WO2017092538A1 - 烟草基因NtTCTP在植物抗马铃薯Y病毒中的应用 - Google Patents

烟草基因NtTCTP在植物抗马铃薯Y病毒中的应用 Download PDF

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WO2017092538A1
WO2017092538A1 PCT/CN2016/104255 CN2016104255W WO2017092538A1 WO 2017092538 A1 WO2017092538 A1 WO 2017092538A1 CN 2016104255 W CN2016104255 W CN 2016104255W WO 2017092538 A1 WO2017092538 A1 WO 2017092538A1
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tobacco
gene
expression vector
nucleotide sequence
nttctp
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French (fr)
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郭玉双
贾蒙骜
夏范讲
张劲松
陈受宜
任学良
赵杰宏
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贵州省烟草科学研究院
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Priority to JP2018529161A priority Critical patent/JP2018537979A/ja
Priority to EP16869848.8A priority patent/EP3385385B1/en
Priority to US15/780,737 priority patent/US20180346922A1/en
Publication of WO2017092538A1 publication Critical patent/WO2017092538A1/zh

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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation

Definitions

  • the invention belongs to the field of plant genetic engineering, in particular to the application of the tobacco gene NtTCTP in plant anti-potato Y virus.
  • TCTP Translationally controlled tumor protein
  • TCTP is involved in a variety of cytological processes such as promoting histamine release, apoptosis, microtubule assembly, ion balance, etc., and with a range of proteins such as polo kinase, tubulin, actin and sodium. Potassium ATPase and the like have interactions.
  • TCTP is involved in cell growth and differentiation, malignant transformation, stress resistance and apoptosis.
  • mice that knocked out the TCTP gene embryonic lethality occurred in mice due to lack of cell differentiation and excessive apoptosis.
  • TCTP In Drosophila, when TCTP expression is interrupted in a specific organ, the number of cells in the organ decreases and defects in cell growth eventually lead to the organ becoming smaller.
  • the expression of TCTP mRNA in plants is closely related to mitosis, and dark can induce the accumulation of TCTP mRNA in the morning glory.
  • TCTP proteins play an important role in regulating long-distance transport of phloem proteins and pollen tube growth. Recent studies have found that TCTP, as a positive regulator of mitotic growth, is able to specifically regulate cell cycle time in Arabidopsis. TCTP regulates cell differentiation through the TOR (Target of rapamycin) growth regulation pathway.
  • the activity of the tobacco gene NtTCTP of the present invention is closely related to the resistance of tobacco to PVY, and the gene sequence is:
  • nucleotide sequence in which the nucleotide sequence set forth in SEQ ID NO: 1 is substituted, deleted and/or increased by one or more nucleotides and which expresses the same functional protein;
  • nucleotide sequence which hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 and expresses the same functional protein under conditions of 0.1 x SSPE containing 0.1% SDS or 0.1 x SSC containing 0.1% SDS In solution, hybridize at 65 ° C and wash the membrane with the solution; or
  • Iv a nucleotide sequence having 90% or more homology with the nucleotide sequence of i), ii) or iii) and expressing the same functional protein.
  • the present invention also provides a protein encoded by the gene NtTCTP, the amino acid sequence of which is shown in SEQ ID NO: 2.
  • the invention also provides the use of the gene NtTCTP in tobacco against potato virus Y (PVY). That is, the tobacco gene NtTCTP was used to regulate tobacco resistance to PVY.
  • the potato virus Y includes a PVY necrotic strain (N strain).
  • the application is to silence the gene NtTCTP in tobacco using RNAi technology.
  • the nucleotide sequence set forth in SEQ ID NO: 3 can be cloned into a plant expression vector, and then the recombinant expression vector can be transferred into tobacco using Agrobacterium-mediated methods to screen transgenic tobacco plants.
  • the plant expression vector is pBin438.
