WO2017128039A1 - 基因组合及其用途 - Google Patents

基因组合及其用途 Download PDF

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WO2017128039A1
WO2017128039A1 PCT/CN2016/072148 CN2016072148W WO2017128039A1 WO 2017128039 A1 WO2017128039 A1 WO 2017128039A1 CN 2016072148 W CN2016072148 W CN 2016072148W WO 2017128039 A1 WO2017128039 A1 WO 2017128039A1
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Prior art keywords
gene
promoter
nucleotide sequence
rice
seq
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PCT/CN2016/072148
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English (en)
French (fr)
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凃巨民
陈浩
罗炬
张翠翠
郑鹏
叶梦楠
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浙江大学
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Priority to PCT/CN2016/072148 priority Critical patent/WO2017128039A1/zh
Priority to US16/073,177 priority patent/US11674146B2/en
Priority to CN202311567386.XA priority patent/CN118028290A/zh
Priority to PCT/CN2016/103934 priority patent/WO2017128791A1/zh
Priority to CN201680080151.XA priority patent/CN109312334B/zh
Publication of WO2017128039A1 publication Critical patent/WO2017128039A1/zh

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    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to the field of transgenic plants.
  • the present invention relates to a combination of genes for controlling expression of a foreign gene of interest in a plant, and a method of cultivating a transgenic plant using the combination of genes.
  • GM crops have been grown in 28 countries around the world, with a production area of 181.5 million hectares in 2014 (James, et al., 2014).
  • GM crops increased crop production value by $133 billion and saved pesticides 500 million kg in the 17 years from 1996 to 2013, only in 2013.
  • GM crops continue to have a significant positive impact on food safety, sustainable development and climate change.
  • Rice is the staple food crop of 50% of the world's population. Although it was announced in 2000 that the transgenic rice line was successfully developed (Tu, et al., 2000), for example, the excellent transgenic restorer line Minghui 63/Bt (Huahui No. 1) and its hybrid rice cultivar Yujing 63/Bt, and In November 2009, after repeated verification by the developer and third parties that there was no biosafety risk, the biosafety certificate issued by the Ministry of Agriculture of China was obtained.
  • these traditional transgenic Bt-resistant rice are endosperm-expressing, that is, the expression product of Bt gene is accumulated in the transgenic rice produced, it is still difficult for the public to recognize its food safety, and its commercial planting is also Therefore, it has not been approved. This situation is not only true in China, but also in other parts of the world and countries such as Europe and Japan. 58% of the people surveyed in Europe in 2006 thought that GM foods should not be encouraged.
  • the technology automatically removes foreign genes from pollen and seeds before and after transgenic flowering, as the computer “unloads” the application software, so that the foreign genes in the transgenic plants are automatically cleared before diffusion and before use (Zhao Degang et al., 2008). ).
  • This technology can be applied directly to asexually propagated crops, but improvements are needed to apply to sexually propagated crops.
  • the main problem is that the exogenous gene of interest in the seeds of sexually propagated crops is no longer retained once removed, thereby rendering the next generation that is propagated through the seed lose the transgenic trait.
  • the inventors have developed a combination of genes capable of shutting down expression of a gene of interest in a particular tissue/organ (eg, endosperm).
  • the technique consists essentially of a locking element that locks the expression of an exogenous gene of interest and a key gene that unlocks the expression of the locked foreign gene of interest.
  • the exogenous gene of interest carried by the former is permanently locked, and the key gene carried by the latter that can open the locked expression of the exogenous gene of interest is expressed only in a specific tissue/organ.
  • the two components are separately transformed and integrated into the parental genome such as rice, and then they can be integrated by hybrid production, so that the expression of the exogenous gene can be opened in the specific tissue/organ.
  • the desired phenotype occurs, and the expression of the exogenous gene of interest is always turned off in a particular tissue/organ (eg, an edible organ, such as a rice endosperm), resulting in a transgenic plant that does not contain any transgenic components in the portion for consumption.
  • tissue/organ eg, an edible organ, such as a rice endosperm
  • the same effect can be obtained by introducing and integrating these two components into the genome of the same recipient plant.
  • the present invention provides a gene combination for controlling expression of a foreign gene of interest in a plant, which consists of a locking element and a key gene, wherein the locking element comprises SEQ ID NO: a nucleotide sequence as shown in 5 or 8, or The nucleotide sequence set forth in SEQ ID NO: 5 or 8 has at least 80% homology and is capable of locking a nucleotide sequence of expression of an exogenous gene of interest operably linked thereto; said key gene comprising SEQ ID NO: a nucleotide sequence represented by 3 or 6, or comprising at least 40% homology to the nucleotide sequence shown in SEQ ID NO: 3 or 6, and capable of releasing the locking element for exogenous purposes
  • the expression of the gene is locked by a nucleotide sequence.
  • the invention provides a locking element for locking expression of a foreign gene of interest linked thereto in a plant, comprising the nucleotide sequence set forth in SEQ ID NO: 5 or 8, or comprising and SEQ ID A nucleotide sequence represented by NO: 5 or 8 has at least 80% homology and is capable of locking a nucleotide sequence of expression of an exogenous gene of interest operably linked thereto.
  • the present invention provides a key gene for releasing expression locking of an exogenous gene of interest in a plant-specific tissue, comprising the nucleoside represented by SEQ ID NO: 3 or 6.
  • the invention provides a method for controlling expression of a foreign gene of interest in a plant, the method comprising introducing a locking element and a key gene into a recipient plant, wherein the locking element comprises SEQ ID NO: a nucleotide sequence represented by 5 or 8, or comprising at least 80% homology to the nucleotide sequence shown in SEQ ID NO: 5 or 8, and capable of locking an exogenous purpose to which it is operably linked a nucleotide sequence for expression of the gene; the key gene comprising the nucleotide sequence set forth in SEQ ID NO: 3 or 6, or comprising at least 40% of the nucleotide sequence set forth in SEQ ID NO: 3 or The homology is and the nucleotide sequence capable of unlocking the expression of the foreign gene of interest by the locking element can be released.
  • the locking element comprises SEQ ID NO: a nucleotide sequence represented by 5 or 8, or comprising at least 80% homology to the nucleotide sequence shown in SEQ ID NO: 5 or 8, and
  • the invention provides a method for controlling expression of a foreign gene of interest in a plant, the method comprising operatively linking the exogenous gene of interest to a locking element, the locking element comprising SEQ ID NO: a nucleotide sequence represented by 5 or 8, or comprising at least 80% homology to the nucleotide sequence set forth in SEQ ID NO: 5 or 8, and capable of locking an exogenous gene of interest operably linked thereto The expressed nucleotide sequence.
  • the present invention provides a method for releasing expression locking of an exogenous gene of interest in a plant, the method comprising introducing a key gene into the plant, the key gene comprising The nucleotide sequence shown in SEQ ID NO: 3 or 6 or comprising at least 40% identical to the nucleotide sequence shown in SEQ ID NO: 3 or A nucleotide sequence that is responsive and capable of releasing the locked expression of the foreign element of interest by the locking element.
  • the present invention provides a method of breeding a transgenic plant, the method comprising: hybridizing a first parent comprising a locking element to a second parent comprising a key gene, thereby obtaining the transgenic plant, wherein
  • the locking element comprises the nucleotide sequence set forth in SEQ ID NO: 5 or 8, or comprises at least 80% homology to the nucleotide sequence set forth in SEQ ID NO: 5 or 8, and is capable of being locked and operable a nucleotide sequence for expression of the ligated exogenous gene of interest;
  • the key gene comprising the nucleotide sequence set forth in SEQ ID NO: 3 or 6, or comprising the nucleotide set forth in SEQ ID NO: 3 or
  • a sequence has at least 40% homology and is capable of releasing a nucleotide sequence that the locking element locks for expression of an exogenous gene of interest.
  • the method further comprises introducing and/or integrating the locking element into the genome of the first parent prior to the hybridizing step, and introducing and/or integrating the key gene into the genome In the genome of the second parent.
  • the invention provides a method of breeding a transgenic plant, the method comprising introducing and/or integrating a locking element and a key gene into the genome of the same plant, thereby obtaining the comprising the locking element And a transgenic plant of a key gene, wherein the locking element comprises the nucleotide sequence set forth in SEQ ID NO: 5 or 8, or comprises at least 80% of the nucleotide sequence set forth in SEQ ID NO: 5 or 8.
  • nucleotide sequence which is homologous and capable of locking expression of an exogenous gene of interest operably linked thereto; the key gene comprising the nucleotide sequence set forth in SEQ ID NO: 3 or 6, or comprising SEQ ID NO
  • the nucleotide sequence shown by 3 or 6 has at least 40% homology and is capable of releasing the nucleotide sequence in which the locking element is locked for expression of the exogenous gene of interest.
  • the invention provides the use of a combination of a locking element and a key gene for regulating expression of a foreign gene of interest in a plant, wherein the locking element is capable of locking expression of an exogenous gene of interest operably linked thereto;
  • the key gene is capable of releasing the expression lock of the locking element for an exogenous gene of interest.
  • the locking element comprises the nucleotide sequence set forth in SEQ ID NO: 5 or 8, or comprises at least 80% of the nucleotide sequence set forth in SEQ ID NO: 5 or 8. And a nucleotide sequence capable of locking the expression of an exogenous gene of interest operably linked thereto; the key gene comprising the nucleotide sequence set forth in SEQ ID NO: 3 or 6, or comprising and SEQ ID a nucleotide sequence represented by NO: 3 or 6 has at least 40% homology and a nucleotide sequence capable of unlocking the expression of the foreign element of interest by the locking element.
  • the gene combination is a locking element comprising a nucleotide sequence of SEQ ID NO: 5 or at least 80% homology to SEQ ID NO: 5, and comprising SEQ ID NO : 3 or a key gene of a nucleotide sequence having at least 40% homology to SEQ ID NO: 3.
  • the gene combination is a locking element comprising a nucleotide sequence of SEQ ID NO: 8 or at least 80% homology to SEQ ID NO: 8, and comprising SEQ ID NO: 6 or a key gene of a nucleotide sequence having at least 40% homology to SEQ ID NO: 6.
  • the locking element is located between the constitutive promoter and the exogenous gene of interest and is operably linked to the constitutive promoter and the exogenous gene of interest.
  • the key gene is operably linked to a tissue-specific promoter.
  • the constitutive promoter is selected from the group consisting of the cauliflower mosaic virus (CaMV) 35S promoter, the nopaline synthase gene Ocs promoter from the T-DNA region of the Agrobacterium tumefaciens Ti plasmid, rice muscle movement
  • the tissue-specific promoter is selected from the group consisting of a rice green tissue-specific promoter ribulose-1,5-bisphosphate carboxylase small subunit rbcS promoter (Kyozuka et al, 1993; Nomura) Et al, 2000), maize phosphoenolpyruvate carboxylase PEPC promoter (Yanagisawa et al, 1989), rice green tissue-specific expression of DX1 promoter (Ye et al, 2012), rice photosystem II10kDa peptide D540 initiation (Cai et al, 2007), the rice leucine-rich leucine repeat-like receptor protein kinase LP2 promoter (Thilmony et al, 2009), and the maize chloroplast C4 Pdk promoter (Jang et al, 1999); preferably,
  • the tissue-specific promoter is the rice green tissue-specific promoter rbcS promoter.
  • the exogenous gene of interest may be an insect resistance gene or a herbicide resistance gene.
  • the insect resistance genes include cry1Ab, cry1Ac, cry1Ab/Ac, cry1C, cry2A and Vip3 insect resistance genes from Bacillus thuringiensis, anf, sep gene from Serratia entomophila, The cmb gene from Clostridium bifermentans, the mtx gene from Bacillus sphaericus, the insecticidal protein gene from Xenorhabdus nematophilus, and Photorhabdus luminescens The tca, tcb gene, and the prl gene from Metarhizium anisopliae are not limited thereto.
  • nucleotide coding sequence and amino acid sequence of the Bt insect-resistant gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • the herbicide resistance gene comprises an EPSP synthase gene resistant to glyphosate, an EPSA mutant gene aroA of Salmonella typhimurium, a bar gene resistant to glufosinate, an ALS mutant gene Ilv against moxazolinone G, the anti-Spirulina AccL-s2 gene, the anti-bromobenzonitrile bxn gene, and the chlorsulfuron-resistant csrl gene, but are not limited thereto.
  • the plant is selected from the group consisting of rice, wheat, barley, oats, corn, corn, sorghum, glutinous rice, sweet potatoes, potatoes, lotus seeds, soybeans, and peanuts.
  • the plant is rice.
  • Locking element Locking element (LOCK) and key gene (KEY)
  • the present inventors invented a set of regulatory elements and gene sequences having a function of regulating gene expression by DNA recombination, and named them as a locking element (LOCK) and a key gene (KEY) according to their respective functions.
  • LOCK locking element
  • KEY key gene
  • the locking element is capable of locking the expression of an exogenous gene of interest to which it is linked.
  • the locking element comprises the nucleotide sequence shown in SEQ ID NO: 5 or 8, or comprises at least 80% homology to the nucleotide sequence shown in SEQ ID NO: 5 or 8, and A nucleotide sequence capable of locking the expression of an exogenous gene of interest operably linked thereto.
  • the locked gene comprises the nucleotide sequence shown in SEQ ID NO: 5 or 8, or consists of the nucleotide sequence shown in SEQ ID NO: 5 or 8.
  • the key gene is capable of releasing the expression lock of the locking element for an exogenous gene of interest.
  • the key gene comprises the nucleotide sequence shown in SEQ ID NO: 3 or 6, Or a nucleotide sequence comprising at least 40% homology to the nucleotide sequence set forth in SEQ ID NO: 3 or 6, and capable of releasing the locked expression of the foreign element of interest by the locking element.
  • the locked gene comprises the nucleotide sequence shown in SEQ ID NO: 3 or consists of the nucleotide sequence shown in SEQ ID NO: 3.
  • the degree of homology between two nucleotide sequences can be determined by algorithms known in the art.
  • the optimal alignment of the sequences used for comparison can be performed by: local homology algorithm (Smith and Waterman Add. APL. Math. 2: 482 (1981)); homology alignment algorithm (Needleman and Wunsch J. Mol. Biol. 48: 443 (1970); Retrieving similarity algorithms (Pearson and Lipman Proc. Natl. Acad Sci. (USA) 85: 2444 (1988)); computer programs of these algorithms and their default parameters (Wisconsin Genetics) GAP, BESTFIT, BLAST, PASTA, and TFASTA, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI) in the software package; or by visual inspection.
  • the locking element may comprise at least 80%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, of the nucleotide sequence set forth in SEQ ID NO: 5 or 8. 97%, 98%, 99%, 99.5%, 99.8% or 100% homology, and is capable of locking the nucleotide sequence of expression of an exogenous gene of interest operably linked thereto.
  • the homology can be calculated using BLAST and its default parameters.
  • the key gene may comprise at least 40%, such as at least 40%, 41%, 42%, 43%, 44%, 45%, of the nucleotide sequence set forth in SEQ ID NO: 3 or 6. 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62% , 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79 %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 100% homology, and a nucleotide sequence capable of releasing the locked expression of the foreign element of interest by the locking element.
  • the gene combination of the invention is a locking element comprising a nucleotide sequence of SEQ ID NO: 5 or at least 80% homology to SEQ ID NO: 5, and comprising SEQ ID NO: 3 or a key gene of a nucleotide sequence having at least 40% homology to SEQ ID NO: 3.
  • the genetic combination of the invention comprises SEQ ID NO: 8 or a locking element of a nucleotide sequence having at least 80% homology to SEQ ID NO: 8, and comprising at least 40% homology to SEQ ID NO: 6 or to SEQ ID NO: The key gene of the nucleotide sequence.
  • the locking element may be: 1) comprising the nucleotide sequence set forth in SEQ ID NO: 5 or 8, or 2) comprising a substitution by the nucleotide sequence set forth in SEQ ID NO: 5 or 8, A nucleotide sequence derived from a deletion or addition of one or more nucleotides and capable of locking the expression of an exogenous gene of interest operably linked thereto.
