CN102453717B - Plant bidirectional promoter BIGDB3 - Google Patents

Plant bidirectional promoter BIGDB3 Download PDF

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CN102453717B
CN102453717B CN 201010521533 CN201010521533A CN102453717B CN 102453717 B CN102453717 B CN 102453717B CN 201010521533 CN201010521533 CN 201010521533 CN 201010521533 A CN201010521533 A CN 201010521533A CN 102453717 B CN102453717 B CN 102453717B
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promoter
vector
plant
pm0a34
bidirectional promoter
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CN102453717A (en
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谢旗
夏然
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中国科学院遗传与发育生物学研究所
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Abstract

本发明公开了一种植物双向启动子BIGDB3。 The present invention discloses a plant bidirectional promoter BIGDB3. 该启动子的核苷酸序列是序列表中序列1所示的核苷酸序列。 The nucleotide sequence of the promoter is a nucleotide sequence shown in the Sequence Listing. 实验证明,将本发明的BIGDB1插入载体pMOA34-G/L中的GUS和LUC之间后,BIGDB1能双向启动GUS和LUC,说明BIGDB1具有双向启动功能。 After the experiments show that the present invention is inserted between BIGDB1 vector pMOA34-G / L in the GUS and LUC, BIGDB1 the LUC and GUS can bidirectional promoter, described BIGDB1 having bidirectional promoter function.

Description

—种植物双向启动子BIGDB3 - species bidirectional promoter BIGDB3

技术领域 FIELD

[0001] 本发明涉及一种植物双向启动子BI⑶B3的克隆与应用。 [0001] The present invention relates to a plant promoter BI⑶B3 bidirectional cloning and application startup.

背景技术 Background technique

[0002] 通过导入外源基因使植物获得新的性状并能稳定遗传是植物基因工程的最终目的。 [0002] The plants obtained by introduction of the exogenous gene a new genetic trait is stable, and the ultimate goal of plant genetic engineering. 花椰菜病毒CaMV 35S启动子作为稳定、高效的外源启动子,一直被人们广泛的使用。 Cauliflower virus CaMV 35S launch, has been widely used as a sub-stable and efficient people exogenous promoter. 然而在转基因过程中由于重复序列的出现会引起基因沉默现象,尤其是进行多个基因导入时,这种现象会更严重,从而严重影响了转基因植株的获得和后代的稳定性。 However, in the course of transgene sequences by repetitive cause gene silencing phenomenon, especially when a plurality of genes is introduced, this phenomenon will be more serious, and thus affect the stability of transgenic plants and progeny. 而在进行两个或两个以上基因导入时,通常需要这些基因的表达量、表达时间和位置的同一性,进而确保转入基因行使正常的功能,使用35S启动子很难达到这样的要求。 And two or more conducting gene introduction, generally require the expression of these genes, the expression of the identity and location of the time, thereby ensuring the normal exercise of transgene function, the 35S promoter is very difficult to achieve such a request. 这些都是植物基因工程中亟待解决的问题。 These are the problems to be solved in plant genetic engineering.

[0003] 对于转基因引起的基因沉默现象人们已经有了比较深入的认识,引起转基因沉默有多种因素,其中最主要的就是重复序列的产生,尤其是多个基因导入时,更人为的造成了多个35S启动子插入的情况,对此至今尚无有效的解决方法。 [0003] For gene silencing transgenic caused people to have more in-depth understanding of transgene silencing caused by a variety of factors, the most important is to generate repeat sequences, especially when more genes into more man-caused a plurality of insertion of the 35S promoter, which has no effective solution. 随着基因组学的发展,多种生物基因组测序已完成,经过生物信息学分析人们发现了双向启动子的存在,这为我们利用植物自身的双向启动子进行转基因操作,从而避免多基因插入引起的基因沉默现象提供了可能。 With the development of genomics, a variety of biological genome sequencing has been completed, through bioinformatic analysis it was discovered that the presence of bidirectional promoter, which transgenic operation with the plant's own bidirectional promoter for us, in order to avoid multiple gene insertion caused gene silencing possible.

发明内容 SUMMARY

[0004] 本发明的目的在于提供一种DNA片段,来源于拟南芥。 [0004] The object of the present invention is to provide a DNA fragment derived from Arabidopsis thaliana.