  • the invention also provides a method for constructing a transgenic tobacco plant, which first constructs the gene
  • the RNAi expression vector of NtTCTP was then transferred into tobacco by Agrobacterium-mediated method to screen transgenic tobacco plants.
  • the starting vector is pBin438.
  • the invention also provides the use of the gene NtTCTP in improving tobacco varieties.
  • the invention also provides the use of the gene NtTCTP in tobacco anti-PVY breeding.
  • the present invention further provides an expression vector carrying an expression cassette for inhibiting the tobacco gene NtTCTP.
  • the expression vector carries a nucleotide sequence as shown in SEQ ID NO: 3.
  • the invention clones the translation control tumor protein gene NtTCTP from tobacco, the gene is 507 bp in length, encodes a protein containing 168 amino acids, and studies the role of the gene in the anti-PVY process of tobacco. Silencing the gene in cultivated tobacco, tobacco showed greater resistance to PVY, reached immunity levels, overexpressed the gene in cultivated tobacco, and tobacco was more sensitive to PVY. This indicates that this gene plays an important role in the anti-PVY process of tobacco and can be used for anti-PVY breeding of plants such as tobacco.
  • RNAi expression vector of the gene NtTCTP is constructed, and the tobacco is genetically transformed by Agrobacterium. The result shows that the gene is silenced in tobacco, and the resistance of the tobacco to PVY is improved, and the resistance can be increased by 50%-80%. .
  • Figure 1 is a result of Western Blot verification of TCTP gene in different tobacco lines in Example 3 of the present invention.
  • FIG. 2 is a diagram showing leaf traits of gene TCTP overexpressing lines O2, O7, gene TCTP silencing strains Ri16, Ri20, and wild tobacco after PVY inoculation in Example 3. It can be seen from the figure that the overexpressed lines O2 and O7 have necrosis, while the silent lines Ri16 and Ri20 have no symptoms.
  • LA Taq DNA polymerase and restriction endonuclease were purchased from TaKaRa.
  • the DNA gel purification and recovery kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.
  • T 4 DNA ligase were purchased from NEB; pGEM-T vector purchased from Promega Biotechnology vector Limited.
  • ABI PRISMTM Optical 96-Well Reaction Plates, Optical Caps were purchased from ABI; Escherichia coli DH5 ⁇ , Agrobacterium tumefaciens EHA105, and plant expression vector pBin 438 were all commercially available. DNA synthesis and sequencing was completed by Huada Gene Technology Services Co., Ltd.
  • the NCBI was used to design a primer for amplifying the full-length gene according to the full-length sequence of the tobacco TCTP gene (Accession NO: KM507327.1) in Genbank, and the restriction sites BamH I and Sal I were added.
  • the designed primer sequences are as follows:
  • TCTPF1 5'-GGATCCATGTTGGTTTATCAGGATCTTCTCT-3’
  • TCTPR1 5'-GTCGACTTAACACTTGACCTCCTTGAGTCCA-3’
  • RNA of 'Shanxiyan' leaves was extracted, the first strand of cDNA was reverse transcribed, and the following reagents were added to a 0.2 ml centrifuge tube for PCR reaction:
  • the PCR reaction procedure was as follows: heating the lid, pre-denaturation at 95 ° C for 5 min; then denaturation at 95 ° C for 30 s, annealing at 55 ° C for 30 s, extension at 72 ° C for 30 s, 35 cycles.
  • the amplified DNA fragment was sequenced and the result is shown in SEQ ID NO: 1.
  • the plant expression vector pBin438 and the full-length amplified PCR product were digested with Sal I and BamH I respectively.
  • the enzyme digestion system was as follows:
  • connection system is as follows:
  • the forward and reverse complements of the 330 bp fragment between the 1 bp and 330 bp positions in the tobacco gene NtTCTP were ligated through an intermediate sequence, and the cleavage sites of BamH I and Sal I were added.
  • the designed sequence was passed through Shanghai.
  • the sequence synthesized is shown in SEQ ID NO: 3.