  • the key gene may be: 1) comprising the nucleotide sequence shown in SEQ ID NO: 3 or 6, or 2) comprising a nucleotide sequence represented by SEQ ID NO: 3 or 6, A nucleotide sequence derived by deleting or adding one or more nucleotides and capable of releasing the locked expression of the foreign element of interest by the locking element.
  • the key gene may: 1) encode the amino acid sequence shown in SEQ ID NO: 4 or 7, or 2) encode, replace or delete in the amino acid sequence shown in SEQ ID NO: 4 or 7.
  • the locking element may: 1) comprise the nucleotide sequence set forth in SEQ ID NO: 5 or 8, or 2) under stringent conditions and the nucleotide sequence set forth in SEQ ID NO: 5 or 8.
  • the complementary sequence hybridizes and is capable of locking the nucleotide sequence of expression of the exogenous gene of interest to which it is operably linked.
  • the key gene 1) comprises the nucleotide sequence shown in SEQ ID NO: 3 or 6, or 2) under stringent conditions and the nucleotide sequence shown in SEQ ID NO: 3 or The complementary sequence hybridizes and is capable of releasing the nucleotide sequence locked by the locking element for expression of the foreign gene of interest.
  • Stringent conditions are used herein to describe hybridization to a nucleotide sequence in 6x sodium chloride/sodium citrate (SSC) at about 45 °C followed by 0.2xSSC/0.1 at about 50-65 °C. One or more washes in %SDS.
  • the "stringent condition” is “highly stringent condition”.
  • the term “highly stringent conditions” refers to, for example, hybridization to a nucleotide sequence in 6x SSC at about 45 °C followed by one or more washes in 0.1 x SSC/0.2% SDS at about 68 °C.
  • the combination of the regulatory gene composed of the locking element and the key gene can control the expression of the exogenous gene of interest in the plant, thereby locking the exogenous gene of interest in the endosperm while realizing the function of the exogenous gene of interest.
  • the locking element is placed between the constitutive promoter and the exogenous gene of interest to thereby lock expression of the exogenous gene of interest in the whole transgenic plant body comprising the locking element;
  • the key gene is operably linked to a tissue-specific promoter, thereby unlocking expression of the locked gene for the foreign gene of interest in a specific tissue, and realizing the function of the exogenous gene of interest.
  • promoter refers to a genetic element that initiates transcription. Promoters can be classified into constitutive promoters, tissue-specific promoters, and inducible promoters according to their mode of action.
  • the "constitutive promoter” refers to a promoter that maintains sustained activity in most or all tissues. Under the control of constitutive promoters, there was no significant difference in gene expression between different tissues and developmental stages.
  • the constitutive promoter for use in the present invention is a promoter derived from a plant or having constitutive activity in a plant.
  • constitutive promoters useful in the present invention include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, the nopaline synthase gene Ocs promoter from the T-DNA region of the Agrobacterium tumefaciens Ti plasmid, rice muscle The kinesin promoter Actin I, the ubiquitin gene promoter Ubi, and the like.
  • the constitutive promoter of the invention is the rice actin promoter Actin I.
  • the tissue-specific promoter refers to a promoter that is active in a particular type of cell or tissue. Under the control of tissue-specific promoters, genes are expressed only in certain specific organs or tissues.
  • the tissue-specific promoter used in the present invention is a promoter derived from a plant or having tissue-specific activity in a plant.
  • a person skilled in the art can express an organ according to a plant species, a desired/undesired exogenous gene of interest/ Tissues (eg, plant tissues/organs for consumption) are selected to select tissue-specific promoters.
  • Tissue-specific promoters useful in the present invention include: a root-specific promoter, a stem-specific promoter, and a leaf-specific promoter.
  • the tissue-specific promoter of the invention is a green tissue-specific promoter of a plant, such as a stem and/or leaf-specific promoter.
  • tissue-specific promoter examples include a rice green tissue-specific promoter ribulose-1,5-bisphosphate carboxylase small subunit rbcS promoter, corn phosphoenolpyruvate carboxylate Chemical enzyme PEPC promoter, rice green tissue-specific expression of DX1 promoter, rice light System II10kDa polypeptide D540 promoter, rice leucine-rich repeat-like receptor protein kinase LP2 promoter, and maize chloroplast C4 Pdk promoter, but are not limited thereto.
  • the tissue-specific promoter of the invention is a rice green tissue-specific promoter rbcS promoter.
  • the promoter is "operably linked" to the locked gene or key gene.
  • operably linked is meant an arrangement of elements wherein the components are configured to perform their normal function. For example, if a promoter affects the transcription of a coding sequence, it is operably linked to the coding sequence.
  • the "exogenous gene of interest” refers to a foreign gene which is not found in a recipient plant in a natural state.
  • a variety of exogenous gene of interest has been introduced into recipient plants for various purposes to increase plant resistance to pests, herbicides, and the like, and to increase and stabilize crop yields and the like.
  • the exogenous gene of interest may be an insect resistance gene or a herbicide resistance gene.
  • the insect resistance genes include cry1Ab, cry1Ac, cry1Ab/Ac, cry1C, cry2A and Vip3 insect-resistant genes from Bacillus thuringiensis, anf, sep gene from Serratia entomophila, The cmb gene from Clostridium bifermentans, the mtx gene from Bacillus sphaericus, the insecticidal protein gene from Xenorhabdus nematophilus, and Photorhabdus luminescens The tca, tcb gene, and the prl gene from Metarhizium anisopliae are not limited thereto.
  • nucleotide coding sequence and amino acid sequence of the Bt insect-resistant gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • the herbicide resistance gene comprises an EPSP synthase gene resistant to glyphosate, an EPSA mutant gene aroA of Salmonella typhimurium, a bar gene resistant to glufosinate, an ALS mutant gene Ilv G against moxazolinone, and an antibiotic.
  • the AccL-s2 gene, the bxn gene of bromoxynil and the csrl gene against chlorsulfuron are not limited thereto.
  • expression of the exogenous gene of interest is controlled by the locking element and the key gene of the present invention.
  • the foreign gene is operably linked to a locking element of the invention.
  • sequence encoding the exogenous gene of interest is also operably linked to appropriate regulatory factors, including promoters, enhancers, terminators, and signal peptides.
  • the invention provides a method of breeding a transgenic plant, the method comprising crossing a first parent comprising a locking element with a second parent comprising a key gene to obtain the transgenic plant.
  • the method for breeding a transgenic plant of the present invention further comprises introducing and integrating the locking element and a gene of interest linked thereto with the target gene into the first parental genome, and The key gene is introduced and integrated into the second parental genome.
  • the invention provides a method of breeding a transgenic plant, the method comprising introducing and integrating a locking element and a key gene into the genome of the same plant to obtain the transgenic plant.
  • Methods for introducing genes into recipient plants include, for example, Agrobacterium-mediated gene transformation, gene gun transformation, pollen tube pathway methods, etc., wherein Agrobacterium-mediated gene transformation is obtained in plant transformation.
  • Agrobacterium-mediated gene transformation is obtained in plant transformation.
  • the specific steps can be found in the attached embodiments.
  • specific exogenous genes such as marker genes for screening, can be isolated and eliminated from the transgenic plants by integration.
  • these transgenic plants after knocking out the marker gene are still included in the scope of the transgenic plants of the present invention.
  • the present invention provides a combination of genes for controlling expression of a foreign gene in a plant not in a specific tissue, and a method of cultivating the transgenic plant using the combination of the genes.
  • the method can be used to cultivate endosperm 0-expressing transgenic anti-aphid rice, but there is no synthesis and accumulation of any transgenic product protein in the rice endosperm produced by this aphid-resistant rice. Therefore, public concerns about the safety of food for GM food crops can be ruled out.
  • the target Bt insect-resistant gene was not expressed in the Japanese stunted rice endosperm of the obtained locking element and key gene, and the Bt protein detection result was 0. However, it is highly expressed in the stem and leaf tissues of the designed expression. Since the target gene expression product was not detected in the rice endosperm, the inventors referred to it as the endosperm 0 expression type Bt transgenic rice, which was distinguished from the traditional endosperm expression type Bt transgenic rice. Further insect-resistant identification results showed that the insect-resistant effect of Japanese wildflowers (30 strains) carrying the locking element and the key gene on the natural outbreak of rice leaf roller was close to zero, and non-transgenic control hybrids (30 strains).
  • the 100% strain rate was significantly different; the insecticidal efficiency of the artificially inoculated larvae of the first instar larvae (the number of insects per strain) also reached the level of resistance to high resistance. Therefore, the successful combination of the gene combination of the present invention and the endosperm 0 expression type transgenic insect-resistant rice will not only break through the deadlock of the traditional endosperm expression type Bt transgenic rice, but also strongly promote the genetically modified food crop. Industrialization process.
  • the invention has broad applicability, and is suitable for a wide range of crops, such as rice, wheat, barley, oats, corn, corn, sorghum, glutinous rice, sweet potato, potato, lotus seed, soybean, and the like for the purpose of production of fruits, seeds, roots and tubers. Peanuts and so on.
  • FIG. 1 Map of pSB130 plasmid vector.
  • the plasmid vector has two T-DNA regions, one of which carries the hygromycin-resistant marker gene Hpt; the other carries a multiple cloning site for the loading of a key gene or a sequence of locked elements to which the gene of interest is ligated.
  • Figure 2 is a flow diagram showing the construction of the expression vector pKey1 of the gene key (Key1; SEQ ID NO: 5) according to one embodiment of the present invention, wherein Figure 2A shows: the first step, the specificity of the recognition site by introducing KpnI and EcoRI The primers were amplified to obtain the Nos fragment and ligated into the corresponding multiple cloning site of pSB130; and in the second step, the Key1 5' fragment was amplified by specific primers introducing the XbaI/SmaI and KpnI recognition sites, and Linked to the corresponding multiple cloning site of pSB130; Figure 2B shows: In the third step, the Key1 3' fragment was amplified by specific primers introducing the SalI and SmaI recognition sites, and ligated into the corresponding multiple cloning site of pSB130.
  • the RbcS fragment was amplified by specific primers introducing HindIII and SalI recognition sites, and ligated into the corresponding multiple cloning site of pSB130 to construct a pKey1 expression vector.
  • Figure 3 is a flow chart showing the construction of the expression vector pLY1 of the locking element (Lock1; SEQ ID NO: 8) and its linked reporter gene (eYFP) according to one embodiment of the present invention, wherein Figure 3A shows: Nos fragment amplification and cloning with pKey1; and second step, using The specific primers inserted into the PstI/XbaI recognition site were amplified to obtain the Lock1 fragment, which was ligated into the corresponding multiple cloning site of pSB130.
  • Figure 3B shows the third step, using specific primers for introducing HindIII and PstI recognition sites. The ActinI fragment was increased and ligated into the corresponding multiple cloning site of pSB130.
  • the RbcS fragment was amplified by specific primers introducing the XbaI and KpnI recognition sites, and ligated into pSB130 corresponding polyclonal. At the site, a pLY1 expression vector was constructed.
  • Figure 4 is a flow chart showing the construction of the expression vector pKey2 of the key gene (Key2; SEQ ID NO: 3) according to another embodiment of the present invention, wherein Figure 4A shows the first step of identifying a site by introducing SacI and EcoRI. The specific primers were amplified to obtain the Nos fragment and ligated into the corresponding multiple cloning site of pSB130; and in the second step, the RbcS fragment was digested with the SalI and HindIII from the pKey1 expression vector and ligated into pSB130.
  • Figure 4B shows:
  • the Key2 3' fragment was amplified by specific primers introducing the SacI and XbaI recognition sites, and ligated into the corresponding multiple cloning site of pSB130;
  • the Key2 5' fragment was amplified by specific primers introducing the SalI and XbaI recognition sites, and ligated into the corresponding multiple cloning site of pSB130 to construct a pKey2 expression vector.
  • Figure 5 Flow chart of construction of expression vector pLY2 of locking element (Lock2; SEQ ID NO: 6) plus reporter gene (eYFP) according to another embodiment of the present invention, wherein Figure 5A shows: first step, using KpnI and EcoRI was digested from pLY1 to obtain a Nos fragment and ligated into the corresponding multiple cloning site of pSB130; and in the second step, a specific primer introduced into the PstI and XbaI recognition sites was used to obtain a Lock2 fragment and ligated into pSB130.
  • Figure 5A shows: first step, using KpnI and EcoRI was digested from pLY1 to obtain a Nos fragment and ligated into the corresponding multiple cloning site of pSB130; and in the second step, a specific primer introduced into the PstI and XbaI recognition sites was used to obtain a Lock2 fragment and ligated into pSB130.
  • Figure 5B shows:
  • the Actin I fragment was digested with p Hin1 by HindIII and PstI and ligated into the corresponding multiple cloning site of pSB130; and the last step, XbaI and KpnI was digested from pLY1 to obtain an eYFP fragment, which was ligated into the corresponding multiple cloning site of pSB130 to construct a pLY2 expression vector.
  • Figure 6 is a flow chart showing the construction of an expression vector pLB of a locking element (Lock1; SEQ ID NO: 8) plus a gene of interest (cry1Ab/1Ac) according to an embodiment of the present invention, wherein Figure 6A shows: The specific primers of the KpnI/SalI and EcoRI recognition sites were amplified to obtain the Nos fragment and ligated into the corresponding multiple cloning site of pSB130; and the second step was amplified by the specific primers introducing the PstI and XbaI recognition sites.
  • the Lock fragment was ligated into the corresponding multiple cloning site of pSB130;
  • Figure 6B shows:
  • the ActinI fragment was amplified by specific primers introduced into the HindIII and PstI recognition sites, and ligated into pSB130.
  • the cry1Ab/1Ac fragment was amplified by specific primers introducing the KpnI and SalI recognition sites, and ligated into the corresponding multiple cloning site of pSB130 to construct pLB expression. Carrier.
  • Figure 7 is a graphical representation of functional verification of a set of locking elements and key genes in accordance with one embodiment of the present invention, wherein pLY1 is a single locking element transformed to obtain a positive resistant callus formed by green tissue under confocal microscopy Observed results, no fluorescence signal was observed under dark field conditions; pLY1+pKey1 was observed by confocal microscopy after green tissue formed by positive resistance callus obtained by locking element transformation and then transformed by key gene and transient expression. A fluorescent signal is present under field conditions.
  • Figure 8 is a graphical representation of functional verification of a set of locking elements and key genes in accordance with another embodiment of the present invention, wherein pLY2 is a single locking element transformed to obtain a positive resistant callus formed by green tissue under confocal microscopy Observed, no fluorescence signal was observed under dark field conditions; pLY2+pKey2 was the result of confocal microscopy of green tissue formed by positive resistance callus obtained by transformation of the locking element and then transformed by key gene and transient expression. The fluorescent signal is presented under the conditions.
  • Figure 9 Copy number analysis of Nipponbare pKey1 and pLB positive transformed lines, wherein: Figure 9A is a copy number analysis of 21 pKey1 positive transformed lines of Nipponbare, and the results show that 13 of them are single copies; Figure 9B shows 22 pLB of Nipponbare Positive transformations were analyzed by copy number and the results showed that 15 of them were single copies.
  • Single-copy transformation lines (three of each of pKey1 and pLB) selected according to important agronomic traits such as seed set rate, other yield traits, plant height and growth period are directly labeled above the sample lanes using the sequence number in this application.
  • FIG. 10 Homozygous situation of a T2 generation line of Nipponbare pLB positive single copy transformation line and its PCR detection results, wherein: M-DL2000 stands for DL2000 DNA Marker; "+” stands for positive plasmid control, “-” stands for non-transgenic Negative control; 1-24 represents 24 independent individuals in one T2 line of Nipponbare pLB positive single copy transformation line.
  • Figure 11 Identification results of insect resistance of 9 outcrosses with 3 pKey1 and 3 pLB single copy transgenic lines, wherein: K11, K12, K13 and LB1, LB2, LB3 are pKey1 and pLB transformants, respectively.