[0005] 本发明提供的DNA片段的核苷酸序列是如下I)或2)或3): [0005] The nucleotide sequence of the DNA fragment of the invention is provided as follows I) or 2) or 3):

[0006] I)序列表中序列I所示的核苷酸序列; [0006] I) a nucleotide sequence I shown in the Sequence Listing;

[0007] 2)在高严谨条件下与所述I)的核苷酸序列杂交且具有启动子活性的核苷酸序列; [0007] 2) under highly stringent conditions to the nucleotide sequence hybridizes I) and a nucleotide sequence having promoter activity;

[0008] 3)与所述I)的核苷酸序列具有65%以上的同源性且具有启动子活性的核苷酸序列。 [0008] 3) the nucleotide sequence I) having 65% or more homology to the nucleotide sequence and having a promoter activity of the promoter.

[0009] 序列表中的序列I由528个脱氧核糖核苷酸组成,是拟南芥基因AT5G67500和AT5G67490的基因间序列。 [0009] Sequence Listing sequence I by the 528 deoxyribonucleotides, inter Arabidopsis AT5G67500 and AT5G67490 gene sequences.

[0010] 上述高严谨条件是指,将杂交膜置于预杂交液(0.25mol/L磷酸钠缓冲液,pH7.2,7% SDS)中,65°C预杂交30min ;弃预杂交液,加入杂交液(0.25mol/L磷酸钠缓冲液,pH7.2,7% SDS,同位素标记的核苷酸片段),65°C杂交12hr ;弃杂交液,加入洗膜液I (20mmol/L磷酸钠缓冲液,ρΗ7.2,5% SDS),65°C洗膜2次,每次30min ;加入洗膜液II (20mmol/L 磷酸钠缓冲液,ρΗ7.2,1% SDS),65°C洗膜30min。 [0010] The highly stringent conditions refers to hybridization membranes placed in prehybridization solution (0.25mol / L sodium phosphate buffer, pH7.2,7% SDS) in, 65 ° C for 30 min Prehybridization; prehybridization solution was discarded, added to the hybridization solution (0.25mol / L sodium phosphate buffer, pH7.2,7% SDS, isotopically labeled nucleotide fragment), 65 ° C hybridization 12hr; disposable hybridization solution was added (20mmol / L phosphoric acid solution the membrane was washed I sodium buffer, ρΗ7.2,5% SDS), 65 ° C the membrane was washed twice, each time for 30 min; was added to wash the membrane II (20mmol / L sodium phosphate buffer, ρΗ7.2,1% SDS), 65 ° C membrane was washed 30min.

[0011] 本发明的DNA 片段(启动子活性片段)还包括与来自序列表中序列I的片段的核苷酸序列互补的核苷酸序列。 [0011] DNA fragments (promoter active fragments) of the present invention further comprises a nucleotide sequence derived from the nucleotide sequence table I fragment complementary to the sequence of SEQ. 这里使用的术语“互补的”系指遵循碱基配对原则产生的之 The term used herein "complementary" refers to base-pairing rules follow the produced

O[0012] 本领域普通技术人员可以很容易地采用已知的方法,例如定向进化和点突变的方法,对本发明的调控片段的核苷酸序列进行突变。 O [0012] ordinary skill in the art can readily using known methods, for example, directed evolution methods and point mutations, regulatory nucleotide sequence fragment of the invention is mutated. 那些经过人工修饰的,具有与本发明分离得到的调控片段的核苷酸序列70%或者更高同源性的核苷酸,只要保持了表达靶基因的启动子活性,均是衍生于本发明的核苷酸序列并且等同于本发明的序列。 Those artificially modified, regulation fragment having a nucleotide sequence of the present invention the isolated polynucleotide of 70% or more homology, as long as the promoter activity to maintain the target gene expression, are derived from nuclear invention identical to the nucleotide sequence and the sequence of the invention.

