CN103710342A - Artificially induced MLE (Mariner-Like Element) transposon kit and application thereof - Google Patents

Artificially induced MLE (Mariner-Like Element) transposon kit and application thereof Download PDF

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CN103710342A
CN103710342A CN 201310698907 CN201310698907A CN103710342A CN 103710342 A CN103710342 A CN 103710342A CN 201310698907 CN201310698907 CN 201310698907 CN 201310698907 A CN201310698907 A CN 201310698907A CN 103710342 A CN103710342 A CN 103710342A
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transposon
transposase
mle
vector
transposition
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周明兵
汤定钦
杨萍
郑丽娜
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浙江农林大学
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Abstract

The invention discloses an artificially induced MLE (Mariner-Like Element) transposon transposition kit and application thereof in plant gene mutation. The transposase of the the MLE transposon is regulated by an inducible promoter, and the expression of the transposase is artificially regulated to control the jumping and rejumping of the transposon; and thus, the problem of mutation stability is solved, the reverse mutation becomes controllable, and purposeful induced transpositional mutation can be performed on different tissues and organs of plants in any plant growth and development period, thereby implementing overall regulation on mutagenesis in the aspects of space-time quantities. Besides, the green fluorescence protein gene is utilized to report the transposition conditions of the MLE transposon so as to simply and effectively screen the plant with variable transposition and track the transposon insertion site.

Description

人工诱导的MLE转座子试剂盒及其应用 Artificially induced MLE transposon kit and its Applications

技术领域 FIELD

[0001] 本发明属于分子生物学领域,具体而言,涉及一种人工诱导的Mariner-LikeElement (MLE)转座子试剂盒及其在植物基因突变中的应用。 The present invention is in the field of molecular biology, in particular, it relates to an artificially induced Mariner-LikeElement (MLE) transposon kit and its application [0001] In a plant gene mutation.

背景技术 Background technique

[0002] 随着新技术新方法的不断发展,分析鉴定基因功能的方法越来越多,大规模研究基因功能主要依赖于构建突变体库,但最直接最有效的方法是构建饱和的基因突变群体,通过突变体分析鉴定基因功能。 [0002] With the development of new methods of new technology, analytical methods to identify gene function more and more large-scale studies of gene function depends on the construction of mutant libraries, but the most direct and effective way is to build a saturated gene mutation groups identified by analysis of the mutant gene function. 因此突变群体的构建是功能基因组学的基础。 So Mutant Population is the basis of functional genomics. 随着农杆菌介导的植物转基因技术的不断完善,通过创造大量的T-DNA独立转化子来建立T-DNA突变体库的方法已成为快速构建插入突变体库的主要方法。 With Agrobacterium mediated plant gene transfer techniques continue to improve, through the creation of a large number of T-DNA method for establishing an independent transformants T-DNA mutant library has become the main method to quickly build insertional mutant library. T-DNA插入突变具有插入稳定、操作简单,但是易产生直接的串联,反向重复和在边界产生缺失,影响随后的分子分析。 T-DNA insertion mutation has an insertion stable, simple, easy to produce it directly in series, and a deletion in the inverted repeats boundaries affect subsequent molecular analysis.

[0003] 转座子是指在基因组上能从同一条染色体的一个位置转移到另一个位置或者从一条染色体转移到另一条染色体上的一段DNA序列,具有分布广泛、拷贝数高、插入位点专一、产生突变稳定、不受植物种类差异的影响、异源转座率高、组织培养诱导激活等优势。 [0003] Transposon refers to the transfer from one location to another location of a chromosome or a chromosome to be transferred from a DNA sequence on another chromosome in the genome, having a wide distribution, high copy number, the insertion site specificity, generating a stable mutation, the difference is not influence plant species, the heterologous transposition rate, tissue culture-induced activation of other advantages. 可以在不了解基因产物的生化性质和表达模式的情况下,分离克隆植物基因,即转座子标签(transposontagging)。 In the case may be biochemical properties and expression patterns do not understand the gene product, isolation and cloning of plant genes, transposon tagging (transposontagging). 其原理是利用植物转座子的插入造成基因突变,以转座子序列为基础,从突变株的基因文库中筛选出带有此转座子的克隆,它必定含有与转座子序列相邻的突变基因的部分序列,再利用这部分序列从野生型基因组中获得完整的基因。 The principle is to use transposon inserted into the plant caused by mutations to the transposon sequence, based on this screening clones with the transposon in mutant gene library, it must contain sequences adjacent to the transposon the partial sequence of the gene mutation, then use this partial sequence to obtain the complete gene from the wild-type genome.