  • WT is a wild type non-transgenic control.
  • Figure 12 ELISA quantitative analysis of Bt protein content in stem and leaf tissues of 9 outcrossed species of Nipponbare 3 pKey1 and 3 pLB single copy transgenic lines, among which: K11, K12, K13 and LB1, LB2, LB3 are single-copy pure parentes selected from pKey1 and pLB transformants, respectively; K11LB1-K13LB3 are heterologous species assembled by the above single-copy pure parent. T51-1 is a positive control.
  • Figure 13 Resistance performance of the locking element and key gene heterozygous K12LB2 to artificially inoculated larvae of the first infested larvae (about 80 worms per strain) under field conditions.
  • Figure 13A shows the resistance of K12LB2 (left) and non-transgenic Nipponbare control (right) to the first instar larvae of the inoculated mites in the tillering stage.
  • the non-transgenic control was caused by the artificial inoculation of Chilo suppressalis in the tillering stage.
  • the heart is so severe that the plant height after heading is greatly reduced, and the effective panicle is also significantly reduced;
  • Figure 13B shows K12LB2 (left) and non-transgenic Nipponbare control (right) for the first week before heading.
  • the larval resistance performance in the figure, can be seen in the non-transgenic control showed a high ratio of white spikes, while the transgenic outcrossed species did not appear white spikes.
  • Figure 14 Resistance of isolated leaves of K12LB2 in vitro to indoor inoculated larvae of the first instar larvae, A: positive control TT51-1; B: non-transgenic Minghui 63 control; C: K12LB2 outcrossing Species; D: non-transgenic Nipponbare control; E: Nipponbare K12 transformed line; F: Nipponbare LB2 transformed line.
  • the picture shows that the resistance of transgenic heterozygous K12LB2 to Chilo suppressalis is consistent with the positive control TT51-1.
  • Figure 15 Resistance performance of isolated stalks of K21LB2 outcrossed stalks to indoor artificially inoculated larvae of the first instar larvae.
  • CK Non-transgenic Japanese clear control. The picture shows that no signs of harm to the stem borer were observed on the stems of the transgenic K12LB2 outcrossed stalks, and all the inoculated mites were dead; while the wormholes and phlegm mites were observed on the control stems. Excreta and live larvae
  • Figure 16 ELISA quantitative analysis of Bt protein content in brown rice and polished rice endosperm of 9 outcrossed species of Nipponbare 3 pKey1 and 3 pLB single copy transgenic lines.
  • K11, K12, K13 and LB1, LB2, LB3 are single copy pure parents selected in pKey1 and pLB transformants, respectively; K11LB1-K13LB3 are heterologous species assembled by the above single copy pure parent.
  • WT was a wild type Nipponbare control; MH63 was a negative control; T51-1 and ZY3 were positive controls.
  • Example 1 Design of the gene combination of the present invention and its feasibility study and verification
  • the complete gene combination is designed to consist of a locking element that locks the expression of the gene of interest and a key gene that can specifically open the locking element.
  • a locking element that locks the expression of the gene of interest
  • a key gene that can specifically open the locking element.
  • two sets of such components have been designed, wherein the DNA sequences of the first set of components are SEQ ID NO: 5 (Lock1) and SEQ ID NO: 3 (Key1), respectively, and the DNA sequence of the second set of components Is SEQ ID NO: 8 (Lock 2) and SEQ ID NO: 6 (Key 2).
  • the locking element is designed to be placed between the constitutive promoter of Actin I and the start codon of the gene of interest, and its function is to prevent the expression of the target gene: as a key gene, it is designed to be placed in RbcS, etc. Under the control of the green tissue-specific expression promoter, its function is to open the expression of the gene of interest locked by the locked element in a specific tissue (such as green tissue).
  • a second set of expression vectors was constructed, in which some functional fragments were obtained from the first set of expression vectors by restriction enzyme digestion, and the other functional fragments still passed specific primers (Table 1). And high-fidelity PCR amplification technology was amplified, and then these functional fragments were ligated into the pSB130 plasmid vector (Fig.
  • the construction of this set of application-type expression vectors is mainly directed to the locking element and the gene fragment of interest.
  • the functional fragment which is common to the verification expression vector is obtained by enzymatic cleavage
  • the locking element in the first expression vector selected by the embodiment of the present application is obtained by specific primers and high-fidelity PCR amplification technology, respectively.
  • two functional fragments of the insect-resistant gene to be ligated, and then these functional fragments are ligated into the pSB130 plasmid vector (Fig. 1) in the designed sequence to obtain the corresponding application-type locking element expression vector pSB130::ActinI::LB1:: NosT (abbreviated as pLB1), a specific construction flow chart of the expression vector is shown in FIG. 6.
  • the pSB130 plasmid vector used in the examples of the present application comprises two "T-DNA” regions, wherein one "T-DNA” region is used to load a locking element or a key gene expression unit which is linked to a gene of interest, and the other is used for loading a moisture-proof mold.
  • Prime marker gene Hpt expression unit The purpose of constructing the double T-DNA expression vector is to focus on the practical application in the future, because the double T-DNA plasmid vector can transform the rice to enable the target gene and the marker gene to have independent integration in the receptor genome, so as to facilitate the marker gene. Separation and rejection by self-crossing in subsequent segregation generations.
  • the verification of the gene combination locking and opening function of the present invention is carried out using a two-step transformation method.
  • the two locking elements pLY1 and pLY2 in the validated expression vector prepared in Example 1 were introduced into rice embryogenic callus for permanent expression by Agrobacterium-mediated method (Liu et al., 1998).
  • the receptor variety used for genetic transformation of the above-described verification type locking elements pLY1 and pLY2 is indica Nipponbare. This variety is a model of rice genetic transformation, and its mature embryo callus is easy to induce and has high genetic transformation efficiency.
  • Induction/subculture medium (per liter): 4.1 g/L N6 (Chu et al, 1975) base salt component + N6 organic component (Table 2) + 2 mg/L 2,4-dichlorophenoxy Acetic acid (2,4-D)+2.0g/L hydrolyzed casein+30g/L sucrose+3g/L agar, pH 5.9
  • Infectious medium per liter: AA basal medium (Table 3) (Toriyama & Hinata, 1985) + 200 ⁇ M acetosyringone, pH 5.9.
  • Co-cultivation medium per liter: CC basal medium (Table 4) (Hiei et al, 1994) + 200 ⁇ M acetosyringone, pH 5.9.
  • Antibacterial medium per liter: induction/subculture medium + 500 mg/L cephalosporin, pH 5.9.
  • Screening medium (per liter): induction/subculture medium + 50 mg/L hygromycin + 500 mg/L cephalosporin, pH 5.9.
  • Regeneration medium (per liter): 4.1g/L N6 base salt component + N6 organic component (Table 2) + 2.0g / L hydrolyzed casein + 30g / L sucrose + 6g / L agar + 2mg / L Kinetin +1 mg/L ⁇ -naphthylacetic acid, pH 5.9.
  • N6 basic salt component (Shanghai Shenggong) and organic components
  • the genetically modified transformation of the expression-type locking element expression vectors pLY1 and pLY2 It is carried out by Agrobacterium-mediated method.
  • the Agrobacterium strain used was EHA105 (BioVector NTCC Inc.), and the recipient cell used was Nipponbare mature embryo-induced embryogenic callus.
  • the specific steps of Agrobacterium transformation are as follows:
  • the nascent callus was cut and transferred to a new callus induction and subculture medium, subcultured under the same conditions, subcultured every 2 weeks until the cytoplasm was thick, the color was bright yellow, the texture was hard and the cell mass was present. Granular embryogenic callus formation.
  • 0.5 ⁇ l of the expression vector plasmid was added to a 1.5 ml centrifuge tube containing 60 ⁇ l of Agrobacterium electroporation competent EHA105, and the head was pipetted and mixed into the electrode cup; after the electric shock, 1 ml of LB liquid medium was quickly added and sucked. After mixing, transfer to the previous 1.5ml centrifuge tube and shake it for 1 hour at 28 °C. After the bacterial solution is resuscitated, draw 100 ⁇ l of the bacterial solution and apply it evenly to the LB solids screen (containing 50 mg/l kanamycin, 25 mg).
  • Agrobacterium infection was carried out according to the method steps reported by Yang et al (2011). The specific procedure is as follows: the Agrobacterium liquid stored at -80 ° C is taken out, and 200 ⁇ l of the solution is uniformly applied to the surface of the LB solid medium containing 25 mg/l of rifampicin and 50 mg/l of kanamycin at 28 ° C. The culture was carried out overnight; then the single colony was selected and expanded with LB liquid medium; then, 200-300 ⁇ l of fresh bacterial solution was taken from the mixture and added to 20 ml of LB liquid culture containing 25 mg/l rifampicin and 50 mg/l kanamycin.
  • the medium was incubated at 28 ° C with shaking (220 rpm) for 16-18 h. After the sufficient amount of the bacterial solution was centrifuged at 4000 rpm for 15 min, the supernatant of the LB medium was discarded; the Agrobacterium (slightly pipetted with a pipette) was resuspended in 20 ml of 0.1 M MgSO 4 solution, and centrifuged at 4000 rpm for 10-15 min.
  • the MgSO 4 supernatant containing antibiotics was discarded; 5 ml of AA infecting medium containing 200 ⁇ M acetosyringone (AS) was added (Table 3) to resuspend Agrobacterium, and then an appropriate amount of AA-AS was added to infect and culture. Base, the OD 600 value of the bacterial liquid is finally adjusted between 0.8-1.0; after the concentration adjustment, the bacterial liquid is dispensed in a sterile 50 ml centrifuge tube, 20-25 ml / tube, and used.
  • AS acetosyringone
  • the embryogenic callus was pre-cultured for about 7 days, and then transferred from the subculture dish to the empty culture dish covered with sterile filter paper, and air-dried on the ultra-clean workbench 10- After about 15 minutes, slowly tumbling the callus with a sterilized teaspoon to make it fully dry; after drying, transfer it to a centrifuge tube containing the bacterial liquid, gently shake it at room temperature for 40 minutes, and centrifuge the tube for super Leave on the net work bench for 10 min; remove the bacterial solution, place the embryogenic callus on sterile filter paper for about 15 minutes, then transfer it to the surface with a sterile filter paper containing AS (200 ⁇ M) CC.
  • the culture medium (Table 4) was cultured at 28 ° C for 50-55 h; the embryogenic callus which was not grown or contaminated by Agrobacterium was selected and transferred to 2,4-D, 500 mg containing 2.0 mg/L. /l Cephthomycin (Cefortaxim) on the N6 antibacterial medium, bacteriostatic culture in the dark room at 28 °C for 3-4 days; then transfer the callus after bacteriostatic culture to 500mg / l cephalosporin and 50mg / L On the screening medium of Hygromycin, culture in a dark room at 28 ° C; picking up well-grown callus every half month for subculture, The concentration of cephalosporin in the culture is adjusted according to the degree of pollution. In general, the concentration can be reduced by half in the third or fourth round. This was subcultured until a rapidly growing, large, and vivid resistant callus was obtained (4-6 rounds of screening and subculture).
  • the resistant callus obtained in the previous step was transferred to N6 regeneration medium, pre-differentiated for one week at 28 °C, then transferred to fresh N6 regeneration medium, and cultured in a light chamber at 25 ° C until green dot formation ( It takes about two weeks).
  • the callus forming the green dot in the above step is a receptor, and the key gene expression vectors pKey1 and pKey2 prepared according to Example 1 were introduced into the green dot by the gene gun-mediated method described in Tu et al 2000).
  • the injured tissue was transiently expressed, and after 24 to 36 hours, the fluorescence signal of the transformed and untransformed (control) green-green callus was observed under a confocal microscope and photographed and stored.
  • the genetic transformation of the application-type locking element and the key gene was carried out by Agrobacterium-mediated method.
  • the selection of the acceptor material, the various media used, the genetic transformation of the vector and the positive resistance callus Both the differentiation and differentiation cultures were identical to the corresponding test procedures described in Example 2 until the positive resistant callus differentiated into green shoots.
  • the obtained green seedlings after washing the medium on the root system, or directly (the simultaneous differentiation of root buds), or after the rooting of the N6 rooting medium (the bud-differentiated type) is transferred into the Yoshida culture solution (Table 5) After cultivation, after the growth state is good and stable, transplant it to the greenhouse until it matures.
  • the supernatant was centrifuged at 4000 rpm for 20 minutes at room temperature (temperature > 18 ° C), shaken by adding 1/10 volume of pre-heated 10X CTAB at 56 ° C, and once again added a volume of 24:1 (chloroform: isoamyl alcohol) and gently shaken. 30min. Centrifuge at 4000 rpm for 20 min at room temperature (temperature > 18 ° C). After that, the supernatant was aspirated with a 5 ml pipette tip, and then a volume of 1X CTAB was added. After the shaking, flocculation of DNA was observed, and the tip was taken out and placed in 1.5 ml.
  • the bottom of the tube was centrifuged and inverted on absorbent paper for 10 min. Then 10 ⁇ L of RNase and 0.5 ml of sterilized 1 M NaCl were added and dissolved in a water bath at room temperature or 56 °C. The dissolved DNA was added to a 2-fold (-20 ° C) volume of -20 ° C pre-cooled 95% ethanol, and the DNA was flocculated and precipitated by shaking. Use the tip of the gun to pick up the flocculent DNA and put it into a 1.5 ml centrifuge tube, add 1.5 ml of 75% ethanol to wash the DNA, then pour the ethanol, add ultrapure water and store at 4 ° C for later use.
  • Detection and pure line identification of positive transgenic plants were performed using PCR techniques. Due to Hpt The marker gene and the locking element or key gene are co-transformed by double T-DNA, and the identification of the T0 generation positive transformation line is based only on the gene of interest. Therefore, only the plants whose target genes are positive after the test are continued to be cultivated to maturity, and the non-positive plants are all eliminated to reduce the workload of subsequent generations.
  • the anti-hygromycin gene amplification primers used were: Hpt-F, 5'-GCTGTTATGCGGCCATTGTC-3' (SEQ ID NO: 9) and Hpt-R, 5'-GACGTCTGTCGAGAAGTTTC-3' (SEQ ID NO: 10);
  • the PCR reaction system 25 ul system was: 1 ul of DNA template to be tested, 2.5 ul of 10 ⁇ PCR buffer, 2 ul of 10 mM dNTP, 0.25 ul of 20 uM primer, 0.5 ul of 2 u/ul tagase, and ddH 2 O to 25 ul.
  • the PCR reaction procedure was: denaturation at 94 ° C for 3 min; subsequent denaturation at 94 ° C for 30 sec, annealing at 55 ° C for 30 sec, extension at 72 ° C for 40 sec for 35 cycles; thereafter, extension at 72 ° C for 5 min at 10 ° C.
  • the amplified products were identified by 1% agarose gel electrophoresis and stored after photographing.
  • the Bt gene amplification primer used was: BtF, 5'-GGCCATACAACTGCTTGAGT-3' (SEQ ID NO: 11); BtR, 5'-GCGTTTCCCATAGTTCCATA-3' (SEQ ID NO: 12), and the amplified fragment was 1 Kb in length.
  • the PCR reaction procedure was: denaturation at 94 ° C for 3 min; followed by denaturation at 94 ° C for 30 sec, annealing at 55 ° C for 30 sec, extension at 72 ° C for 1 Min for 35 cycles; thereafter, extension at 72 ° C for 5 min at 10 ° C.
  • the amplified products were identified by 1% agarose gel electrophoresis and stored after photographing.
  • the PCR reaction system is shown in Table 6.
  • the key gene amplification primer used was: KeyF, 5'-AACGAGTGATGAGGTTCGCA-3' (SEQ ID NO: 13); KeyR, 5'-ACCCGGCAAAACAGGTAGTT-3' (SEQ ID NO: 14), and the amplified fragment was 672 bp in length.