[0013] 这里使用的术语“同源性”指与天然核酸序列的序列相似性。 [0013] The term used herein "homology" refers to sequence similarity to the native nucleic acid sequence. “同源性”包括与本发明的调控片段的核苷酸序列具有优选地75%或更高,更优选地85%或更高,甚至更优选地90%或更高,以及最优选地95%或更高同一性的核苷酸序列。 "Homology" regulatory nucleotide sequence comprises a fragment of the invention preferably has a 75% or higher, more preferably 85% or higher, even more preferably 90% or higher, and most preferably 95 % or more identical nucleotide sequences. 同源性可以用肉眼或计算机软件进行评价。 Homology may be evaluated visually or by computer software. 使用计算机软件,两个或多个序列之间的同源性可以用百分比(%)表示,其可以用来评价相关序列之间的同源性。 Using computer software, the homology between two or more sequences may be expressed as a percentage (%), which can be used to evaluate the correlation between the homology sequences.

[0014] 含有上述的DNA片段的重组载体也属于本发明的保护范围之内。 [0014] The recombinant vector containing the DNA fragment is also within the scope of the present invention.

[0015] 上述重组载体是上述的DNA片段插入载体pM0A34-G/L的多克隆位点制成的重组载体; [0015] The recombinant vector is a recombinant vector formed pM0A34-G / L of the multiple cloning site of the DNA fragment into a vector;

[0016] 上述载体pM0A34-G/L具体可以是将⑶S基因和LUC基因分别插入pM0A34载体的多克隆位点得到的载体。 [0016] The vector pM0A34-G / L may be a specific gene ⑶S and LUC genes were inserted into the vector multiple cloning site pM0A34 resulting vector.

[0017] 含有上述的DNA片段的转基因细胞系也属于本发明的保护范围之内。 [0017] transgenic cell line containing the DNA fragment is also within the scope of the present invention.

[0018] 含有上述的DNA片段的重组菌也属于本发明的保护范围之内。 [0018] The recombinant bacteria containing the above DNA fragment is also within the scope of the present invention.

[0019] 上述的DNA片段在作为启动子中的应用也属于本发明的保护范围之内。 [0019] In the above-mentioned DNA fragment as a promoter is also within the scope of application of the present invention.

[0020] 进一步讲,上述启动子是双向启动子。 [0020] Further, the above-mentioned promoter is a bidirectional promoter.

[0021] 上述应用具体可以是上述的DNA片段在驱动目的基因在植物体内表达中的应用。 [0021] The application may be an application specific DNA fragment in the above-described drive the gene expression in plants.

[0022] 上述植物为双子叶植物或单子叶植物,如拟南芥或烟草。 [0022] The plant is a dicot or a monocot, such as Arabidopsis or tobacco.

[0023] 实验证明,将本发明的BI⑶BI插入载体PM0A34-G/L中的⑶S和LUC之间后,BI⑶BI能双向启动⑶S和LUC,说明BI⑶BI具有双向启动功能。 [0023] The experiments show that the present invention is inserted between BI⑶BI vector PM0A34-G / L in the rear ⑶S and LUC, BI⑶BI ⑶S bidirectional promoter and can LUC, having described BI⑶BI bidirectional promoter function.

附图说明 BRIEF DESCRIPTION

[0024] 图1为pM0A34-G/L-BI⑶B3植物双元载体图谱。 [0024] FIG. 1 is a pM0A34-G / L-BI⑶B3 plant binary vector map.

[0025] 图2为转基因拟南芥GUS染色结果,A是刚萌发的种子,B是去掉种皮的刚萌发的种子,C是胚芽,D是子叶展开的幼苗,E是三片真叶的幼苗,F是花序。 [0025] FIG. 2 is a transgenic Arabidopsis GUS staining, A is just germinating seeds, B to remove the seed coat is just germinating seeds, C is the germ, D is cotyledons of seedlings, E three true leaves seedlings, F is the inflorescence.

[0026] 图3为转基因拟南芥LUC分析结果,A、B、C是不同的转基因株系。 [0026] FIG. 3 is a result of analysis of transgenic Arabidopsis LUC, A, B, C are different transgenic lines.

具体实施方式 Detailed ways

[0027] 下面结合具体实施例对本发明作进一步说明,但本发明并不限于以下实施例。 [0027] The following embodiments in conjunction with specific embodiments of the present invention is further illustrated, but the present invention is not limited to the following embodiments.

