggctcg agccagcggc aagcaagaga agatcaagca cgacatactg 1980

tcgcggttca tcgagctggg ggaggccggc ggcgacgagg ggggcggcag cttcggggac 2040

gacaagagcc tccgcgacgt ggtgctcaac ttcgtgatcg ccgggcgtga cacgacggcg 2100

acgacgctgt cgtggttcac gtacatggcg atgacgcacc cagccgtcgc cgacaagctc 2160

cggcgcgagc tggccgcgtt cgaggctgag cgcgcgcgcg aggagggcgt cgcgctcgcc 2220

gacgccgccg gcgaggcgtc attcgcggcg cgcgtggcgc agttcgcgtc gctgctgagc 2280

tacgacgcgg tggggaagct ggtgtacctg cacgcctgcg tgacggagac gctccgcctc 2340

tacccggcgg tgccgcagga ccccaagggg atcgtggagg acgacgtgct ccccgacggc 2400

accaaggtgc gcgccggcgg gatggtgacg tacgtgccct actcgatggg gaggatggag 2460

tacaactggg gccccgacgc ggcgagcttc cggccggagc ggtggctcag cggcgacggc 2520

ggcgcgttcc gcaacgcctc gccgttcaag ttcaccgcgt tccaggccgg gccgcggatc 2580

tgcctcggca aggactccgc ctacctccag atgaagatgg cgctcgccat cctcttccgc 2640

ttctacacct tcgacctcgt cgaggaccac cccgtcaagt accggatgat gaccatcctc 2700

tccatggctc acggcctcaa ggtccgcgtc tccacctccg tctgaccccc gccgccgctc 2760

gccggcagcc gcgccgccgc cgcccgtatc gcttaccgga gtagtaaata agcctatgta 2820

atctggtttg aatttgaaat ttgaatgtac catgtttgat tctaggattt gttggtccta 2880

gaccctgctt gaaacggtgc gaatttcatc taaatggttg agaaatttta tcgaaagctg 2940

ttccattcta cgctacaaat ggtgggactg gatttaaaca ttggcgacgt ggacaaggct 3000

agtggactga gactctgag 3019

<210>4

<211>3177

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

aagctttccc tggcaacagg ctgcacggtc cgacgcgttt cgaaatttaa atgagctcat 60

tttgatactc ttgattgatt gtttatttag ttactcggta tctcagtaac agatgagaga 120

tttattcagc aaatgctccg gtttgctcga agattgtaat aagtgtgggc aagaatcagg 180

atcaatccat aagagcagta ttttcatgct cttttgatct tggtttcaga cttatttcag 240

tgttgacatt ggttatttct caattcattc gagtatttgt tgttacatca caaaggataa 300

gttctataga aaaaatcttc cttttcaagt gatgttcttt aaatttctgt agaattgtgc 360

cctgcaattt ctcaaatctt tgatagccgg cttatttgta ttgactggaa aataaattag 420

ttgtcaataa ctagaaacat tagagatgca aagtattgaa tatatcttgg caattgtatt 480

tcatattgtc tgtttctgtg agaatgtttt aactagatgg caactgattt ttgggacaaa 540

atcgcttcta caatggctta tgtaactcgt actcgtcgat ggcatttctc atatttggat 600

gtttgtcaat ggtatttctt ggattttctc ttcattgaaa tagcctattc agatgaaata 660

gaaaccaact actttgggtt cacaatttat atttctttcg aggatgcccc atttcatttt 720

agttgtcatc aaagactctg tctctttaca ttaataatac aatatcgaca gaaaatcgta 780

accctggttt cagttggtga caatttaaca gaattcagat ggatatggct ctgatattag 840

aaggtggcat acctttagtc gctgcaaatg cttcagttat ctgaacaaaa caacgaactt 900

ctggctgagc aggggaaaaa aaatactgtg gcattcattt tgtgtttacg taacgattct 960

tttctaggtg gacagatcac aaaaagaaaa ctaaagctaa gatccaactc ctaagggtgt 1020

taggttaggg acaccatatg aatgagacaa tcttaattct tggtcacaca aagattgtct 1080

caaggttggt agcatcagtg cccaatatat cacctaacta tgccatccaa aatgctacat 1140

agcatctctt gtagacactg gtacccttca tgaagagccc catggaggaa gctcatgcaa 1200

tgccagtgac atcattcttc ccagtagcag gaatccacaa gctcatagct atcttccttg 1260

ttgtcctctc atggatcttg gtccacaagt ggagcctgag gaaccagaaa gggccaagat 1320

catggccaat catcggcgcg acagtggagc aactgaagaa ctaccacagg atgcatgact 1380