  • the synthetic fragment was digested with BamH I and Sal I and ligated with the same digested pBin438 vector to construct a final silencing vector.
  • the tobacco leaf discs were first pre-incubated for 2-3 days and then infested with Agrobacterium tumefaciens EHA105 containing the plant expression vector. After 3-4 days of co-cultivation, the leaf discs were taken out, sterilized in a liquid medium containing a sterilizing agent for 2-3 hours, and then transferred to a screening medium for screening and differentiation culture, and differentiation was started about 3 weeks.
  • bud When the resistant bud grows to a height of 3-4 cm, it is cut from the base of the explant and inoculated onto a rooting medium for rooting culture. After the root system of the plantlets is developed, domestication and transplanting are carried out. A large number of transplanted resistant plants were obtained. Plants positive for PCR detection were transplanted into large pots until the seeds were harvested.
  • Basic medium MS medium, pH 5.8, 0.8% agar.
  • Screening medium minimal medium + 25 mg / L Km + 100 mg / L cephalosporin.
  • Differentiation medium basic medium + 1.0 mg / L 6-BA + 0.1 mg / L NAA, pH 5.8, 0.75% agar.
  • Rooting medium MS medium + 0.5 mg/L NAA, pH 5.8, 0.75% agar. , pH 5.8, 0.8% agar.
  • the transgenic material was obtained, and the TCTP gene overexpressing strains O2 and O7; the TCTP gene silencing strains Ri16 and Ri20.
  • the transgenic tobacco lines O2, O7 of the overexpressed gene NtTCTP were constructed in Example 2.
  • the PVY strain is a PVY necrotic strain (N strain).
  • PVY inoculum Fresh diseased leaves were added to the crusher, crushed with a small amount of phosphate buffer, and the diseased leaf residue was filtered with gauze, and fresh juice was taken to prepare P VY inoculum.
  • the inoculation period is the tobacco 4 leaf stage, select 2-3 pieces of young leaves on the tobacco plants to be inoculated, gently rub with sandpaper to produce micro-injury, then use the brush to pick up the PVY inoculum and gently brush On the blade after rubbing.
  • the disease level is investigated according to the following criteria:
  • Grade 0 The whole plant is disease-free or has extremely slight mosaics.
  • Grade 1 1/10 to 1/3 of the leaves showed slight small vein necrosis or spotted streak symptoms and slight deformation, the other leaves of the whole plant were asymptomatic, and the plants were not significantly dwarfed.
  • Grade 2 1/3 to 1/2 of the leaves showed spotted streaks or vein necrosis, most of the small leaf veins of the diseased leaves were necrotic, causing a large area of yellowing and dead leaves, and the plant dwarfed to the normal plant height 2 /3 or more.
  • Grade 3 1/2 to 2/3 of the leaves showed more severe vein necrosis or more than 2/3 of the leaves showed spotted streak necrosis, a large area of a single leaf withered, and a small number of leaves fell off. Or the whole plant leaves begin to turn yellow.
  • Grade 4 The whole plant leaves showed severe vein necrosis or leaf deformation and atrophy. The leaves of 2/3 of the leaves were wilting and falling off, and the diseased plants were dwarfed to 1/2 to 1/3 of the normal tobacco plants, or the whole plants were withered.
  • the disease was investigated 14 days after inoculation.
  • the silent strains Ri16 and Ri20 showed immunity, no disease, the disease index was 0, and the disease resistance was greatly improved, while the overexpressing lines O2 and O7 showed better than the wild type control (WT). More severe symptoms of necrosis, the condition is more serious (Figure 2).
  • the disease index of strain O2 was 96.97
  • the disease index of O7 was 91.92
  • the disease index of control wild tobacco was 85.86.
  • the activity of the tobacco gene NtTCTP provided by the present invention is closely related to the resistance of tobacco to PVY.
  • the tobacco was genetically transformed by Agrobacterium-mediated method. The results showed that the gene was silenced in tobacco and increased the resistance of tobacco to PVY, and the resistance could be increased by 50%-80%.