  • the PCR reaction procedure was: denaturation at 94 ° C for 3 min; followed by denaturation at 94 ° C for 30 sec, annealing at 55 ° C for 30 sec, extension at 72 ° C for 1 Min for 35 cycles; thereafter, extension at 72 ° C for 5 min at 10 ° C.
  • the amplified products were identified by 1% agarose gel electrophoresis and stored after photographing.
  • the PCR reaction system is shown in Table 1.
  • 21 pKey1 and 22 pLB positive transformed lines were identified from 41 pKey1 and 50 pLB independent transformed lines of Nipponbare, respectively.
  • Transgenic copy number detection was performed using Southern hybridization techniques. First, the total rice DNA was digested with restriction endonuclease HindIII overnight at 37 °C (8-12 h); the digested DNA was separated by 0.8% (w/v) agarose gel electrophoresis (40V). , 12h), after which the electrophoreticly separated DNA blot was transferred to a nylon membrane (GE Healthcare, UK). Preparation of hybridization probes and Southern hybridization and hybridization signal detection were carried out according to the instructions provided by Roche DIG-High Prime DNA Labeling and Detection Starter Kit II (Switzerland), wherein the hybrid probe preparation primers were respectively :
  • pKEY1 Key-1U20, 5'-ATGTCCAATTTACTGACCGT-3' (SEQ ID NO: 15), and
  • pLB Bt-577U24, 5'-AGGCTGATTGGAAACTACACCGAC-3' (SEQ ID NO: 17), and
  • Fig. 1 The copy number analysis results of the pKey1 and pLB positive transformed lines of Nipponbare are shown in Fig. 1. As can be seen from Fig. 1, 13 of the 21 pKey1 positive transformants (9A) in Nipponbare were single-copy, while 15 of the 22 pLB-positive transformed lines (Fig. 9B) were single-copy.
  • PCR amplification assays and screens for single copy transgenic lines were performed in T2 lines.
  • T2 generation seeds and plants were obtained from the pKEY1 and pLB single-copy transformation lines of each recipient variety after self-crossing for two generations. After that, PCR amplification was used to screen out the key pairs and LB. Pure system.
  • Figure 2 is the homozygous situation of one T2 generation strain of Nipponbare pLB single copy transformation line and its PCR detection results. As shown in Fig. 10, 24 individuals of the T2 line showed positive LB gene amplified fragments, indicating that the T2 line was a transgenic LB gene. PCR amplification assays of other pLB and pKey1 transgenic lines were performed accordingly.
  • the field resistance of the pKey1/pLB transformed heterozygous species to the stem borer was evaluated by the artificial insect method, and the artificial insects were divided into two at the highest tillering stage and one week before heading.
  • the eggs were purchased from Jiangxi Shennong Technology Co., Ltd., and the purchased eggs were placed in a glass test tube with a length of 12 cm and a diameter of 2.0 cm and a wet cotton ball on the bottom, an egg block/tube, a cotton plug, and a nozzle. Put a black cloth on the outside. Eggs hatch at 26-28 ° C, 80% humidity and 16 hours light / 8 Hour in dark conditions.
  • the hatched first-instar larvae were inoculated into the base of the rice blast with the test tube within 6 hours after hatching, the base of the test tube was inserted into the paddy soil, the tube mouth was close to the rice straw, and then the black cloth was opened and the tampon was removed.
  • the amount of insects was 1 egg block/strain, about 80 head worms.
  • the number of dead hearts or white spikes of the damage of the stem borer was investigated, and at the same time, the total number of tillers or the total number of effective spikes of the test plants were investigated.
  • the field resistance of the pKey1/pLB transformation line outcrossed species to the rice leaf roller was evaluated by the natural insect inoculation method.
  • the number of damaged leaves and the degree of damage of leaves per plant damaged by Cnaphalocrocis medinalis were investigated 3-5 days after the occurrence of the hazard peak.
  • the statistical analysis of the field resistance evaluation index of the pKey1/pLB transformation line heterozygous species to the stem borer and the rice leaf roller was performed by SPSS12.0 (SPSS, Chicago, USA) software, and the t test of the LSD method was used to test The difference in the number of heart or white spikes of the pKey1/pLB transformed outcrossed species was significantly different from that of the wild type control.
  • the natural insects the damage caused by the leafhoppers in the closed net room
  • the identification of the artificial insects showed that the nine outcrossed species were highly resistant to the rice leaf roller (Fig. 11);
  • the heterozygous species K11LB1, K11LB2 and K11LB3 also showed good resistance to the adult mites (1 egg/strain) (Fig. 11).
  • Further quantitative analysis of the Bt protein content of the leaves and stems of the 9 outcrossed species in the tillering, heading and filling stages revealed that 3 of the 9 outcrossed species (K11LB3, K12LB3 and K13LB3) were in the leaves.
  • the Bt protein content was significantly lower than the positive control at three different growth stages, and the Bt protein content in the other six combined leaves was close to or higher than the positive control (Fig. 12); likewise, the three outcrosses in the stem The Bt protein content in the stem was also the lowest among the 9 outcrosses, and the highest KTLB1 and K12LB2 outcrossed species had Bt protein content of 40-50% of the positive control (Fig. 12).
  • Table 7 Resistance performance of K12LB2 outcrossed species to inoculated sorghum in the tillering stage and 7 days before heading in field conditions
  • the indoor insect resistance evaluation is carried out for the stem borer, and the eggs are also from the Jiangxi Shennong Technology Company.
  • the hatching method of the eggs is the same as before.
  • the methods used for the evaluation of insect resistance include the stem method and the leaf method.
  • the stem method is to take the stems of the rice after jointing and cut into 12cm long strips and leaf sheaths. Each insect tube is placed in two stem segments of the same plant and 20 first-instar larvae. The insect tube was sealed with a cotton plug, placed at 27 ° C and 80% relative humidity, and the larval mortality was counted after 6 days.
  • the leaf method is to take the blade leaves and the second leaf leaves at the booting stage, cut into 8 cm long leaf segments, and place them in a petri dish with filter paper moistened with 2 ml of distilled water. Each culture dish has 4 segments and 15 heads.
  • the larvae of the third instar larvae were wrapped with PARAFILM four times to prevent larvae from drilling out. Thereafter, they were housed at room temperature (25 ° C), and the mortality of the larvae was counted after 6 days.
  • Table 8 Resistance of isolated leaves and stems of K12LB2 in vitro to artificially inoculated stem borer
  • Example 6 Determination of Bt protein content in stems, leaves and endosperm of heterozygous seeds using applied locking elements and key gene transformation lines
  • the inventors further determined the Bt protein content of the brown rice and polished rice of the nine key gene-locked gene outcrossed species described in Example 5.
  • the working fluid is determined to include an extract/diluent and an eluent.
  • the extract/diluent was prepared by diluting the stock solution (35 ml) supplied with the kit to 175 ml with double distilled water.
  • the eluent was prepared by adding the phosphate attached to the kit to 1 L of double distilled water and 5 ml of Tween-20, followed by stirring to completely dissolve it. Both working solutions were stored at 4 ° C and preheated to room temperature before use.
  • Each replicate was set up with 3 replicates, 1 strain/repeat, and about 20 mg of fresh tissue per plant was taken and ground to a homogenate with 500 ⁇ l of extraction/diluted solution.
  • the homogenate was placed in a 1.5 ml centrifuge tube and allowed to stand for 30 min, then the supernatant was aspirated, centrifuged at 4000 rpm for 3 min, and diluted with a dilution/diluent to determine the Bt protein concentration.
  • the dilution ratios for each period and tissue samples are shown in Table 9.
  • the ELISA reaction process is carried out according to the instructions of the kit. The specific steps are as follows:
  • Absorbance values were measured using a MK3 type microplate reader (Thermo Labsystems Co., Shanghai, China) and read at a wavelength of 450 nm.
  • a standard curve is drawn using the concentration and absorbance of the standard sample, the concentration of the test sample is read on the standard curve, and the protein content in the rice tissue is calculated based on the dilution factor.
  • the present invention provides a nucleotide sequence of an exogenous gene of interest Bt, a locking element Lock1/Lock2 and a key gene Key1/Key2 for constructing a gene combination, and a deduced amino acid sequence thereof, wherein the Bt gene cry1Ab/cry1Ac is from Chinese agricultural science The academician Fan Yunliu academician synthesized and provided it; the locking element Lock1/Lock2 and the key gene Key1/Key2 were artificially synthesized.
  • the sequences listed in the sequence listing are as follows.

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Abstract

一种用于控制外源基因不在植物特定组织中表达的新的基因组合，以及应用该基因组合培育转基因植物的方法，该方法可用于培育例如0表达型转基因水稻，即由所述水稻所产生的稻米胚乳中没有任何转基因产物蛋白的合成和积累。

Description

基因组合及其用途 技术领域
本发明涉及转基因植物领域。具体地，本发明涉及一种用于控制植物中的外源目的基因的表达的基因组合，以及应用该基因组合培育转基因植物的方法。
背景技术
转基因技术自上世纪八十年代发明以来取得了举世瞩目的成就。培育的转基因作物已在全球28个国家种植，2014年生产面积已达1.815亿公顷(James，et al.，2014)。据英国PG经济公司的布鲁克斯和巴菲特提供的最新即时信息表明，自1996至2013年的17年间，转基因作物提高了作物生产价值1330亿美元，节省农药活性成分5亿kg，仅在2013年就减少二氧化碳排放量28亿kg，相当于1240万辆汽车一年的尾气排放量，帮助缓解了一些在世界上最贫穷的国家超过165万小农场和他们的家庭共计6500万人的贫困(James，et al.，2014)。因此他们得出的结论是转基因作物继续对食品安全、可持续发展和气候变化产生显著的正影响。目前，已有超过10个粮食和纤维作物被批准商业化种植，范围从大宗的粮食、经济作物如玉米、大豆和棉花等到小宗的水果、蔬菜作物如木瓜、茄子和南瓜等。
水稻是全球50％人口的主粮作物。尽管已于2000年宣告成功研制出转基因水稻系(Tu，et al.，2000)，例如优良的转基因恢复系明恢63/Bt(华恢1号)及其杂交稻汕优63/Bt，并于2009年11月在经过研制方和第三方反复验证其无生物安全性风险的情况下，获得我国农业部颁发的生物安全证书。然而，由于这些传统的转Bt基因抗虫水稻是胚乳表达型的，即所生产的转基因大米中有Bt基因的表达产物积累，依然很难使公众认可它的食用安全性，其商业化种植也因此一直未获批准。这种状况不仅在中国是如此，在世界其它一些地区和国家如欧洲和日本也是如此。2006年在欧洲调查的人群中有58％的人认为转基因食品“不应该被鼓励”。
为了解决人们对转基因植物生态安全性和食品安全性的恐惧和担忧，许多安全生物技术先后被研制出来，如转基因花粉不育技术 (Paoletti and Pimentel，1996)、种子不育或无籽技术(Daniell，2002；Li，1998)、叶绿体转基因技术(Bock，2001)、“终结者”种子技术(Oliver，et al.，1998)和外源基因删除技术(Luo，et al.，2007)等。在这些技术中，影响较大的是美国康涅狄格大学李义教授及其学生罗克明等人研发的外源基因删除技术。该技术是在转基因开花前后自动除去花粉和种子中的外源基因，如同计算机“卸载”应用软件一样，使转基因植物中的外源基因在扩散前和人们使用前被自动清除(赵德刚等，2008)。该技术可直接应用于无性繁殖作物，但还需改进才能应用于有性繁殖作物。主要的问题是有性繁殖作物种子中的外源目的基因一旦被删除就不再保有，从而使通过种子繁殖的下一代丢失转基因性状。
因此，在转基因粮食、蔬菜和水果等作物上，迫切需要能够在可食用的组织或器官中关闭目的基因表达，但又不至于在通过种子繁殖的下一代中丢失转基因性状的新型生物技术体系。
发明内容
出于以上目的，本发明人研发了能够在特定组织/器官(例如，胚乳中)关闭目的基因表达的基因组合。该技术主要由能锁定外源目的基因表达的锁定元件和能开启被锁定的外源目的基因表达的钥匙基因两个组件构成。在独立存在条件下，前者携带的外源目的基因是永久锁定的，而后者携带的能开启被锁定的外源目的基因表达的钥匙基因仅在特定组织/器官中是表达的。将这两个组件分别转化和整合到诸如水稻的父母本基因组中，然后通过杂交生产就可以将其综合到一起，也就可以获得能在所述特定组织/器官中开启外源目的基因表达，以致出现期望的表型，而在特定组织/器官(例如食用器官，如水稻胚乳)中则始终关闭该外源目的基因表达，从而产生在供食用的部分中不含任何转基因成分的转基因植物。同样地，将这两个组件分别导入和整合到同一个受体植株的基因组中，也能获得有同样的效果。同时，不存在由于目的基因DNA在繁殖过程中被删除而不能遗传的问题，因此，拥有广泛的适用性。
具体地，在第一个方面，本发明提供了一种用于控制植物中的外源目的基因的表达的基因组合，其由锁定元件和钥匙基因组成，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与 SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
一方面，本发明提供了一种用于在植物中锁定与其连接的外源目的基因的表达的锁定元件，其包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列。
相应地，一方面，本发明提供了一种用于在植物特定组织中解除所述锁定元件对于外源目的基因的表达锁定的钥匙基因，其包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
在第二方面，本发明提供了一种用于控制植物中的外源目的基因的表达的方法，所述方法包括将锁定元件和钥匙基因导入受体植物中，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
一方面，本发明提供了一种用于控制外源目的基因在植物中表达的方法，所述方法包括将所述外源目的基因可操作连接至锁定元件，所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列。
相应地，一方面，本发明提供了一种用于解除所述锁定元件对外源目的基因在植物中的表达锁定的方法，所述方法包括将钥匙基因导入所述植物中，所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同 源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
在第三方面，本发明提供了一种培育转基因植物的方法，所述方法包括将包含锁定元件的第一亲本与包含钥匙基因的第二亲本杂交，从而获得所述的转基因植物，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
在具体实施方式中，所述方法还包括在所述杂交步骤之前，将所述锁定元件导入和/或整合到所述第一亲本的基因组中，以及将所述钥匙基因导入和/或整合到所述第二亲本的基因组中。
在可选的实施方式中，本发明提供了一种培育转基因植物的方法，所述方法包括将将锁定元件和钥匙基因导入和/或整合到同一植株的基因组中，从而获得包含所述锁定元件和钥匙基因的转基因植物，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
在第四方面，本发明提供了锁定元件和钥匙基因的组合用于调控植物中的外源目的基因的表达的用途，其中所述锁定元件能够锁定与其可操作连接的外源目的基因的表达；所述钥匙基因能够解除所述锁定元件对于外源目的基因的表达锁定。
在该方面的具体实施方式中，所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少 40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
根据本发明的一些具体实施方式，所述基因组合为包含SEQ ID NO：5或与SEQ ID NO：5所具有至少80％的同源性的核苷酸序列的锁定元件，和包含SEQ ID NO：3或与SEQ ID NO：3所具有至少40％的同源性的核苷酸序列的钥匙基因。
根据本发明的另一些具体实施方式，所述基因组合为包含SEQ ID NO：8或与SEQ ID NO：8所具有至少80％的同源性的核苷酸序列的锁定元件，和包含SEQ ID NO：6或与SEQ ID NO：6所具有至少40％的同源性的核苷酸序列的钥匙基因。
根据本发明的各个方面，所述锁定元件位于组成型启动子和所述外源目的基因之间，并与所述组成型启动子和所述外源目的基因可操作连接。
根据本发明的各个方面，所述钥匙基因与组织特异性启动子可操作连接。
根据本发明的各个方面，所述组成型启动子选自花椰菜花叶病毒(CaMV)35S启动子、来自根癌农杆菌Ti质粒T-DNA区域的胭脂碱合成酶基因Ocs启动子、水稻肌动蛋白启动子Actin I、玉米泛素(ubiquitin)基因启动子Ubi；优选地，所述组成型启动子是水稻肌动蛋白启动子Actin I。
根据本发明的各个方面，所述组织特异性启动子选自水稻绿色组织特异性启动子核酮糖-1，5-二磷酸羧化酶小亚基rbcS启动子(Kyozuka et al，1993；Nomura et al，2000)、玉米磷酸烯醇式丙酮酸羧化酶PEPC启动子(Yanagisawa et al，1989)、水稻绿色组织特异表达DX1启动子(Ye et al，2012)、水稻光系统II10kDa多肽D540启动子(Cai et al，2007)、水稻富亮氨酸重复序列类受体蛋白激酶LP2启动子(Thilmony et al，2009)，以及玉米叶绿体C4 Pdk启动子(Jang et al，1999)；优选地，所述组织特异性启动子是水稻绿色组织特异性启动子rbcS启动子。
根据本发明的各个方面，所述外源目的基因可以是抗虫基因或抗除草剂基因。所述抗虫基因包括来自苏云金芽胞杆菌(Bacillus thuringiensis)的cry1Ab、cry1Ac、cry1Ab/Ac、cry1C、cry2A和Vip3抗虫基因、来自嗜虫沙雷氏菌(Serratia entomophila)的anf、sep基因， 来自双酶梭菌(Clostridium bifermentans)的cmb基因、来自球形芽孢杆菌(Bacillus sphaericus)的mtx基因、来自嗜线虫致病杆菌(Xenorhabdus nematophilus)的杀虫蛋白基因、来自发光光杆状菌(Photorhabdus luminescens)的tca、tcb基因，以及来自金龟子绿僵菌(Metarhizium anisopliae)的prl基因，但不限于此。
根据本发明，所述Bt抗虫基因的核苷酸编码序列和氨基酸序列分别如SEQ ID NO：1和SEQ ID NO：2中所示。
根据本发明的各个方面，所述抗除草剂基因包括抗草甘膦的EPSP合成酶基因、鼠伤寒沙门氏菌EPSP突变基因aroA、抗草丁膦的bar基因、抗米唑啉酮的ALS突变基因Ilv G、抗稀禾定的AccL-s2基因、抗溴苯腈的bxn基因和抗绿磺隆的csrl基因，但不限于此。
根据本发明的各个方面，所述植物选自水稻、小麦、大麦、燕麦、玉米、粟米、高粱、薏米、红薯、马铃薯、莲子、大豆和花生。优选地，所述植物为水稻。
本文中使用以下定义来进一步定义和描述本公开。除非在特定情况下另有限定，否则这些定义适用于本说明书中通篇所用的术语。
除非另有定义，本文所用的所有技术和科学术语的含义均与本发明所属领域的普通技术人员通常理解的一样。如发生矛盾，则以本说明书，包括本文给出的定义为准。
锁定元件(LOCK)和钥匙基因(KEY)
本发明人经DNA重组发明了一组具有调控基因表达功能的调控元件和基因序列，并且根据其各自的功能分别命名为锁定元件(LOCK)和钥匙基因(KEY)。
所述锁定元件能够锁定与其连接的外源目的基因的表达。具体地，所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列。优选地，所述锁定基因包含SEQ ID NO：5或8所示的核苷酸序列，或由SEQ ID NO：5或8所示的核苷酸序列组成。
所述钥匙基因能够解除所述锁定元件对于外源目的基因的表达锁定。具体地，所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列， 或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。优选地，所述锁定基因包含SEQ ID NO：3所示的核苷酸序列，或由SEQ ID NO：3所示的核苷酸序列组成。
可通过本领域中已知的算法测定两条核苷酸序列之间同源性的程度。用于比较的序列的最佳比对可通过以下进行：局部同源性算法(Smith和Waterman Add.APL.Math.2：482(1981))；同源性比对算法(Needleman和Wunsch J.Mol.Biol.48：443(1970))；检索类似性算法(Pearson和Lipman Proc.Natl.Acad Sci.(USA)85：2444(1988))；这些算法的计算机程序及其默认参数(Wisconsin Genetics软件包中的GAP、BESTFIT、BLAST、PASTA，和TFASTA，Genetics Computer Group (GCG)，575 Science Dr.，Madison，WI)；或通过目视检查。
根据本发明，所述锁定元件可以包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％，例如至少91％，92％，93％，94％，95％，96％，97％，98％，99％，99.5％，99.8％或100％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列。所述同源性可以使用BLAST及其默认参数计算。
根据本发明，所述钥匙基因可以包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％，例如至少40％，41％，42％，43％，44％，45％，46％，47％，48％，49％，50％，51％，52％，53％，54％，55％，56％，57％，58％，59％，60％，61％，62％，63％，64％，65％，66％，67％，68％，69％，70％，71％，72％，73％，74％，75％，76％，77％，78％，79％，80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，99.5％，99.8％或100％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。所述同源性可以使用BLAST及其默认参数计算。
在一个具体实施方式中，本发明的基因组合为包含SEQ ID NO：5或与SEQ ID NO：5所具有至少80％的同源性的核苷酸序列的锁定元件，和包含SEQ ID NO：3或与SEQ ID NO：3所具有至少40％的同源性的核苷酸序列的钥匙基因。
在另一个具体实施方式中，本发明的基因组合为包含SEQ ID NO： 8或与SEQ ID NO：8所具有至少80％的同源性的核苷酸序列的锁定元件，和包含SEQ ID NO：6或与SEQ ID NO：6所具有至少40％的同源性的核苷酸序列的钥匙基因。
根据本发明，所述锁定元件可以：1)包含SEQ ID NO：5或8所示的核苷酸序列，或者2)包含由SEQ ID NO：5或8所示的核苷酸序列经过取代、缺失或添加一个或多个核苷酸而衍生的，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列。
根据本发明，所述钥匙基因可以：1)包含SEQ ID NO：3或6所示的核苷酸序列，或者2)包含由SEQ ID NO：3或6所示的核苷酸序列经过取代、缺失或添加一个或多个核苷酸而衍生的，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
根据本发明，所述钥匙基因可以：1)编码SEQ ID NO：4或7所示的氨基酸序列，或者2)编码在SEQ ID NO：4或7所示的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸而衍生的，并且能够解除所述锁定元件对于外源目的基因的表达锁定的氨基酸序列。
根据本发明，所述锁定元件可以：1)包含SEQ ID NO：5或8所示的核苷酸序列，或者2)在严格条件下与SEQ ID NO：5或8所示的核苷酸序列的互补序列杂交，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列。
根据本发明，所述钥匙基因：1)包含SEQ ID NO：3或6所示的核苷酸序列，或者2)在严格条件下与SEQ ID NO：3或6所示的核苷酸序列的互补序列杂交，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
“严格条件”在本文中用于描述在约45℃下在6x氯化钠/柠檬酸钠(SSC)中与核苷酸序列的杂交，随后为在约50-65℃下在0.2xSSC/0.1％SDS中的一次或多次洗涤。优选地，所述“严格条件”为“高度严格条件”。术语“高度严格条件”指例如在约45℃下在6xSSC中与核苷酸序列的杂交，随后为在约68℃下在0.1xSSC/0.2％SDS中的一次或多次洗涤。
根据本发明，由所述锁定元件和所述钥匙基因构成的调控基因组合可以控制外源目的基因在植物体中的表达，从而在实现外源目的基因功能的同时，锁定外源目的基因在胚乳、块根、块茎和果肉等储藏组织或器官中的表达，以便获得无转基因表达产物的胚乳、块根、块 茎和果肉等，但又不影响其通过有性繁殖过程遗传给下一代的能力。