[0028] 下述实施例中,如无特殊说明,均为常规方法。 [0028] In the following examples, Unless otherwise specified, all conventional methods.

[0029] 实施例1、启动子的获得及其检测 [0029] Example 1, to obtain the promoter and detection

[0030] 一、启动子的获得 [0030] First, the promoters get

[0031] 根据生物信息学分析的结果,拟南芥基因组中存在一些潜在的双向启动子序列。 [0031] According to the results of the bioinformatics analysis, Arabidopsis genome some potential bidirectional promoter sequence. 以拟南芥基因组DNA为模板,利用正向引物(BI⑶B3-F:5/ -GAGCTCTAGAGCGGCCGCTGTTAGATCAGATGTGTCGGCC-3')和反向引物(BIO)BS-Rj' -ACGCGTCGACGGTTGATCGGAGATTGAGAGAGATC-3')扩增,扩增产物进行琼脂糖电泳分离,回收并连接到pEASY-Tl (全式金公司)克隆载体上,转化大肠杆菌XL1-BLUE (Agilent Technologies-Stratagene Products)中,经测序后确定克隆载体正确,扩增产物(命名为BIGDB3)的核苷酸序列如序列表中序列I所示。 Arabidopsis thaliana genomic DNA as a template, the forward primer (BI⑶B3-F: 5 / -GAGCTCTAGAGCGGCCGCTGTTAGATCAGATGTGTCGGCC-3 ') and reverse primer (BIO) BS-Rj' -ACGCGTCGACGGTTGATCGGAGATTGAGAGAGATC-3 ') amplified PCR products were separated by agarose electrophoresis, recovered and ligated into the pEASY-Tl (long form CICC) cloning vector, transformed into E. coli XL1-BLUE (Agilent Technologies-Stratagene products), the cloning vector after sequencing to determine correct amplification product (designated nucleotide sequence BIGDB3) as sequence table I below.

[0032] 二、重组表达载体的构建 [0032] Second, constructing a recombinant expression vector

[0033] 1、带标记基因的中间载体(pM0A34-G/L)的构建 [0033] 1, with the intermediate vector construct marker gene (pM0A34-G / L) of

[0034] 以pCambial300_221(CAMBIA公司)载体为模版,利用引物GUS-F:5 ' -ACGCGTCGACATGTTACG TCCTGTAGAAACCCCAAC-3 ' 和GUS-R:5' -CAGCCGACCGGTTCATTGTTTGCCTCC CTGCTGC-3'扩增出1812bp 的⑶S 基因,经测序后,通过SalI和AgeI双酶切连接到pM0A34载体上(记载该材料的文献是Barrell,PJandConner,AJ(2006)Minimal T-DNA vectors suitable for agricultural deployment oftransgenic plants,公众可从中科院遗传与发育生物学研究所获得),构建成pM0A34_G中间载体。 [0034] In pCambial300_221 (CAMBIA company) vector as a template, using primers GUS-F: 5 '-ACGCGTCGACATGTTACG TCCTGTAGAAACCCCAAC-3' and GUS-R: 5 '-CAGCCGACCGGTTCATTGTTTGCCTCC CTGCTGC-3' The amplified 1812bp ⑶S gene by sequencing after, connected by AgeI and SalI digested vector to pM0A34 (the material is described in the literature Barrell, PJandConner, AJ (2006) Minimal T-DNA vectors suitable for agricultural deployment oftransgenic plants, from the public CAS Genetics and Developmental Biology obtaining Institute) to construct intermediate vector pM0A34_G.

[0035]将 pGL3_Enhancer Vector (Promaga 公司)经XbalI (补平)和XhoI 双酶切后的产物LUC基因与中间载体pM0A34-G经SpeI (补平)和XhoI双酶切后的载体进行连接,获得携带两个标记基因GUS和LUC的表达载体pM0A34-G/L。 [0035] The pGL3_Enhancer Vector (Promaga Corporation) by product after XbalI (blunted) and XhoI double digestion and LUC genes intermediate vector pM0A34-G by Spel (blunted) to connect the carrier and XhoI double digestion, to obtain It carries two marker genes GUS and LUC expression vector pM0A34-G / L.