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Abstract

提供了烟草基因NtTCTP在植物抗马铃薯Y病毒(PVY)中的应用。该烟草基因NtTCTP全长507bp，编码一个含168个氨基酸的蛋白，其活性与烟草抗PVY的抗性密切相关。在烟草中沉默该基因，烟草表现出对PVY更强的抗性，可达到免疫水平，在烟草中超量表达该基因，烟草对PVY更敏感。该基因可用于烟草等植物抗PVY育种。

Description

烟草基因NtTCTP在植物抗马铃薯Y病毒中的应用
相关申请的交叉引用
本发明要求2015年12月1日提交、申请号为201510858859.0的中国专利申请的优先权，其所公开的内容作为参考全文并入本申请。
技术领域
本发明属于植物基因工程领域，具体地说，涉及烟草基因NtTCTP在植物抗马铃薯Y病毒中的应用。
背景技术
翻译控制肿瘤蛋白(Translationally controlled tumor protein，TCTP)，也称p21或p23，是真核生物中普遍表达和分布的一种蛋白。其最早发现于哺乳动物肿瘤细胞中，并在转录后水平受到调控。
多种生物中的大量研究发现，TCTP参与多种细胞学过程，如促进组胺释放，细胞凋亡，微管组装、离子平衡等，并与一系列蛋白，如polo激酶、tubulin、actin和钠钾ATP酶等有相互作用。动物中TCTP参与细胞生长分化，恶性转化，抗逆以及细胞凋亡等。敲除TCTP基因后的老鼠，由于缺少细胞分化和过度细胞凋亡，导致老鼠出现胚胎致死现象。果蝇中，某一特异器官中TCTP表达被中断后，该器官细胞数目下降和细胞生长出现缺陷最终导致该器官变小。植物中TCTP mRNA表达与有丝分裂密切相关，黑暗可以诱导牵牛中TCTP mRNA积累。另外，TCTP蛋白在调节韧皮部蛋白的长距离运输和花粉管生长的过程中发挥着重要作用。最近研究发现，拟南芥中，TCTP作为有丝分裂生长的正调控因子，能够特异调控细胞周期的时间。TCTP通过TOR(Target of rapamycin)生长调控途径调节细胞分化。卷心菜中的研究发现，TCTP不仅调控生长发育，同时受外界环境诱导。这些已有研究表明，TCTP不仅能够调控有机体生长发育，而且具有植 物专属功能，因此，研究该类基因对PVY的抗性研究，具有重要意义。
挖掘植物基因组中抗马铃薯Y病毒(potato virus Y,PVY)基因，是植物抗病毒育种的关键。
发明内容
本发明的目的是提供烟草基因NtTCTP在植物抗马铃薯Y病毒中的应用。
为了实现本发明目的，本发明的烟草基因NtTCTP，其活性与烟草抗PVY的抗性密切相关，基因序列为：
i)SEQ ID NO:1所示的核苷酸序列；或
ii)SEQ ID NO:1所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列；或
iii)在严格条件下与SEQ ID NO:1所示序列杂交且表达相同功能蛋白质的核苷酸序列，所述严格条件为在含0.1％SDS的0.1×SSPE或含0.1％SDS的0.1×SSC溶液中，在65℃下杂交，并用该溶液洗膜；或
iv)与i)、ii)或iii)的核苷酸序列具有90％以上同源性且表达相同功能蛋白质的核苷酸序列。
本发明还提供所述基因NtTCTP编码的蛋白，其氨基酸序列如SEQ ID NO:2所示。
本发明还提供所述基因NtTCTP在烟草抗马铃薯Y病毒(PVY)中的应用。即，将烟草基因NtTCTP用于调节烟草对PVY的抗性。所述马铃薯Y病毒包括PVY坏死株系(N株系)。
所述应用是利用RNAi技术沉默烟草中的基因NtTCTP。例如，可将如SEQ ID NO:3所示的核苷酸序列克隆至植物表达载体上，然后采用农杆菌介导的方法，将重组表达载体转入烟草中，筛选转基因烟草植株。优选所述植物表达载体为pBin438。