在具体的实施方式中，将所述锁定元件置于组成型启动子和外源目的基因之间，从而在包含所述锁定元件的整株转基因植物体中锁定外源目的基因的表达；同时，将所述钥匙基因与组织特异地启动子可操作地连接，从而在特定组织中解除所述锁定基因对于外源目的基因的表达锁定，实现外源目的基因功能。
启动子
根据本发明，所述“启动子”是指起始转录作用的基因元件。启动子按照其作用方式可以分为组成型启动子、组织特异性启动子和诱导型启动子等。
根据本发明，所述“组成型启动子”是指在多数或全部组织中保持持续的活性的启动子。在组成型启动子调控下，不同组织器官和发育阶段的基因表达没有明显差异。优选地，用于本发明的组成型启动子是源自植物或在植物中具有组成型活性的启动子。
可用于本发明的组成型启动子的实例包括但不限于：花椰菜花叶病毒(CaMV)35S启动子、来自根癌农杆菌Ti质粒T-DNA区域的胭脂碱合成酶基因Ocs启动子、水稻肌动蛋白启动子Actin I、玉米泛素(ubiquitin)基因启动子Ubi等。优选地，本发明的组成型启动子是水稻肌动蛋白启动子Actin I。
根据本发明，所述组织特异性启动子是指在特定类型的细胞或组织中具有活性的启动子。在组织特异性启动子调控下，基因只在某些特定的器官或组织部位表达。优选地，用于本发明的组织特异性启动子是源自植物或在植物中具有组织特异性活性的启动子本领域技术人员可以根据植物种类、期望/不期望外源目的基因表达的器官/组织(例如供食用的植物组织/器官)来选择组织特异性启动子。
可用于本发明的组织特异性启动子包括：根特异启动子、茎特异启动子、叶特异启动子。优选地，本发明所述的组织特异性启动子是植物的绿色组织特异性启动子，例如茎和/或叶特异性启动子。具体地，根据本发明的组织特异性启动子的实例包括水稻绿色组织特异性启动子核酮糖-1，5-二磷酸羧化酶小亚基rbcS启动子、玉米磷酸烯醇式丙酮酸羧化酶PEPC启动子、水稻绿色组织特异表达DX1启动子、水稻光 系统II10kDa多肽D540启动子、水稻富亮氨酸重复序列类受体蛋白激酶LP2启动子，以及玉米叶绿体C4 Pdk启动子，但不限于此。优选地，本发明的组织特异性启动子是水稻绿色组织特异性启动子rbcS启动子。
根据本发明，所述启动子与所述锁定基因或钥匙基因是“可操作连接的”。所述“可操作连接”是指元件的排列，其中所述组分经配置以发挥其正常功能。例如，如果启动子影响编码序列的转录，那么它是与编码序列可操作连接的。
外源目的基因
根据本发明，所述“外源目的基因”是指在天然状态下不存在于受体植物中的外来基因。现已出于各种目的，将多种外源目的基因导入到受体植物中，以提高植物对害虫、除草剂等的抗性，并提高和稳定作物产量等。
根据本发明，所述外源目的基因可以是抗虫基因或抗除草剂基因。所述抗虫基因包括来自苏云金芽胞杆菌(Bacillus thuringiensis)的cry1Ab、cry1Ac、cry1Ab/Ac、cry1C、cry2A和Vip3类抗虫基因、来自嗜虫沙雷氏菌(Serratia entomophila)的anf、sep基因，来自双酶梭菌(Clostridium bifermentans)的cmb基因、来自球形芽孢杆菌(Bacillus sphaericus)的mtx基因、来自嗜线虫致病杆菌(Xenorhabdus nematophilus)的杀虫蛋白基因、来自发光光杆状菌(Photorhabdus luminescens)的tca、tcb基因，以及来自金龟子绿僵菌(Metarhizium anisopliae)的prl基因，但不限于此。
根据本发明，所述Bt抗虫基因的核苷酸编码序列和氨基酸序列分别如SEQ ID NO：1和SEQ ID NO：2中所示。
根据本发明，所述抗除草剂基因包括抗草甘膦的EPSP合成酶基因、鼠伤寒沙门氏菌EPSP突变基因aroA、抗草丁膦的bar基因、抗米唑啉酮的ALS突变基因Ilv G、抗稀禾定的AccL-s2基因、抗溴苯腈的bxn基因和抗绿磺隆的csrl基因，但不限于此。
根据本发明，所述外源目的基因的表达受到本发明的锁定元件和钥匙基因的控制。在具体的实施方式中，所述外源基因与本发明的锁定元件可操作地连接。
如果需要，编码外源目的基因的序列也可操作地连接至合适的调控因子，包括启动子、增强子、终止子和信号肽等。
转基因植物
本发明提供了一种培育转基因植物的方法，所述方法包括将包含锁定元件的第一亲本与包含钥匙基因的第二亲本杂交，从而获得所述转基因植物。
在具体实施方式中，本发明所述的培育转基因植物方法还包括在所述杂交步骤之前，将所述锁定元件和与其操作链接的目的基因导入和整合到所述第一亲本基因组中，以及将所述钥匙基因导入和整合到所述第二亲本基因组中。
在可选的实施方式中，本发明提供了一种培育转基因植物的方法，所述方法包括将将锁定元件和钥匙基因导入和整合到同一植株的基因组中，从而获得所述转基因植物。
将基因导入受体植物的方法是本领域中已知的，包括例如农杆菌介导基因转化、基因枪转化、花粉管通道法等，其中，由农杆菌介导的基因转化在植物转化中得到了广泛的使用，具体步骤可参见后附的实施例。
在本发明的进一步考虑的实施方式中，可以通过整合的方式，将特定的外源基因，例如用于筛选的标记基因从转基因植物中分离和剔除。然而，这些剔除标记基因后的转基因植物仍包含在本发明所述的转基因植物的范畴之内。
本发明的优点
本发明提供了一种一种用于控制植物中的外源基因不在特定组织中表达的基因组合，以及应用该基因组合培育转基因植物的方法。以水稻和Bt抗虫基因为例，该方法可用于培育胚乳0表达型转基因抗螟虫的水稻，但这种抗螟虫水稻所生产的稻米胚乳中没有任何转基因产物蛋白的合成和积累。因此，可排除公众对转基因粮食作物食用安全性的担忧。
已有的试验结果显示，目的Bt抗虫基因在所获得的锁定元件和钥匙基因的日本晴异交种稻米胚乳中不表达，其Bt蛋白检测结果为0， 但在设计表达的茎、叶组织中则高效表达。由于稻米胚乳中检测不到目的基因表达产物，本发明人将其称之为胚乳0表达型转Bt基因抗虫水稻，以与传统的胚乳表达型转Bt基因抗虫水稻相区别。进一步的抗虫性鉴定结果表明，携带锁定元件和钥匙基因的日本晴异交种(30株)对自然爆发的稻纵卷叶螟的抗虫效果接近0危害，与非转基因对照杂种(30株)的100％受害株率相比呈极显著差异；其对人工接种的二化螟一龄幼虫(1个卵块的虫量/株)的抗虫效率也都达到抗至高抗水平。因此，本发明的基因组合和胚乳0表达型转基因抗虫水稻的成功选育将不仅有望突破传统的胚乳表达型转Bt基因水稻不被公众接受的僵局，同时，也将有力地推进转基因粮食作物的产业化进程。
本发明具有广适性，即适用于以果实、种子、块根、块茎为生产目的的广大作物，如水稻、小麦、大麦、燕麦、玉米、粟米、高粱、薏米、红薯、马铃薯、莲子、大豆和花生等等。本发明的这些和其他方面将参照以下附图和详细说明而得到更好的理解。
附图说明
图1：pSB130质粒载体图谱。该质粒载体有两个T-DNA区，其中，一个携带抗潮霉素的标记基因Hpt；另一个携带多克隆位点，用于钥匙基因或连接了目的基因的锁定元件序列的装载。
图2：根据本发明的一个实施方式的基因钥匙(Key1；SEQ ID NO：5)的表达载体pKey1构建的流程图，其中图2A显示：第一步，用引入KpnI和EcoRI识别位点的特异引物扩增获得Nos片段，并将其连入pSB130相应的多克隆位点上；和第二步，用引入XbaI/SmaI和KpnI识别位点的特异引物扩增获得Key1 5′片段，并将其连入pSB130相应的多克隆位点上；图2B显示：第三步，用引入SalI和SmaI识别位点的特异引物扩增获得Key1 3′片段，并将其连入pSB130相应的多克隆位点上；和最后一步，用引入HindIII和SalI识别位点的特异引物扩增获得RbcS片段，并将其连入pSB130相应的多克隆位点上，从而构建出pKey1表达载体。
图3：根据本发明的一个实施方式的锁定元件(Lock1；SEQ ID NO：8)及其相连接的报告基因(eYFP)的表达载体pLY1构建的流程图，其中图3A显示：第一步，Nos片段扩增和克隆同pKey1；和第二步，用引 入PstI/XbaI识别位点的特异引物扩增获得Lock1片段，并将其连入pSB130相应的多克隆位点上；图3B显示：第三步，用引入HindIII和PstI识别位点的特异引物扩增获得ActinI片段，并将其连入pSB130相应的多克隆位点上；和最后一步，用引入XbaI和KpnI识别位点的特异引物扩增获得RbcS片段，并将其连入pSB130相应的多克隆位点上，从而构建出pLY1表达载体。
图4：根据本发明的另一个实施方式的钥匙基因(Key2；SEQ ID NO：3)的表达载体pKey2构建的流程图，其中图4A显示：第一步，用引入SacI和EcoRI识别位点的特异引物扩增获得Nos片段，并将其连入pSB130相应的多克隆位点上；和第二步，用SalI和HindIII从pKey1表达载体上酶切分离获得RbcS片段，并将其连入pSB130相应的多克隆位点上；图4B显示：第三步，用引入SacI和XbaI识别位点的特异引物扩增获得Key2 3′片段，并将其连入pSB130相应的多克隆位点上；和最后一步，用引入SalI和XbaI识别位点的特异引物扩增获得Key2 5′片段，并将其连入pSB130相应的多克隆位点上，从而构建出pKey2表达载体。
图5：根据本发明的另一个实施方式的锁定元件(Lock2；SEQ ID NO：6)加报告基因(eYFP)的表达载体pLY2构建的流程图，其中图5A显示：第一步，用KpnI和EcoRI从pLY1酶切分离获得Nos片段，并将其连入pSB130相应的多克隆位点上；和第二步，用引入PstI和XbaI识别位点的特异引物获得Lock2片段，并将其连入pSB130相应的多克隆位点上；图5B显示：第三步，用HindIII和PstI从pLY1酶切分离获得ActinI片段，并将其连入pSB130相应的多克隆位点上；和最后一步，用XbaI和KpnI从pLY1酶切分离获得eYFP片段，并将其连入pSB130相应的多克隆位点上，从而构建出pLY2表达载体。
图6：根据本发明的一个实施方式的锁定元件(Lock1；SEQ ID NO：8)加目的基因(cry1Ab/1Ac)的表达载体pLB构建的流程图，其中图6A显示：第一步，用引入KpnI/SalI和EcoRI识别位点的特异引物扩增获得Nos片段，并将其连入pSB130相应的多克隆位点上；和第二步，用引入PstI和XbaI识别位点的特异引物扩增获得Lock片段，并将其连入pSB130相应的多克隆位点上；图6B显示：第三步，用引入HindIII和PstI识别位点的特异引物扩增获得ActinI片段，并将其连入pSB130 相应的多克隆位点上；和最后一步，用引入KpnI和SalI识别位点的特异引物扩增获得cry1Ab/1Ac片段，并将其连入pSB130相应的多克隆位点上，从而构建出pLB表达载体。
图7：根据本发明的一个实施方式的一组锁定元件和钥匙基因的功能验证的图示，其中，pLY1为单一的锁定元件转化获得的阳性抗性愈伤形成的绿色组织在共聚焦显微镜下的观察结果，暗场条件下未呈现荧光信号；pLY1+pKey1为锁定元件转化获得的阳性抗性愈伤形成的绿色组织再经钥匙基因转化和瞬时表达后在共聚焦显微镜下的观察结果，暗场条件下呈现荧光信号。
图8：根据本发明的另一个实施方式的一组锁定元件和钥匙基因功能验证的图示，其中pLY2为单一的锁定元件转化获得的阳性抗性愈伤形成的绿色组织在共聚焦显微镜下的观察结果，暗场条件下未呈现荧光信号；pLY2+pKey2为锁定元件转化获得的阳性抗性愈伤形成的绿色组织再经钥匙基因转化和瞬时表达后在共聚焦显微镜下的观察结果，暗场条件下呈现荧光信号。
图9：日本晴pKey1和pLB阳性转化系的拷贝数分析，其中：图9A为日本晴的21个pKey1阳性转化系拷贝数分析，结果显示其中的13个为单拷贝；图9B为日本晴的22个pLB阳性转化系拷贝数分析，结果显示其中的15个为单拷贝。根据结实率、其它产量性状、株高和生育期等重要农艺性状表现选择出的单拷贝转化系(pKey1和pLB各三个)在本申请中采用顺序号直接标注于样本泳道的上方。
图10：日本晴pLB阳性单拷贝转化系的1个T2代株系的纯合情况及其PCR检测结果，其中：M-DL2000代表DL2000DNA Marker；“+”代表阳性质粒对照，“-”代表非转基因阴性对照；1-24代表日本晴pLB阳性单拷贝转化系的1个T2代株系中24个独立的单株。
图11：日本晴3个pKey1和3个pLB单拷贝转基因系组配的9个异交种的抗虫性鉴定结果，其中：K11、K12、K13和LB1、LB2、LB3分别为pKey1和pLB转化体中筛选的单拷贝纯系亲本；K11LB1-K13LB3为上述单拷贝纯系亲本所组配的异交种。WT为野生型非转基因对照。
图12：日本晴3个pKey1和3个pLB单拷贝转基因系组配的9个异交种的茎、叶组织中Bt蛋白含量的ELISA定量分析结果，其中： K11、K12、K13和LB1、LB2、LB3分别为pKey1和pLB转化体中筛选的单拷贝纯系亲本；K11LB1-K13LB3为上述单拷贝纯系亲本所组配的异交种。T51-1为阳性对照。
图13：锁定元件和钥匙基因异交种K12LB2在大田条件下对人工接种的二化螟一龄幼虫(1个卵块约80头虫/株)的抗性表现。图13A为K12LB2(左侧)和非转基因日本晴对照(右侧)对分蘖期人工接种的二化螟一龄幼虫的抗性表现，图中，非转基因对照由于分蘖期人工接种的二化螟造成的枯心严重，以致抽穗后的株高大幅度降低，有效穗也明显减少；图13B为K12LB2(左侧)和非转基因日本晴对照(右侧)对抽穗前一星期人工接种的二化螟一龄幼虫的抗性表现，图中，可以看到非转基因对照上出现高比率的白穗，而转基因异交种上则没有出现白穗。
图14：K12LB2异交种离体叶片对室内人工接种的二化螟一龄幼虫的抗性表现，图中A：阳性对照TT51-1；B：非转基因明恢63对照；C：K12LB2异交种；D：非转基因日本晴对照；E：日本晴K12转化系；F：日本晴LB2转化系。图片显示转基因异交种K12LB2对二化螟的抗性表现与阳性对照TT51-1一致。
图15：K21LB2异交种离体茎秆对室内人工接种的二化螟一龄幼虫的抗性表现。CK：非转基因日本晴对照。图片显示转基因K12LB2异交种离体茎秆上没有观察到二化螟危害的迹象，接种的二化螟全部死亡；而对照茎秆上观察到二化螟危害的虫孔、啃噬槽及其排泄物，并有存活的幼虫
图16：日本晴3个pKey1和3个pLB单拷贝转基因系组配的9个异交种的糙米和精米胚乳中Bt蛋白含量的ELISA定量分析结果。图中，K11、K12、K13和LB1、LB2、LB3分别为pKey1和pLB转化体中筛选的单拷贝纯系亲本；K11LB1-K13LB3为上述单拷贝纯系亲本所组配的异交种。WT为野生型日本晴对照；MH63为阴性对照；T51-1和ZY3为阳性对照。
通过参考以下非限定性实施例可更好地理解本发明，提供所述非限定性实施例作为本发明的示例。介绍以下实施例以更充分地说明本发明的实施方式，且决不应解释为对本发明广泛范围的限制。
具体实施方式
实施例1：本发明的基因组合的设计及其可行性研究与验证
1.基因组合的设计
如前所述，完整的基因组合在设计上是由能锁定目的基因表达的锁定元件(Lock)和能组织特异开启锁定元件的钥匙基因(Key)两个组件组成。为了使用的方便，共设计了两套这样的组件，其中，第一套组件的DNA序列分别是SEQ ID NO：5(Lock1)和SEQ ID NO：3(Key1)，第二套组件的DNA序列是SEQ ID NO：8(Lock2)和SEQ ID NO：6(Key2)。作为锁定元件在设计上是被设置在ActinI等组成型表达启动子和目的基因起始密码子之间，其功能是阻止目的基因的表达：而作为钥匙基因在设计上则是被置于RbcS等绿色组织特异型表达启动子的控制之下，其功能是在特定组织(如绿色组织)里开启被锁定元件锁定的目的基因的表达。
为了验证上述设计是否具备预期的锁定和开启功能，我们先利用增强型黄色荧光蛋白基因eYFP对各个组件进行了测试。然后在获得肯定结果的前提下，我们再利用抗虫的Bt杂种蛋白基因cry1Ab/1Ac进一步对其开展了应用性研究。
2.用于锁定和开启功能验证的验证型表达载体构建
为了构建第一套锁定元件和钥匙基因的表达载体，利用引入了限制性核酸内切酶识别位点的特异引物(表1)和高保真PCR技术扩增所有功能片段，然后依设计顺序将这些功能片段分别连入pSB130质粒载体(图1)，从而获得相应的基因钥匙表达载体pSB130：：RbcS：：Key1：：NosT(简称pKey1)和锁定元件表达载体pSB130：：ActinI：：Lock1：：eYFP：：NosT(简称pLY1)，其具体构建流程分别示于图2和图3。
以构建成功的第一套表达载体作为基础，构建第二套表达载体，其中部分功能片段可以通过酶切方法从第一套表达载体中获得，另一部分功能片段则依然通过特异引物(表1)和高保真PCR扩增技术扩增出来，然后依设计顺序将这些功能片段连入pSB130质粒载体(图1)，从而获得相应的基因钥匙表达载体pSB130：：RbcS：：Key2：：NosT(简称pKey2)和锁定元件表达载体pSB130：：ActinI：：Lock2：：eYFP：：NosT(简称pLY2)，这两个表达载体的具体构建流程图分别示于图4和图5。
3.用于抗虫目的应用型表达载体构建
由于验证型和应用型表达载体在基因钥匙部分完全相同，而主要 差别仅在于与锁定元件所连接的目的基因片段的不同，因此，这套应用型表达载体的构建主要是针对锁定元件和目的基因片段进行。构建时，一方面通过酶切获得部分与验证型表达载体通用的功能片段，另一方面通过特异引物和高保真PCR扩增技术分别获得本申请实施例选用的第一套表达载体中的锁定元件以及待连接的抗虫基因两个功能片段，然后依设计顺序将这些功能片段连入pSB130质粒载体(图1)，便可获得相应的应用型锁定元件表达载体pSB130：：ActinI：：LB1：：NosT(简称pLB1)，该表达载体的具体构建流程图示于图6。
本申请实施例所用的pSB130质粒载体包含两个“T-DNA”区，其中一个“T-DNA”区用于装载连接目的基因的锁定元件或钥匙基因表达单位，另一个用于装载抗潮霉素标记基因Hpt表达单位。构建双T-DNA表达载体的目的在于着眼于今后的实际应用，因为双T-DNA质粒载体转化水稻后能使目的基因和标记基因在受体基因组中有机会发生独立整合，以便于标记基因能在随后的分离世代中通过自交方式分离和剔除。
Figure PCTCN2016072148-appb-000001
Figure PCTCN2016072148-appb-000002
实施例2：外源目的基因表达锁定与开启功能的验证
本发明的基因组合锁定与开启功能的验证采用两步转化法进行。第一步，先利用农杆菌介导法(Liu et al.，1998)将按照实施例1中制备的验证型表达载体中的两个锁定元件pLY1和pLY2导入水稻胚性愈伤组织进行永久表达，之后，经过连续多轮(3-5轮，12-14天/轮)抗生素筛选，抗性愈伤预分化(7-9天)和分化培养，直到形成绿点(7-9天)；第二步，再利用基因枪介导法(Tu et al.，2000)将验证型表达载体中的两个钥匙基因pKey1和pKey2轰入有绿点的阳性愈伤进行瞬时表达，24-36h后在共聚焦显微镜下观察绿点所在的阳性愈伤的发光情况，并以第一步中有绿点的pLY1或pLY2阳性转化系为对照，如果所设计的基因组合具有完备的锁定和开锁功能，则对照完全不发光，而经第二步基因枪转化的绿色阳性愈伤能够发光；反之，则否。详细试验步骤叙述如下：
1.受体材料
用于上述验证型锁定元件pLY1和pLY2遗传转化的受体品种为粳稻日本晴。该品种是水稻遗传转化的模式品种，其成熟胚愈伤组织诱导容易，遗传转化效率高。