[0004] 以转座子作标签分离基因有以下优点:(1)转座子插入引起的突变会由于转座子的切离而回复。 [0004] In transposon gene isolated as labels has the following advantages: mutation (1) due to the transposon insertion due to cut away from the transposon reply. (2)由于转座子总是沿其邻近的位点转座,因此只要转座子在染色体上定位后,就很容易确定与其连锁的突变基因的相对位置。 (2) Since transposon always along adjacent transposition sites, so long as the transposon located on chromosome, it is easy to determine the relative position of its linked mutations. (3)由于转座子的不断跳跃,一旦把转座子导入目标植物,通过自交、杂交、回交等手段便可得到一个较大的含不同插入位点的转座子群体。 (3) Due to the continuous transposon jumping, once the transposon into the target plants, by selfing, backcross other means can be obtained a larger population with different transposon insertion sites. (4)把转座子导入异源植物`不受转化条件的限制。 (4) introducing the transposon `not limit the heterologous transformed plant conditions. 目前利用转座子标签法成功克隆植物基因目前仅限于Ac/Ds和Spm/dSpm这两类转座子,这两类转座子均存在着转座活性不高,突变效率不理想和优先转座到与之相临近位置的问题,是目前利用转座子构建随机饱和突变体群的主要障碍。 Now successfully cloned using transposon tagging of plant genes at present limited to Ac / Ds and Spm / dSpm transposons these two types, which types are there transposon transposition activity is not high, and preferentially mutated transfer efficiency over position adjacent to the seat problem therewith is the main obstacle to the use of transposon construct a random mutant unsaturated group. 因此进一步研究、挖掘新的转座子,改进试剂盒,达到最佳标签条件,是当前转座子标签法研究的主要任务。 Therefore further research, and tap new transposon, improvement kit to achieve the best conditions for the label, is the main task of the current study transposon tagging turn.

[0005] Mariner-Like 转座子(Mariner-LikeElements, MLE)是DNA 转座兀件中一个重要家族,最早是在研究毛里塔尼亚果蚬(Dr ο soph i 1 amaur isti ana)白眼基因的一个不稳定突变时发现的。 [0005] Mariner-Like transposon (Mariner-LikeElements, MLE) is a DNA transposition Wu an important family member, if early in the study Mauritania clams (Dr ο soph i amaur isti ana 1) a gene unstable eyed found when the mutation. 此后在其他动物以及植物基因组中也发现了大量MLE转座子的存在。 Since then other animal and plant genomes also discovered the existence of a large number of MLE transposons. MLE转座子通过DNA的切除/粘贴机制来实现转座。 MLE by DNA excision transposon / transposase paste mechanism is achieved. 与其它转座子相比较,MLE转座子有以下三个显著特点:(1)结构简单,由两端反向重复序列(TerminallnvertedRepeats, TIRs)和编码转座酶的基因组成,反向重复序列长度一般为10-40bp。 Compared to other transposons, transposons MLE three following significant features: (1) simple structure, inverted repeats (TerminallnvertedRepeats, TIRs) and the two ends of the gene encoding the transposase enzyme composition, inverted repeats length is generally 10-40bp. 转座酶编码序列长度一般为1000-1500bp,由DNA结合域和转座催化域组成。 A transposase encoding sequence length is generally 1000-1500bp, composed of DNA binding domain and the catalytic domain of the transposase. 既能催化短至200bp,仅包括TIRs及侧翼序列的DNA片段转座,也能催化携带有目的基因(如抗性基因),长至6000bpDNA片段的转座,因此MLE转座子既可以开发成基因诱变的突变工具,也可以开发成转运基因的转基因工具。 Catalytic both short 200bp, a DNA fragment comprising only the transposase TIRs and flanking sequences, can also catalyze carries the gene (e.g. resistance gene), as long as transposition 6000bpDNA fragments, thus MLE transposons may be developed into tool mutant gene mutagenesis can be developed into a transgenic tool transporter gene. (2)MLE转座子在基因组插入位点接近随机,靶向序列为TA,统计结果表明,MLE转座子80%插入位点位于基因5 '端UTR的下游和基因编码序列,这一特点使MLE转座子在应用于基因标签上更优越于其他类型的转座子。 (2) MLE transposon insertion site in the genome of nearly random, the TA targeting sequence, statistics show, 80% MLE transposon insertion site of a gene 5 'end of the downstream gene coding sequence UTR, a feature MLE transposon applied so superior to other types of transposon gene signature. (3)异源转座率高,绝大部分MLE转座子转座不依赖于宿主因子,只要有活性转座酶和能被转座酶识别的TIRs存在,就能实现转座,因此从宿主克隆的转座子导入非宿主基因组后,依然能够保持转座活性。 (3) a heterologous transposition rate, most of the MLE Transpositional not dependent on host factors, as long as the active transposase and can be recognized by transposase exists TIRs, transposition can be achieved, and therefore from after cloning the transposon introduced into a host non-host genome, still able to maintain transposition activity. 因此开发出基于超活性MLE转座子的高效试剂盒,将为大规模构建植物突变体库和研究基因功能提供新的工具,使植物基因的分离和鉴定更加简单易行。 Therefore, the development of ultra-efficient kit is based on the active MLE transposons, will construct a large-scale plant mutant library and study gene function to provide new tools that enable the separation and identification of plant genes more simple. 查询以往专利,尚未有利用植物MLE转座子构建转座子试剂盒的报道。 Patent inquiries in the past, has not yet reported the use of plant MLE transposons transposons building kit.