本发明还提供一种转基因烟草植株的构建方法，先构建所述基因 NtTCTP的RNAi表达载体，然后采用农杆菌介导的方法，将重组表达载体转入烟草中，筛选转基因烟草植株。优选出发载体为pBin438。
本发明还提供所述基因NtTCTP在改良烟草品种中的应用。
本发明还提供所述基因NtTCTP在烟草抗PVY育种中的应用。
本发明进一步提供一种表达载体，所述表达载体携带有抑制烟草基因NtTCTP的表达盒。
优选地，所述表达载体携带有如SEQ ID NO:3所示的核苷酸序列。
本发明从烟草中克隆得到翻译控制肿瘤蛋白基因NtTCTP，该基因全长507bp，编码一个含168个氨基酸的蛋白，并研究了该基因在烟草抗PVY过程中的作用。在栽培烟草中沉默该基因，烟草表现出对PVY更强的抗性，可达到免疫水平，在栽培烟草中超量表达该基因，烟草对PVY更敏感。说明该基因在烟草抗PVY过程中具有重要作用，可用于烟草等植物抗PVY育种。
本发明构建了基因NtTCTP的RNAi表达载体，通过农杆菌介导对烟草进行遗传转化，结果表明，该基因在烟草中沉默后，提高烟草对PVY的抗性，抗性可提高50％-80％。
附图说明
图1为本发明实施例3中不同烟草株系中TCTP基因的Western Blot验证结果。
图2为本发明实施例3中PVY接种后，基因TCTP过表达株系O2、O7；基因TCTP沉默株系Ri16、Ri20以及野生烟草的叶片性状图。从图中可以看出，超量表达株系O2、O7叶脉已坏死，而沉默株系Ri16和Ri20叶脉无任何症状。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验 手册(Sambrook J & Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。
实施例1 烟草基因NtTCTP的克隆
一、材料与方法
1.实验材料
1.1栽培烟草品种‘山西烟’，由贵州省烟草科学研究院育种工程中心提供。
1.2试剂
LA Taq DNA聚合酶、限制性内切酶购自TaKaRa公司。DNA凝胶纯化回收试剂盒购自天根生化科技(北京)有限公司。T4DNA连接酶购自NEB公司；pGEM-T vector载体购自Promega生物技术有限公司。ABI PRISMTM Optical 96-Well Reaction Plates,Optical Caps购自ABI公司；Escherichia coli DH5α、根癌农杆菌(Agrobacterium tumefaciens EHA105)、植物表达载体pBin438均为市售商品。DNA合成及测序由华大基因科技服务有限公司完成。
2.实验方法
登录NCBI，根据Genbank中烟草TCTP基因(Accession NO:KM507327.1)的全长序列设计扩增全长基因的引物，并添加酶切位点BamH Ⅰ和Sal Ⅰ。设计的引物序列如下：
TCTPF1：5’-GGATCCATGTTGGTTTATCAGGATCTTCTCT-3’
TCTPR1：5’-GTCGACTTAACACTTGACCTCCTTGAGTCCA-3’
2.2烟草基因NtTCTP全长扩增
提取‘山西烟’叶片总RNA，反转录合成cDNA第一链，在0.2ml离心管中加入下列试剂，进行PCR反应：
Figure PCTCN2016104255-appb-000001
Figure PCTCN2016104255-appb-000002
PCR反应程序为：加热盖，95℃预变性5min；然后95℃变性30s，55℃退火30s，72℃延伸30s，35个循环。
扩增得到的DNA片段经测序，结果如SEQ ID NO:1所示。
实施例2 烟草基因NtTCTP转基因株系的构建