2.水稻转化及生长所使用的培养基及组成成分
2.1.诱导/继代培养基(每升)：4.1g/L N6(Chu et al，1975)基础盐组分+N6有机组分(表2)+2mg/L 2，4-二氯苯氧乙酸(2，4-D)+2.0g/L水解酪蛋白+30g/L蔗糖+3g/L琼脂，pH 5.9
2.2.侵染培养基(每升)：AA基础培养基(表3)(Toriyama & Hinata，1985)+200μM乙酰丁香酮，pH 5.9。
2.3.共培养培养基(每升)：CC基础培养基(表4)(Hiei et al，1994)+200μM乙酰丁香酮，pH 5.9。
2.4.抑菌培养基(每升)：诱导/继代培养基+500mg/L头孢霉素，pH 5.9。
2.5.筛选培养基(每升)：诱导/继代培养基+50mg/L潮霉素+500mg/L头孢霉素，pH 5.9。
2.6.再生培养基(每升)：4.1g/L N6基础盐组分+N6有机组分(表2)+2.0g/L水解酪蛋白+30g/L蔗糖+6g/L琼脂+2mg/L激动素+1mg/Lα-萘乙酸，pH 5.9。
2.7.水稻生根培养基(每升)：2.05g/L N6基础盐组分+1/2的N6有机组分(表2)+1.0g/L水解酪蛋白+15g/L蔗糖+3g/L琼脂，pH 5.9。
表2：N6基础盐组分(上海生工)和有机组分
Figure PCTCN2016072148-appb-000003
表3：AA培养基配方
Figure PCTCN2016072148-appb-000004
表4：CC培养基配方
Figure PCTCN2016072148-appb-000005
3.锁定元件转化
如前所述，验证型锁定元件表达载体pLY1和pLY2的遗传转化采 用农杆菌介导法进行。所用的农杆菌菌株为EHA105(BioVector NTCC Inc.)，所用的受体细胞为日本晴成熟胚诱导的胚性愈伤组织。农杆菌转化的具体步骤如下：
3.1水稻胚性愈伤诱导
取成熟的日本晴种子若干，去壳后，挑选粒型饱满、胚完好的糙米放入预先高压灭菌的烧杯中，先用70％酒精浸泡1min，并不间断地振荡，以除去表面杂质；再用20％的次氯酸钠浸泡灭菌20min(可置于摇床上振荡)；再用无菌蒸馏水冲洗4～5次，以便稀释并除去糙米表面残留的次氯酸钠。将无菌处理过的糙米接种于含有2.0mg/L 2，4-D的MS愈伤诱导和继代培养基(表2)上，28℃暗培养2周左右，直至成熟胚的盾片处产生合适大小的初生愈伤。之后，切下初生愈伤，并转移到新的愈伤诱导和继代培养基上，在相同条件下继代培养，每2周继代一次，直至细胞质浓、颜色鲜黄、质地硬实和细胞团呈颗粒状的胚性愈伤形成为止。
3.2农杆菌EHA105菌株的遗传转化和摇菌培养
取0.5μl表达载体质粒加入到含有60μl农杆菌电击感受态EHA105的1.5ml离心管中，待枪头吸打混匀后移入电极杯中；电击后，迅速加入1ml的LB液体培养基，吸打混匀后移入先前的1.5ml离心管中，于28℃振荡仪摇上1h；菌液复苏后，吸取100μl菌液，均匀涂布于LB固体筛选(含50mg/l的卡那霉素、25mg/l的利福平)培养基(上海生工)表面，28℃培养2天；菌落PCR验证阳性克隆后，对阳性克隆进行摇菌培养，所得菌液于50％的甘油浓度和-80℃条件下保存备用。
3.3水稻愈伤的农杆菌侵染和筛选继代
农杆菌侵染按Yang et al(2011)报道的方法步骤进行。其具体程序是：取出保存于-80℃的农杆菌菌液，从中吸取200μl均匀涂布于到含有25mg/l利福平和50mg/l卡那霉素的LB固体培养基表面，于28℃条件下培养过夜；再从中挑单菌落用LB液体培养基扩大培养；之后，从中吸取200-300μl的新鲜菌液接入到20ml含有25mg/l利福平和50mg/l卡那霉素的LB液体培养基中，28℃振荡(220rpm)培养16-18h。取足量的菌液于4000rpm下离心15min之后，弃去LB培养基上清液；加入20ml 0.1M MgSO4溶液重新悬浮农杆菌(用移液枪轻轻吹打)，于4000rpm下离心10-15min，弃去含有抗生素的MgSO4上清液；再加入 5ml含有200μM乙酰丁香酮(Acetosyringone，AS)的AA侵染培养基(表3)重新悬浮农杆菌，再加入适量的AA-AS侵染培养基，使菌液的OD600值最终调整在0.8-1.0之间；浓度调整后，用无菌的50ml离心管分装菌液，20-25ml/管，待用。
农杆菌侵染前，先将胚性愈伤组织预培养7天左右，再将其从继代培养皿中转移至覆有无菌滤纸的空培养皿中，在超净工作台上风干10-15min左右，期间用灭菌过的小勺缓缓翻滚愈伤组织使之充分干燥；待其干燥后，移入盛有菌液的离心管，在室温下轻轻摇晃40min，将该离心管于超净工作台上静置10min；倒除菌液，将胚性愈伤组织置于无菌滤纸上干燥15min左右，然后，将其转移至表面以无菌滤纸覆盖的含有AS(200μM)的CC共培养培养基(表4)上，于28℃暗培养50-55h；挑选表面农杆菌未大量生长或未污染的胚性愈伤组织，移至含有2.0mg/L的2，4-D，500mg/l头孢霉素(Cefortaxim)的N6抑菌培养基上，28℃暗室中抑菌培养3-4d；再将抑菌培养后的愈伤组织移至含有500mg/l头孢霉素和50mg/L潮霉素(Hygromycin)的筛选培养基上，28℃暗室培养；每隔半月挑选生长状态良好的愈伤组织作继代，并根据污染的程度来调整培养中头孢霉素的浓度，一般情况下第三或第四轮继代的时候可考虑浓度减半。这样继代培养直至获得生长迅速、量大和颜色鲜活的抗性愈伤组织为止(4-6轮筛选和继代培养)。
3.4抗性愈伤组织分化培养
将上一步获得的抗性愈伤组织转移至N6再生培养基上，28℃暗室预分化一周，再移至新鲜的N6再生培养基上，于25℃光室进行分化培养，直至绿点形成(约需两周时间)为止。
4.基因钥匙转化
以上一步形成绿点的愈伤组织为受体，利用Tu et al 2000)描述的基因枪介导法将按照实施例1制备的验证型用钥匙基因表达载体pKey1和pKey2导入形成了绿点的愈伤组织进行瞬时表达，经24-36h小时恢复培养后，在共聚焦显微镜下观察转化和未转化(对照)的有绿点的愈伤组织的荧光信号，并拍照记录和保存。
5.实验结果
试验结果示于图7和图8。从图中可以看出，在共聚焦显微镜下，仅经锁定元件表达载体pLY1和pLY2转化获得的阳性抗性愈伤形成的 绿色组织未能观察到黄色荧光信号，而只有当该绿色组织再经过钥匙基因表达载体pKey1和pKey2复转化和瞬时表达后才能观察到黄色荧光信号。这些试验结果因此证实所设计的两套基因组合都能正常和有效地行驶锁定和开启功能。
实施例3：应用型锁定元件和钥匙基因转化系的获得及其异交种生产
应用型锁定元件和钥匙基因的遗传转化采用农杆菌介导法进行。除了使用按照实施例1制备的应用型锁定元件表达载体pLB1和基因钥匙表达载体pKey1之外，受体材料的选用、所使用的各种培养基、载体的遗传转化和阳性抗性愈伤的预分化和分化培养均与实施例2中所述相应的试验步骤相同，直至阳性抗性愈伤分化成绿苗。所获得的绿苗，洗净根系上的培养基后，或直接(根芽同时分化型)，或经前述N6生根培养基壮根后(芽先分化型)移入Yoshida培养液(表5)中过渡培养，待其生长状态良好与稳定后，再移栽到温室，直至成熟。
表5：Yoshida营养液配方(Yoshida，1976)
Figure PCTCN2016072148-appb-000006
经过上述步骤的遗传转化，先后获得pKey1独立转化体42份和pLB1独立转化体59份。之后，分别针对这两种独立转化体进行了纯系选育及其它们之间的异交种生产和抗虫性鉴定。具体如下文所述。
实施例4：应用型锁定元件和钥匙基因转化系的分子分析
1.DNA抽提
1.1DNA小样抽提
从按照实施例3获得的转基因植株上取下2-3cm长的新鲜叶片置于碾钵中，加入500ul的1.5×CTAB提取液，并碾磨至匀浆，转移匀浆至1.5ml离心管中，置于56℃水浴保温20min，之后加入500ul的氯仿∶异戊醇(24∶1)，并充分混匀(上下颠倒数次)，室温下离心5min(14,000r/min)后取300ul上清液，加入到600ul的的无水乙醇(-20℃预冷)中，混匀后于-20℃中放置30min以上，室温离心5min(14,000r/min)后弃去上清，用75％乙醇浸洗DNA沉淀，之后，弃去乙醇，室温风干DNA沉淀，最后用200ul无菌水溶解，待用。
1.2DNA大样抽提
取3-5片(3-5g)成株期水稻叶片，加入液氮迅速研磨至粉末状，转移至经预先冷冻的50ml离心管中，立即加入15-20ml煮沸预热的1.5XCTAB，摇匀后将其置入56℃水浴30min。然后加入一倍体积24∶1(三氯甲烷∶异戊醇)，轻柔旋摇30min至溶液分三层，上中下依次为黄色、绿色、黑色。常温(温度＞18℃)4000rpm离心20min取上清，加入1/10体积56℃预热的10X CTAB摇匀，再次加入一倍体积24∶1(三氯甲烷∶异戊醇)并轻柔旋摇30min。常温(温度＞18℃)4000rpm离心20min，之后，用5ml枪头吸出上清，再加入一倍体积1X CTAB，旋摇后会出现絮状DNA沉淀，利用枪头将其挑出置于1.5ml离心管管底，倒转于吸水纸上沥干10min。然后加入10μL RNase及0.5ml已灭菌的1M NaCl，室温或56℃水浴溶解。将溶解后的DNA加入2倍(-20℃)体积-20℃预冷的95％乙醇，旋摇使DNA絮状沉淀析出。利用枪头挑取絮状DNA放进1.5ml离心管中，加1.5ml 75％乙醇清洗DNA，然后倒去乙醇，加入超纯水于4℃保存备用。
2.阳性转基因植株的检测和纯系鉴定
阳性转基因植株的检测和纯系鉴定利用PCR技术进行。由于Hpt 标记基因和锁定元件或钥匙基因是通过双T-DNA共转化的，T0代阳性转化系的鉴定仅基于目的基因进行。因此检测后仅保留目的基因为阳性的植株继续栽培至成熟，非阳性的植株则全部清除，以减少后续世代的工作量。
所用的抗潮霉素基因扩增引物为：Hpt-F，5′-GCTGTTATGCGGCCATTGTC-3′(SEQ ID NO：9)和Hpt-R，5′-GACGTCTGTCGAGAAGTTTC-3′(SEQ ID NO：10)；PCR反应体系(25ul体系)为：待测样品DNA模板1ul，10×PCR buffer 2.5ul，10mM dNTP 2ul，20uM引物0.25ul，2u/ul Tag酶0.5ul，加ddH2O至25ul。PCR反应程序为：94℃变性3min；接着94℃变性30sec，55℃退火30sec，72℃延伸40sec，共35个循环；之后，经72℃延伸5min于10℃保持。扩增产物经1％琼脂糖凝胶电泳鉴定，拍照后保存。
所用的Bt基因扩增引物为：BtF，5′-GGCCATACAACTGCTTGAGT-3′(SEQ ID NO：11)；BtR，5′-GCGTTTCCCATAGTTCCATA-3′(SEQ ID NO：12)，扩增片段长度为1Kb。PCR反应程序为：94℃变性3min；接着94℃变性30sec，55℃退火30sec，72℃延伸1Min，共35个循环；之后，经72℃延伸5min于10℃保持。扩增产物经1％琼脂糖凝胶电泳鉴定，拍照后保存。PCR反应体系见表6。
所用的钥匙基因扩增引物为：KeyF，5′-AACGAGTGATGAGGTTCGCA-3′(SEQ ID NO：13)；KeyR，5′-ACCCGGCAAAACAGGTAGTT-3′(SEQ ID NO：14)，扩增片段长度为672bp。PCR反应程序为：94℃变性3min；接着94℃变性30sec，55℃退火30sec，72℃延伸1Min，共35个循环；之后，经72℃延伸5min于10℃保持。扩增产物经1％琼脂糖凝胶电泳鉴定，拍照后保存。PCR反应体系见表1。
表6：PCR反应体系
Figure PCTCN2016072148-appb-000007
这样，从日本晴的41份pKey1和50份pLB独立转化系中分别鉴定出21份pKey1和22份pLB阳性转化系。
3.转基因拷贝数检测
转基因拷贝数检测利用Southern杂交技术进行。首先，用限制性内切酶HindIII对水稻总DNA在37℃条件下酶切过夜(8-12h)；将酶切后的DNA通过0.8％(w/v)的琼脂糖凝胶电泳分离(40V，12h)，之后，将电泳分离的DNA印迹转移到尼龙膜(GE Healthcare，UK)上。杂交探针的制备和Southern杂交及杂交信号检测均按罗氏地高辛试剂盒[Roche DIG-High Prime DNA Labeling and Detection Starter Kit II(Switzerland)]提供的说明书进行，其中杂交探针制备引物分别为：
pKEY1：Key-1U20，5′-ATGTCCAATTTACTGACCGT-3′(SEQ ID NO：15)，和
       Key-800L20，5′-GCTTCAAAAATCCCTTCCAG-3′(SEQ ID NO：16)；
pLB：Bt-577U24，5′-AGGCTGATTGGAAACTACACCGAC-3′(SEQ ID NO：17)，和
     Bt-1161L24，5′-ACAGCGGATGGCAAGTTAGAAGAG-3′(SEQ ID NO：18)。
日本晴的pKey1和pLB阳性转化系的拷贝数分析结果示于图1。从图1可见，在日本晴的21份pKey1阳性转化系(9A)中，有13份是单拷贝的，而在22份pLB阳性转化系(图9B)中，有15份是单拷贝的。
4.单拷贝转基因纯系的PCR扩增检测与筛选
单拷贝转基因纯系的PCR扩增检测与筛选在T2代株系中进行。分别将各受体品种的pKEY1和pLB单拷贝转化系自交两代后获取T2代种子和植株，之后，经PCR扩增检测筛选出分别带有key1和LB的 纯系。图2是日本晴pLB单拷贝转化系的1个T2代株系的纯合情况及其PCR检测结果。如图10所示，该T2代株系的24个单株都显示阳性LB基因扩增片段，表明该T2代株系为转LB基因纯系。其它pLB和pKey1转基因纯系的PCR扩增检测均依此进行。
实施例5：应用型锁定元件和钥匙基因转化系异交种的抗虫性鉴定
1、材料种植
将经过实施例4中所述的PCR扩增检测筛选的且结实率和其它农艺性状好的日本晴pKey1单拷贝转基因纯系K11、K12和K13分别与pLB单拷贝转基因纯系LB1、LB2和LB3进行异交，获得拥有pKey1和pLB全套转化系的9个异交种。
2014年，含野生日本晴对照、3个pKey1和3个pLB转化系亲本以及由它们配置的9个异交种共16份供试材料于6月25日播种，15天后，利用佑隆Bt胶体金免疫检测试纸，按说产品明书中描述的步骤，对所有9个pKey1/PLB转化系异交种供试幼苗进行阳性检测，经检测确认的阳性杂种植株以及野生型对照和亲本材料幼苗，按2行/小区，15株/行，共3次重复，于7月28日移栽到转基因专用隔离网室泥池中，宽窄行种植，宽行40cm，窄行26.7cm，株距19.8cm。整个试验区周围种植2行非转基因水稻植株作为保护行。全生育期不施用杀虫剂，水肥管理同当地大田。
2015年，从上一年种植的9个pKey1/pLB转化系异交种中选择茎杆中Bt蛋白表达量最高的1个异交种重复进行抗虫性鉴定试验，并以野生型日本晴作对照。供试材料于5月25日播种，并经佑隆Bt胶体金免疫检测试纸检测后，阳性杂种植株和野生型对照秧苗按2行/小区，12株/行，共3次重复，于6月25日移栽到转基因专用隔离网室泥池中，插植规格和栽培管理同2014年。
2、田间抗虫性评价
pKey1/pLB转化系异交种对二化螟的田间抗虫性评价利用人工接虫法进行，人工接虫分两次在最高分蘖期和抽穗前1星期进行。虫卵购自江西神农科技公司，购回的虫卵置入长12cm和直径2.0cm、且底部置有湿润棉球的玻璃试管中，1个卵块/管，塞上棉塞，并在管口外面扎上黑布。虫卵孵化在26-28℃温度、80％湿度和16小时光照/8 小时黑暗条件下进行。孵化的一龄幼虫于孵化后6小时内连同试管接种到稻蔸基部，试管基部插入稻田土中，管口紧靠稻杆，然后打开黑布并拔除棉塞。接虫量为1个卵块/株，约80头虫。两周后，调查二化螟危害的枯心数或白穗数，同时，调查供试单株的总分蘖数或总有效穗数。
pKey1/pLB转化系异交种对稻纵卷叶螟的田间抗虫性评价利用自然接虫法进行。稻纵卷叶螟危害的单株受害叶片数和叶片受害程度调查在危害高峰出现后的3-5天进行。
pKey1/pLB转化系异交种对二化螟和稻纵卷叶螟的田间抗性评价指标的统计分析用SPSS12.0(SPSS，Chicago，USA)软件完成，LSD法的t测验被用来检验pKey1/pLB转化系异交种的枯心数或白穗数与野生型对照差异的显著性。
2014年自然发虫(封闭网室盆栽时发生卷叶螟危害)和人工接虫鉴定结果表明：这9个异交种对稻纵卷叶螟具有高效的抗性(图11)；其中的3个异交种K11LB1、K11LB2和K11LB3对人工接虫的二化螟(1个卵块/株)也具有较好的抗性(图11)。进一步对这9个异交种的叶片和茎杆的Bt蛋白含量于分蘖期、抽穗期和灌浆期进行ELIZA定量分析发现，9个异交种中有3个(K11LB3、K12LB3和K13LB3)叶片中的Bt蛋白含量在三个不同的生育时期明显低于阳性对照，其余6个组合叶片中的Bt蛋白含量接近或高于阳性对照(图12)；同样地，在茎杆中这3个异交种茎杆中的Bt蛋白含量也是9个异交种中最低的，最高的K12LB1和K12LB2两个异交种的Bt蛋白含量则达到阳性对照的40-50％(图12)。
2015年选用茎杆中Bt蛋白含量最高的K12LB2异交种重复进行了大田人工接虫鉴定，结果示于表7。从表7可以看出，分蘖期接种的二化螟在K12L2异交种上为害造成的枯心率不足1％，极显著低于非转基因对照的31.9-45.6％；而抽穗前一星期接种的二化螟在K12LB2异交种上为害造成的白穗率也不足2％，极显著低于非转基因对照的43.1-54.6％。图13所示是K12LB2异交种与对照对人工接种的二化螟的抗虫性比较照片，其差异十分明显。
表7：K12LB2异交种在大田条件下对分蘖期和抽穗前7天人工接种的二化螟的抗性表现
Figure PCTCN2016072148-appb-000008
3、室内抗虫性评价
室内抗虫性评价是针对二化螟进行，虫卵同样来商自江西神农科技公司，虫卵孵化方法同前。抗虫性评价采用的方法有茎杆法和叶片法。
茎杆法是取拔节后的水稻茎杆，切成12cm长带节和叶鞘的茎段，每个养虫管放入同一单株的2个茎段和20头一龄三化螟幼虫。养虫管用棉塞封口后，置于27℃和80％的相对湿度下饲养，6天后统计幼虫的死亡率。
叶片法是取孕穗期的剑叶和倒二叶叶片，切成8cm长的叶段，置入垫有用2ml蒸馏水湿润的滤纸的培养皿中，每个培养皿放4段叶段和15头一龄三化螟幼虫，并用PARAFILM缠4次封口，以防幼虫钻出。之后，置于室温下(25℃)饲养，6天后统计幼虫的死亡率。
每个养虫管或培养皿统计为1次重复，每样本设置3次重复。不同Key/Lock转化系异交种对二化螟的抗性用成组茎秆和叶片数据的t测验进行比较。结果表明：在室内人工接种条件下，K12LB2异交种的离体叶片和茎秆对二化螟的抗虫表现也十分优异(图14和15)，其中，K12LB2异交种离体叶片喂养的二化螟幼虫死亡率达100％，它的离体茎秆喂养的二化螟幼虫死亡率也达到95％以上，而对照的离体叶片和茎秆喂养的二化螟幼虫死亡率则都不超过20％(表8)。
表8：K12LB2异交种离体叶片和茎秆对人工接种的二化螟的抗性表现
Figure PCTCN2016072148-appb-000009
实施例6：应用型锁定元件和钥匙基因转化系异交种茎杆、叶片和胚乳中Bt蛋白含量的测定
为了验证基因组合是否具有胚乳特异性关闭目的基因的功能，本发明人对实施例5中所述的9个钥匙基因锁定基因异交种的糙米和精米进一步进行了Bt蛋白含量测定.