ggcttgtcga gtacttgtcg aaggacagga cggtgaccgt cgacatgcct ttcacctcct 1440

acacctacat tgccgacccg gtgaacgtcg agcatgtcct gaagaccaac ttcaccaatt 1500

accccaaggg tgaagtctac aggtcttaca tggatgtgct gctcggtgat ggcatattca 1560

atgccgacgg cgagatgtgg aggaagcaaa ggaagacggc gagcttcgag tttgcctcca 1620

agaacttgag agacttcagc actgtggtgt tcagggagta ctccctgaag ctatcaagca 1680

ttctgagcca agcgtgcaag gccggcagag ttgtagacat gcaggaattg ttcatgagga 1740

tgacactgga ctcgatctgc aaggtcgggt ttggggttga gatcgggacg ctgtcacctg 1800

atctcccgga gaacagcttt gcccaggcct tcgacgctgc caacatcatc gtcacgctgc 1860

ggttcatcga tcctctgtgg cgtctcaaga agttcttgca cgtcggatca gaggctctcc 1920

tcgagcagag catgaagctg gttgatgact tcacctacag cgtgatccgc cgccgcaagg 1980

ctgagatctt gcaggctcga gccagcggca agcaagagaa gatcaagcac gacatactgt 2040

cgcggttcat cgagctgggg gaggccggcg gcgacgaggg gggcggcagc ttcggggacg 2100

acaagagcct ccgcgacgtg gtgctcaact tcgtgatcgc cgggcgtgac acgacggcga 2160

cgacgctgtc gtggttcacg tacatggcga tgacgcaccc agccgtcgcc gacaagctcc 2220

ggcgcgagct ggccgcgttc gaggctgagc gcgcgcgcga ggagggcgtc gcgctcgccg 2280

acgccgccgg cgaggcgtca ttcgcggcgc gcgtggcgca gttcgcgtcg ctgctgagct 2340

acgacgcggt ggggaagctg gtgtacctgc acgcctgcgt gacggagacg ctccgcctct 2400

acccggcggt gccgcaggac cccaagggga tcgtggagga cgacgtgctc cccgacggca 2460

ccaaggtgcg cgccggcggg atggtgacgt acgtgcccta ctcgatgggg aggatggagt 2520

acaactgggg ccccgacgcg gcgagcttcc ggccggagcg gtggctcagc ggcgacggcg 2580

gcgcgttccg caacgcctcg ccgttcaagt tcaccgcgtt ccaggccggg ccgcggatct 2640

gcctcggcaa ggactccgcc tacctccaga tgaagatggc gctcgccatc ctcttccgct 2700

tctacacctt cgacctcgtc gaggaccacc ccgtcaagta ccggatgatg accatcctct 2760

ccatggctca cggcctcaag gtccgcgtct ccacctccgt ctgaggatcc cccccgccgc 2820

cgctcgccgg cagccgcgcc gccgccgccc gtatcgctta ccggagtagt aaataagcct 2880

atgtaatctg gtttgaattt gaaatttgaa tgtaccatgt ttgattctag gatttgttgg 2940

tcctagaccc tgcttgaaac ggtgcgaatt tcatctaaat ggttgagaaa ttttatcgaa 3000

agctgttcca ttctacgcta caaatggtgg gactggattt aaacattggc gacgtggaca 3060

aggctagtgg actgagactc tgagtctaga atgcgcatgc gatcgcgact gctgaaccga 3120

tacaaccgtc ctggtgaaag actgtgtaca tcggaagcct tgggactgca ggaattc 3177

<210>5

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

aggtcgggtt tggggtt 17

<210>6

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

gatgttggca gcgtcgaa 18

<210>7

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

agcttcgggg acgacaaga 19

<210>8

<211>15

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gcgccggagc ttgtc 15

Claims

1. A promoter of Oryza longistaminata (Oryza longistaminata) OlCYP704B2 gene, wherein the promoter has any one of the following nucleotide sequences:

(1) a nucleotide sequence shown as SEQ ID NO. 1;

(2) the nucleotide sequence shown as SEQ ID NO.1 is obtained by replacing, inserting or deleting one or more bases of the nucleotide sequence and has the function of driving the gene to express in the plant anther;

2. An expression cassette comprising the promoter of Oryza longata (Oryza longistaminata) OlCYP704B2 gene according to claim 1.