1.过表达基因NtTCTP载体的构建
1.1将植物表达载体pBin438、全长扩增PCR产物分别用Sal Ⅰ和BamH Ⅰ双酶切，酶切体系如下：
Figure PCTCN2016104255-appb-000003
1.2切产物经1％的琼脂糖凝胶电泳检测，回收PCR酶切产物和载体pBin438的大片段，连接。回收产物按下列比例进行定向连接。连接体系如下：
Figure PCTCN2016104255-appb-000004
16℃连接16小时或更长，以增加连接效率。
1.3重组质粒转化根癌农杆菌
1)取0.5～1.0μL DNA，加入到根癌农杆菌感受态细胞中，轻轻混合，冰浴5min，迅速投入液氮中冷冻5min；
2)37℃水浴融化，加入800μL YEB液体培养基，28℃，100r/min， 振荡培养4h；
3)4 000r/min，离心5min，倒去大部分上清液，余100μL左右，悬浮菌体；
4)涂布在含有40mg/L利福平(Rif)、50mg/L链霉素(Sm)和100mg/L卡那霉素(Km)的固体YEB培养基上，28℃培养48～96h。
2.沉默载体的构建
将烟草基因NtTCTP中从第1bp～330bp位之间330bp的片段的正向序列和反向互补序列通过中间序列连接，并添加BamH Ⅰ和Sal Ⅰ的酶切位点，设计好的序列通过上海生工合成，合成的序列如SEQ ID NO:3所示。合成片段经BamH Ⅰ和Sal Ⅰ酶切，与经过同样酶切的pBin438载体连接，构建成最终的沉默载体。
3.根癌农杆菌介导的烟草的遗传转化
先将烟草叶盘预培养2-3天，然后用含有植物表达载体的根癌农杆菌EHA105分别进行侵染。共培养3-4天后，将叶盘取出，在含有除菌剂的液体培养基中除菌2-3h，然后再转到筛选培养基进行筛选及分化培养，约3周左右开始分化出抗性芽。待抗性芽长至3-4cm高时，将其从外植体基部切下，接种到生根培养基上进行生根培养。待小植株根系发达后，进行驯化移栽。得到大量移栽成活的抗性植株。将PCR检测呈阳性的植株移栽到大花盆中，直到收获种子。
基本培养基：MS培养基，pH5.8，0.8％琼脂。
筛选培养基：基本培养基+25mg/L Km+100mg/L头孢菌素。
分化培养基：基本培养基+1.0mg/L 6-BA+0.1mg/L NAA，pH5.8，0.75％琼脂。
生根培养基：MS培养基+0.5mg/L NAA，pH5.8，0.75％琼脂。，pH5.8，0.8％琼脂。
获得了转基因材料，TCTP基因超量表达株系O2、O7；TCTP基因沉默株系Ri16、Ri20。
实施例3 转基因烟草株系对PVY抗性的研究
1.材料与方法
1.1实验材料
实施例2中构建得到的过表达基因NtTCTP的转基因烟草株系O2、O7。
实施例3中构建得到的基因NtTCTP沉默的转基因烟草株系Ri16、Ri20。
PVY毒株为PVY坏死株系(N株系)。
1.2实验方法
1.2.1PVY接种物制备：将新鲜的病叶加入在打碎机中，加少量磷酸缓冲液打碎，用纱布过滤去病叶残体，取新鲜的汁液制成P VY接种物。
1.2.2PVY接种：接种时期为烟草4叶期，在待接种的烟株上选择2-3片幼嫩叶片用砂纸轻轻摩擦，产生微伤，然后用毛刷蘸取PVY接种物轻轻刷在摩擦后的叶片上。
1.2.3数据调查：选择发病最重的时间，调查PVY的发病率和病级。
病级按如下标准调查：
0级：全株无病或出现极轻微花叶。
1级：1/10到1/3的叶片表现轻微小叶脉坏死或出现点刻状条斑症状以及轻微变形，整株其他叶片无症状，植株无明显矮化。
2级：1/3到1/2的叶片表现点刻状条斑或脉坏死，病叶大部分小叶脉坏死，造成叶片较大面积的黄化、枯死，植株矮化为正常株高的2/3以上。
3级：1/2到2/3的叶片表现较严重的脉坏死或2/3以上的叶片出现点刻状条斑坏死，单叶较大面积的枯死，少部分叶片脱落。或整株叶片开始变黄。