1.ELISA试剂盒和水稻样品
日本晴pKey1/pLB转化系异交种茎秆、叶片和胚乳中Bt蛋白含量用一龙公司(EnviroLogixTM Inc.，Portland，USA)酶联免疫吸附(ELISA)试剂盒：QualiPlateTM Kit for Cry1Ab/1Ac(Catalog Number AP003 CRBS)测定。水稻检测的时期是分蘖期、抽穗期和灌浆期，取样的组织有叶片(分蘖期、抽穗期和灌浆期)、茎秆(分蘖期、抽穗期和灌浆期)和胚乳(灌浆期)。
2.Bt蛋白含量测定工作液和样品的制备
测定工作液包括抽提液/稀释液和洗脱液。抽提液/稀释液是将试剂盒提供的原液(35ml)用双蒸水稀释至175ml而成。洗脱液是将试剂盒附带的磷酸盐加入1L双蒸水和5ml吐温-20，然后搅拌使之完全溶解制成。两种工作液都在4℃保存，使用前预热至室温。
每个异交种设3个重复，1株/重复，每株取约20mg的新鲜组织，用500μl的抽提/稀释液研磨成匀浆。将匀浆装入1.5ml的离心管中静置30min，然后吸取上清夜，4000rpm离心3min，用抽提/稀释液稀释一定的倍数后测定其中的Bt蛋白浓度。各个时期和组织样品的稀释倍数见表9。
表9：Bt蛋白样品的稀释倍数
Figure PCTCN2016072148-appb-000010
3.ELISA反应和蛋白含量计算
ELISA反应过程参照试剂盒的说明书进行，具体步骤如下：
(1)加50μl空白样品、Cry1Ab/1Ac标准样品和待测样品到96孔板的孔中，1个样品/孔，然后用封口胶将96孔板密封后低速(200rpm)振荡15min；
(2)加50μlCry1Ab/1Ac酶标抗体到上述加样的96孔板的每一孔中，加样的顺序与上一步相同，同样于密封后低速(200rpm)振荡1-2h；
(3)倒去上清夜，向96孔板的每个孔中加入300μl洗脱液，剧烈振荡1min，倒去洗脱液，倒扣在吸水纸上将残余液体沥干；
(4)将步骤3重复两次；
(5)向96孔板的每个孔中加入100μl底物，含有Bt蛋白的样品对应的孔中将呈现蓝色，将各个孔密封后避光，低速振荡(200rpm)30min；
(6)向96孔板的每个孔中加入100μl反应终止液，将蓝色转换成黄色，立即读取吸光度值。
吸光度值用MK3型酶标仪(Thermo Labsystems Co.，Shanghai，China)测定，在450nm波长下读取。用标准样品的浓度和吸光度绘制标准曲线，在标准曲线上读出测试样品的浓度，再根据稀释倍数计算出水稻组织中的蛋白含量。蛋白含量的计算公式为：Bt蛋白含量(μg/g鲜重)＝测试样品浓度(μg/g)×稀释倍数×抽提液重量(g)/组织鲜重(mg)。蛋白浓度在不同异交种、不同时期和组织之间的差异用成组数据的t测验进行比较。
结果显示9个异交种的Bt蛋白含量检测值都与阴性对照日本晴、明恢63以及pKey1和pLB转基因亲本的检测值无明显差异(图16)，该结果因此初步证实本发明人设计的基因组合确实可以完全关闭目的基因在稻米胚乳中的表达，且符合最初的设计目标要求。
序列表和注释
本发明提供了用于构建基因组合的外源目的基因Bt、锁定元件Lock1/Lock2和钥匙基因Key1/Key2的核苷酸序列及其推导的氨基酸序列，其中，Bt基因cry1Ab/cry1Ac由中国农科院范云六院士人工合成并提供使用；锁定元件Lock1/Lock2和钥匙基因Key1/Key2为人工合成。序列表中所列的各序列具体如下。
Figure PCTCN2016072148-appb-000011
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本发明不限于本文所述的具体实施方式的范围。实际上，除本文所述那些之外的本发明的各种变式对于参阅上述说明书的本领域技术人员将是显而易见的。这些变式应落入后附权利要求书的范围之内。
还应理解，对于核酸或多肽所给出的所有的碱基大小或氨基酸大小，和所有的分子量或分子质量数值都是近似值，并且提供以用于说明。

Claims (25)

  1. 一种用于控制植物中的外源目的基因的表达的基因组合，其由锁定元件和钥匙基因组成，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
  2. 权利要求1的基因组合，其中所述锁定元件位于组成型启动子和所述外源目的基因之间，并与所述组成型启动子和所述外源目的基因可操作连接。
  3. 权利要求2的基因组合，其中所述组成型启动子选自花椰菜花叶病毒35S启动子、来自根癌农杆菌Ti质粒T-DNA区域的胭脂碱合成酶基因Ocs启动子、水稻肌动蛋白启动子Actin I和玉米泛素基因启动子Ubi；优选地，所述组成型启动子是水稻肌动蛋白启动子Actin I。
  4. 权利要求1或2的基因组合，其中所述钥匙基因与组织特异性启动子可操作连接。
  5. 权利要求4的基因组合，其中所述组织特异性启动子选自水稻绿色组织特异性启动子核酮糖-1，5-二磷酸羧化酶小亚基rbcS启动子、玉米磷酸烯醇式丙酮酸羧化酶PEPC启动子、水稻绿色组织特异表达DX1启动子、水稻光系统II10kDa多肽D540启动子、水稻富亮氨酸重复序列类受体蛋白激酶LP2启动子，以及玉米叶绿体C4 Pdk启动子；优选地，所述组织特异性启动子是水稻绿色组织特异性启动子rbcS启动子。
  6. 权利要求1-5中任一项所述的基因组合，其中所述外源目的基因是抗虫基因或抗除草剂基因，其中所述抗虫基因选自来自自苏云金芽胞杆菌(Bacillus thuringiensis)的cry1Ab、cry1Ac、cry1Ab/Ac、cry1C、cry2A和Vip3抗虫基因、来自嗜虫沙雷氏菌(Serratia entomophila)的anf、sep基因，来自双酶梭菌(Clostridium bifermentans)的cmb基因、来自球形芽孢杆菌(Bacillus sphaericus)的mtx基因、来自嗜线虫致病杆 菌(Xenorhabdus nematophilus)的杀虫蛋白基因、来自发光光杆状菌(Photorhabdus luminescens)的tca、tcb基因，以及来自金龟子绿僵菌(Metarhizium anisopliae)的prl基因；和/或所述抗除草剂基因选自抗草甘膦的EPSP合成酶基因、鼠伤寒沙门氏菌EPSP突变基因aroA、抗草丁膦的bar基因、抗米唑啉酮的ALS突变基因Ilv G、抗稀禾定的AccL-s2基因、抗溴苯腈的bxn基因和抗绿磺隆的csrl基因。
  7. 权利要求1-6中任一项所述的基因组合，其中所述植物选自水稻、小麦、大麦、燕麦、玉米、粟米、高粱、薏米、红薯、马铃薯、莲子、大豆和花生；优选地，所述植物是水稻。
  8. 一种用于控制植物中的外源目的基因的表达的方法，所述方法包括将锁定元件和钥匙基因导入受体植物中，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
  9. 一种培育转基因植物的方法，所述方法包括将包含锁定元件的第一亲本与包含钥匙基因的第二亲本进行杂交，从而获得包含所述锁定元件和钥匙基因的转基因植物，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
  10. 权利要求9的方法，其还包括在所述杂交步骤之前，将所述锁定元件导入和整合到所述第一亲本的基因组中，以及将所述钥匙基因导入和整合到所述第二亲本的基因组中。
  11. 一种培育转基因植物的方法，所述方法包括将将锁定元件和钥匙基因导入和整合到同一植株的基因组中，从而获得所述转基因植物，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含 与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
  12. 权利要求8-11中任一项的方法，其中使所述锁定元件位于组成型启动子和所述外源目的基因之间，并与所述组成型启动子和所述外源目的基因可操作连接。
  13. 权利要求12的方法，其中所述组成型启动子选自花椰菜花叶病毒35S启动子、来自根癌农杆菌Ti质粒T-DNA区域的胭脂碱合成酶基因Ocs启动子、水稻肌动蛋白启动子Actin I、玉米泛素基因启动子Ubi；优选地，所述组成型启动子是水稻肌动蛋白启动子Actin I。
  14. 权利要求8-12中任一项的方法，其中使所述钥匙基因与组织特异性启动子可操作连接。
  15. 权利要求14的方法，其中所述组织特异性启动子选自水稻绿色组织特异性启动子核酮糖-1，5-二磷酸羧化酶小亚基rbcS启动子、玉米磷酸烯醇式丙酮酸羧化酶PEPC启动子、水稻绿色组织特异表达DX1启动子、水稻光系统II10kDa多肽D540启动子、水稻富亮氨酸重复序列类受体蛋白激酶LP2启动子，以及玉米叶绿体C4 Pdk启动子；优选地，所述组织特异性启动子是水稻绿色组织特异性启动子rbcS启动子。
  16. 权利要求8-15中任一项所述的方法，其中所述外源目的基因是抗虫基因或抗除草剂基因，其中所述抗虫基因选自来自苏云金芽胞杆菌(Bacillus thuringiensis)的cry1Ab、cry1Ac、cry1Ab/Ac、cry1C、cry2A和Vip3抗虫基因、来自嗜虫沙雷氏菌(Serratia entomophila)的anf、sep基因，来自双酶梭菌(Clostridium bifermentans)的cmb基因、来自球形芽孢杆菌(Bacillus sphaericus)的mtx基因、来自嗜线虫致病杆菌(Xenorhabdus nematophilus)的杀虫蛋白基因、来自发光光杆状菌(Photorhabdus luminescens)的tca、tcb基因，以及来自金龟子绿僵菌(Metarhizium anisopliae)的prl基因；和/或所述抗除草剂基因选自抗草甘膦的EPSP合成酶基因、鼠伤寒沙门氏菌EPSP突变基因aroA、抗草丁膦的bar基因、抗米唑啉酮的ALS突变基因Ilv G、抗稀禾定的AccL-s2基因、抗溴苯腈的bxn基因和抗绿磺隆的csrl基因。
  17. 权利要求8-16中任一项所述的方法，其中所述植物选自水稻、小麦、大麦、燕麦、玉米、粟米、高粱、薏米、红薯、马铃薯、莲子、大豆和花生；优选地，所述植物是水稻。
  18. 锁定元件和钥匙基因的基因组合用于调控植物中的外源目的基因的表达的用途，其中所述锁定元件能够锁定与其可操作连接的外源目的基因的表达；所述钥匙基因能够解除所述锁定元件对于外源目的基因的表达锁定。
  19. 如权利要求18所述的用途，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
  20. 如权利要求18或19所述的用途，其中使所述锁定元件位于组成型启动子和所述外源目的基因之间，并与所述组成型启动子和所述外源目的基因可操作连接。
  21. 权利要求20的用途，其中所述组成型启动子选自花椰菜花叶病毒35S启动子、来自根癌农杆菌Ti质粒T-DNA区域的胭脂碱合成酶基因Ocs启动子、水稻肌动蛋白启动子Actin I、玉米泛素基因启动子Ubi；优选地，所述组成型启动子是水稻肌动蛋白启动子Actin I。
  22. 权利要求18-21中任一项的用途，其中使所述钥匙基因与组织特异性启动子可操作连接。
  23. 权利要求22的用途，其中所述组织特异性启动子选自水稻绿色组织特异性启动子核酮糖-1，5-二磷酸羧化酶小亚基rbcS启动子、玉米磷酸烯醇式丙酮酸羧化酶PEPC启动子、水稻绿色组织特异表达DX1启动子、水稻光系统II10kDa多肽D540启动子、水稻富亮氨酸重复序列类受体蛋白激酶LP2启动子，以及玉米叶绿体C4 Pdk启动子；优选地，所述组织特异性启动子是水稻绿色组织特异性启动子rbcS启动子。
  24. 权利要求18-23中任一项所述的用途，其中所述外源目的基因是抗虫基因或抗除草剂基因，其中所述抗虫基因选自来自苏云金芽胞杆菌(Bacillus thuringiensis)的cry1Ab、cry1Ac、cry1Ab/Ac、cry1C、cry2A 和Vip3抗虫基因、来自嗜虫沙雷氏菌(Serratia entomophila)的anf、sep基因，来自双酶梭菌(Clostridium bifermentans)的cmb基因、来自球形芽孢杆菌(Bacillus sphaericus)的mtx基因、来自嗜线虫致病杆菌(Xenorhabdus nematophilus)的杀虫蛋白基因、来自发光光杆状菌(Photorhabdus luminescens)的tca、tcb基因，以及来自金龟子绿僵菌(Metarhizium anisopliae)的prl基因；和/或所述抗除草剂基因选自抗草甘膦的EPSP合成酶基因、鼠伤寒沙门氏菌EPSP突变基因aroA、抗草丁膦的bar基因、抗米唑啉酮的ALS突变基因Ilv G、抗稀禾定的AccL-s2基因、抗溴苯腈的bxn基因和抗绿磺隆的csrl基因。
  25. 权利要求18-24中任一项所述的用途，其中所述植物选自水稻、小麦、大麦、燕麦、玉米、粟米、高粱、薏米、红薯、马铃薯、莲子、大豆和花生；优选地，所述植物是水稻。
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