3. The expression cassette of claim 2, further comprising a rice OsCYP704B2 gene or a rice OsCYP704B2 gene mutant, wherein the sequence of the rice OsCYP704B2 gene is shown in SEQ ID No. 2.

4. The expression cassette of claim 3, wherein the rice OsCYP704B2 gene mutant has a nucleotide sequence with the nucleotide sequence shown in SEQ ID NO.2, wherein the 660 th site A is mutated into C, and/or the 1032 th site G is mutated into A.

5. The expression cassette according to claim 3 or 4, wherein the expression cassette comprises a mutant of the rice OsCYP704B2 gene and a 3' -UTR of the rice OsCYP704B2 gene;

the rice OsCYP704B2 gene mutant is a nucleotide sequence which enables the 660 th A of a nucleotide sequence shown as SEQ ID NO.2 to be mutated into C, the 1032 th G to be mutated into A, the 348 th G to be mutated into C, the 525 th A to be mutated into G, the 717 th G to be mutated into C, the 888C to be mutated into G, the 1206G to be mutated into C, the 1335 th C to be mutated into G, the 1422G to be mutated into C and the 1428 th G to be mutated into C;

preferably, the sequence of the expression cassette is shown as SEQ ID NO. 3.

6. The expression cassette according to any one of claims 3 to 5, wherein the expression cassette is obtained by modifying the sequence shown as SEQ ID No.3 by any one or more of the following:

(4) adding a multiple cloning site at the 3' end of the sequence shown as SEQ ID NO. 3;

preferably, the nucleotide sequence of the expression cassette is shown as SEQ ID NO. 4.

7. A biomaterial comprising a promoter according to claim 1 or an expression cassette according to any one of claims 2 to 6, wherein the biomaterial comprises a vector, a host cell or an engineered bacterium.

8. Use of any one of the promoter of claim 1 or the expression cassette of any one of claims 2 to 6 or the biomaterial of claim 7:

(1) the application of the driving gene in the expression of plant anther;

(2) the application in preparing transgenic plants;

(3) the application in plant fertility restoration;

(4) the application in preparing invisible male nuclear sterile plants;

(5) the application in plant genetic breeding or germplasm improvement;

(6) the application in plant hybrid seed production.

9. The method for detecting the rice carrying the expression cassette of any one of claims 4 to 6 is characterized in that a genome of rice to be detected is used as a template, PCR amplification is carried out by adopting the following primer combination (1) or (2), an amplification product is cut by HaeIII enzyme, and whether the rice to be detected carries the expression cassette of any one of claims 4 to 6 is judged according to the fragment type of the amplification product:

(1) forward primer F1: AGGTCGGGTTTGGGGTT, respectively;

reverse primer R1: GATGTTGGCAGCGTCGAA, respectively;

(2) forward primer F2: AGCTTCGGGGACGACAAGA, respectively;

reverse primer R2: GCGCCGGAGCTTGTC are provided.

10. The method for identifying the OsCYP704B2 wild type, the OsCYP704B2 mutant and the OsCYP704B2 fertility restoration transgenic event is characterized in that a genome of rice to be detected is used as a template, a primer combination in the following (1) or (2) is adopted for PCR amplification, a HaeIII enzyme digestion amplification product is adopted, and the rice OsCYP704B2 genotype is judged according to the band type of the enzyme digestion product:

(1) forward primer F1: AGGTCGGGTTTGGGGTT, respectively;

reverse primer R1: GATGTTGGCAGCGTCGAA, respectively;

(2) forward primer F2: AGCTTCGGGGACGACAAGA, respectively;

reverse primer R2: GCGCCGGAGCTTGTC, respectively;

the OsCYP704B2 fertility restorer transgenic event is the transformation of the expression cassette of any one of claims 4 to 6 or the biological material of claim 7.