4级：全株叶片表现严重的脉坏死或叶片变形、萎缩，2/3左右的叶片萎蔫脱落，病株矮化为正常烟株的1/2至1/3，或整株凋萎。
病情指数DI＝[∑(各级病株株数×该病级值)/(调查总株数×最高级值)]×100
2.实验结果
(1)NtTCTP基因在不同烟草株系中的表达验证
通过Western Blot验证表明，2个沉默株系Ri16和Ri20中TCTP基因都没有表达，说明已经成功沉默TCTP基因，而2个过表达株系O2和O7表达量均超过对照(WT)，说明TCTP基因过表达也构建成功(图1)。
(2)NtTCTP基因过表达和沉默株系抗病性鉴定结果
接种14天后调查病情，沉默株系Ri16和Ri20均表现免疫，未发病，病情指数为0，抗病性有很大提高，而过表达株系O2和O7均表现出比野生型对照(WT)更严重的坏死症状，病情更为严重(图2)。其中，株系O2的病情指数为96.97，O7的病情指数为91.92，对照野生烟草的病情指数为85.86。
以上结果表明，在烟草中沉默TCTP基因能够提高植物对PVY的抗病性，该基因具有重要的应用价值。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供的烟草基因NtTCTP，其活性与烟草抗PVY的抗性密切相关。通过构建基因NtTCTP的RNAi表达载体，采用农杆菌介导法对烟草进行遗传转化，结果表明，该基因在烟草中沉默后，提高烟草对PVY的抗性，抗性可提高50％-80％。
Figure PCTCN2016104255-appb-000005
Figure PCTCN2016104255-appb-000006

Claims (10)

  1. 烟草基因NtTCTP在烟草抗马铃薯Y病毒中的应用；其中，烟草基因NtTCTP为：
    i)SEQ ID NO:1所示的核苷酸序列；或
    ii)SEQ ID NO:1所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列；或
    iii)在严格条件下与SEQ ID NO:1所示序列杂交且表达相同功能蛋白质的核苷酸序列，所述严格条件为在含0.1％SDS的0.1×SSPE或含0.1％SDS的0.1×SSC溶液中，在65℃下杂交，并用该溶液洗膜；或
    iv)与i)、ii)或iii)的核苷酸序列具有90％以上同源性且表达相同功能蛋白质的核苷酸序列。
  2. 根据权利要求1所述的应用，其特征在于，利用RNAi技术沉默烟草中的基因NtTCTP。
  3. 根据权利要求2所述的应用，其特征在于，将如SEQ ID NO:3所示的核苷酸序列克隆至植物表达载体上，然后采用农杆菌介导的方法，将重组表达载体转入烟草中，筛选转基因烟草植株；优选所述植物表达载体为pBin438。
  4. 根据权利要求1-3任一项所述的应用，其特征在于，所述马铃薯Y病毒包括马铃薯Y病毒坏死株系N株系。
  5. 一种转基因烟草植株的构建方法，其特征在于，先构建基因NtTCTP的RNAi表达载体，然后采用农杆菌介导的方法，将重组表达载体转入烟草中，筛选转基因烟草植株。
  6. 根据权利要求5所述的方法，其特征在于，所用出发载体为pBin438。
  7. 烟草基因NtTCTP在改良烟草品种中的应用。
  8. 烟草基因NtTCTP在烟草抗PVY育种中的应用。
  9. 一种表达载体，其特征在于，所述表达载体携带有抑制烟草基因NtTCTP的表达盒。
  10. 根据权利要求9所述的表达载体，其特征在于，所述表达载体携带有如SEQ ID NO:3所示的核苷酸序列。
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