[0036] 2、表达载体pM0A34-G/L-BI⑶B3的构建 [0036] 2, the expression vector pM0A34-G / L-BI⑶B3 of

[0037] 用SalI和NotI双酶切步骤一验证正确的克隆载体和步骤I得到的植物表达载体PM0A34-G/L,回收后经T4DNA连接酶连接得到pM0A34-G/L_BI⑶B3植物表达载体(图1)。 [0037] digested with SalI and NotI to verify a correct step and the step of cloning vectors I plant expression vector obtained PM0A34-G / L, connected by T4DNA ligase to give recovered pM0A34-G / L_BI⑶B3 plant expression vector (FIG. 1 ).

[0038] 3、表达载体pM0A34-G/L-BI⑶B3 (反向)的构建 [0038] 3 expression vector pM0A34-G / L-BI⑶B3 (reverse) Construction

[0039] 将pM0A34-G/L-BI⑶B3中BI⑶B3序列调转方向,构建成pM0A34_G/L_BI⑶B3 (反向)载体。 [0039] The pM0A34-G / L-BI⑶B3 BI⑶B3 the sequence reverses direction, constructed pM0A34_G / L_BI⑶B3 (reverse) vector.

[0040] 以步骤一中带有BI⑶B3基因正确序列的克隆载体为模板,利用引物BI⑶B3-F2:5 ' -GAGC GTCGACTGTTAGATCAGATGTGTCGGCC-3 '和BI⑶B3-R2:5 ' -ACGCGCGGCCGCGGTTGATCGGAGATTGAGAGAGATC-3',获得BI⑶B3反向片段,经测序后用SalI和NotI双酶切连接到步骤二的步骤I得到的植物表达载体PM0A34-G/L,得到pM0A34-G/L_BI⑶BI (反向)植物表达载体。 [0040] In step a cloning vector in correct sequence with BI⑶B3 gene as a template, with primers BI⑶B3-F2: 5 '-GAGC GTCGACTGTTAGATCAGATGTGTCGGCC-3' and BI⑶B3-R2: 5 '-ACGCGCGGCCGCGGTTGATCGGAGATTGAGAGAGATC-3', reverse is obtained BI⑶B3 fragment was sequenced after connection digested with SalI and NotI step II into a plant expression vector obtained I PM0A34-G / L, to obtain a plant expression pM0A34-G / L_BI⑶BI (reverse) vector.

[0041] 三、转化植物并培养 [0041] Third, the transformed plant and cultured

[0042] 1、导入农杆菌 [0042] 1, into Agrobacterium

[0043] 通过电击转化的方法将步骤二中的pM0A34-G/L-BI⑶B3阳性克隆导入农杆菌EHA105中,挑取重组农杆菌单克隆菌落转入3-4ml LB培养液中(含100 μ g/ml壮观霉素和50 μ g/ml利福平),28°C摇菌过夜。 [0043] By the method of electroporation in step two pM0A34-G / L-BI⑶B3 positive clones into Agrobacterium strain EHA105, Agrobacterium monoclonal recombinant colonies were picked into 3-4ml LB broth (containing 100 μ g / ml spectinomycin and 50 μ g / ml rifampicin), 28 ° C shaking overnight bacteria. 培养得到的菌液转接于500ml LB (含100 μ g/ml壮观霉素和50 μ g/ml利福平),28°C摇菌过夜。 Transfer the culture broth obtained in 500ml LB (containing 100 μ g / ml spectinomycin and 50 μ g / ml rifampicin), 28 ° C shaking overnight bacteria. 当菌液0D600值为1.0,收集菌液入离心管,4000rpm、20min,收集菌体。 When the value is 1.0 0D600 bacteria, bacteria solution was collected into a centrifuge tube, 4000rpm, 20min, cells were collected. 用新鲜配制的如下溶液重悬菌体:溶剂为水,溶质是壬基酚聚氧乙烯醚(NP-40)和蔗糖,其中,壬基酚聚氧乙烯醚(NP-40) (Fluka 74385)的浓度为0.05%和蔗糖的质量百分浓度为5%。 A freshly prepared solution of resuspended cells as follows: the solvent is water, the solute is a nonylphenol polyoxyethylene ether (NP-40), and sucrose, wherein, nonylphenol polyoxyethylene ether (NP-40) (Fluka 74385) a concentration of mass percent concentration of 0.05% and 5% sucrose.

[0044] 2、转化拟南芥 [0044] 2, to transform Arabidopsis

[0045] 选取抽苔期的野生型col-0生态型拟南芥(美国俄亥俄州立大学的生物资源中心(ABRC))健壮植株,通过真空渗透法将步骤I得到的农杆菌导入拟南芥花中,用塑料膜覆盖I天,保持湿润。 [0045] Select bolting of wild-type Arabidopsis thaliana ecotype col-0 (U.S. Ohio State University Center for Biological Resources (the ABRC)) healthy plants by vacuum infiltration method step I introduced into Agrobacterium obtained Floral , washed with a plastic film covering I days, kept moist. 然后将处理过的拟南芥植株放于22〜25°C的光照条件下使其正常生长。 The treated Arabidopsis plants placed under light conditions 22~25 ° C is normally grown. 收获种子,干燥条件下保存待用。 Harvest seeds, dry conditions save for later use.

[0046] 四、基因表达检测[0047] 1、转基因拟南芥中⑶S的表达分析 [0046] Fourth, the detection of gene expression [0047] 1, analysis of transgene expression in Arabidopsis ⑶S

[0048] 选取步骤三中10个不同株系的转基因拟南芥种子,于1/2MS培养基上萌发并生长,分别取刚萌发的种子、去掉种皮的刚萌发的种子、胚芽、子叶展开的幼苗、三片真叶的幼苗以及花序进行⑶S染色分析,⑶S染色方法参见Jefferson RA, Kavanagh TA, BevanMW(1987)GUS fusions:b-glucuronidase as a sensitive and veratile gene fusionmaker in higher plants.EMBO J 6:3901_7,分析结果如图2,转基因植株中⑶S基因得到表达。 [0048] step 3 to select 10 different transgenic lines of Arabidopsis seeds, germinated on 1 / 2MS medium and grown, were taken just germinating seeds, seed coat removed just germinating seeds, embryos, cotyledons seedlings, seedlings three true leaves and inflorescences ⑶S for staining, staining ⑶S see Jefferson RA, Kavanagh TA, BevanMW (1987) GUS fusions: b-glucuronidase as a sensitive and veratile gene fusionmaker in higher plants.EMBO J 6 : 3901_7, the analysis results in FIG. 2, the transgenic plants ⑶S gene is expressed.

[0049] 2、转基因拟南芥中LUC的表达分析 [0049] 2, LUC expression analysis of transgenic Arabidopsis

[0050] 选取⑶S基因有表达的5个不同株系的转基因拟南芥,于1/2MS培养基上萌发并生长,取生长2周幼苗进行LUC发光分析,分析结果如图3,转基因植株中LUC基因得到表达。 [0050] Select ⑶S gene expression of 5 different strains of transgenic Arabidopsis thaliana, germinated and grown on 1 / 2MS medium, taken two weeks seedlings were grown LUC emission analysis and the results shown in Figure 3, the transgenic plants LUC gene was expressed.

Claims (2)

1.序列表中序列I所示的DNA片段在作为拟南芥启动子中的应用;所述启动子是双向启动子。 1 in the Sequence Listing I DNA fragment shown as a sub-applications in Arabidopsis thaliana; the promoter is a bidirectional promoter.
2.如权利要求1所述的应用,其特征在于:所述应用是序列表中序列I所示的DNA片段在驱动目的基因在拟南芥体内表达中`的应用。 2. The use according to claim 1, wherein: the application is expressed in vivo in the Sequence Listing I DNA fragment shown driving the gene in Arabidopsis' applications.
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WO2002016655A2 (en) 2000-08-24 2002-02-28 The Scripps Research Institute Stress-regulated genes of plants, transgenic plants containing same, and methods of use

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WO2002016655A2 (en) 2000-08-24 2002-02-28 The Scripps Research Institute Stress-regulated genes of plants, transgenic plants containing same, and methods of use

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关翠萍.中国番茄黄化曲叶病毒双向启动子的鉴定.《自然科学进展》.2005,第15卷(第8期),
江海湃.水稻HTD1基因启动子在转基因拟南芥中的表达特征.《核农学报》.2009,第23卷(第3期),

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