发明内容 SUMMARY

[0006]目前利用转座子标签法成功克隆植物基因目前仅限于Ac/Ds和Spm/dSpm这两类转座子,这两类转座子均存在着转座活性不高,突变效率不理想和优先转座到与之相临近位置的问题,是目前利用转座子构建随机饱和突变体群的主要障碍。 [0006] It was successfully cloned using transposon tagging of plant genes at present limited to Ac / Ds and Spm / dSpm transposons these two types, which types are there transposon transposition activity is not high, efficiency over the mutation and priority transposition therewith to a position adjacent the problem is the major obstacle to the use of transposon construct a random mutant unsaturated group. 因此进一步研究、挖掘新的转座子,改进试剂盒,达到最佳标签条件,是当前转座子标签法研究的主要任务。 Therefore further research, and tap new transposon, improvement kit to achieve the best conditions for the label, is the main task of the current study transposon tagging turn.

[0007] 本发明一方面涉及一种MLE转座子试剂盒,其特征在于包括含有核苷酸序列为SEQIDN0.2的miniPpMLE21的载体以及含有核苷酸序列为SEQIDN0.3的转座酶序列的载体。 [0007] In one aspect the present invention relates to a kit MLE transposons, comprising the nucleotide sequence comprising the SEQIDN0.2 miniPpMLE21 carrier and comprising a nucleotide sequence of the transposase sequence is SEQIDN0.3 carrier.

[0008] 在本发明的一个优选实施方式中,其中含有核苷酸序列为SEQIDN0.2的miniPpMLE21的载体是将miniPpMLE21转座子构建到pBINm-gfp5_ER载体上,获得转座子供体载体pBINm-gfp5_ER-miniPpMLE21。 [0008] In a preferred embodiment of the present invention, wherein the nucleotide sequence comprises a vector SEQIDN0.2 miniPpMLE21 is miniPpMLE21 transposon constructs to pBINm-gfp5_ER vector, transposon donor vector is obtained pBINm-gfp5_ER -miniPpMLE21.

[0009] 在本发明的另一个优选实施方式中,所述的含有核苷酸序列为SEQIDN0.3的转座酶序列的载体是转座酶序列构建到PCAMBIA1301载体上。 [0009] In another preferred embodiment of the present invention, said vector containing a nucleotide sequence of a transposase sequences SEQIDN0.3 transposase sequence is built onto PCAMBIA1301 carrier.

[0010] 本发明另一方面还涉及上述MLE转座子试剂盒在诱变转基因植物中的应用。 [0010] The present invention further relates to the use the above-described aspect MLE transposon mutagenesis kit in transgenic plants.

[0011] 在一个优选实施方式中,所述的应用包括如下步骤: [0011] In a preferred embodiment, the application comprising the steps of:

[0012] (1)将miniPpMLE21转座子构建到pBINm-gfp5_ER载体上,获得转座子供体载体pBINm-gfp5-ER-miniPpMLE21,转化拟南芥,通过卡那霉素筛选阳性植株; [0012] (1) Construction of a transposon to miniPpMLE21 pBINm-gfp5_ER vector, transposon donor vector is obtained pBINm-gfp5-ER-miniPpMLE21, transformed Arabidopsis plants were screened by positive kanamycin;

[0013] (2)转座酶序列(SEQIDN0.3)构建到pCAMBIA1301载体,获得转座酶表达载体pCAMBIA1301-Tp,转化携带有PpMLE21转座子的拟南芥,通过卡那霉素和潮霉素共同筛选阳性植株; [0013] (2) transposase sequence (SEQIDN0.3) Construction of vector pCAMBIA1301 to obtain a transposase expression vector pCAMBIA1301-Tp, PpMLE21 transposon carries transformed Arabidopsis by hygromycin and kanamycin Su jointly screened positive plants;

[0014] (3)通过提高培养环境的温度,诱导转座酶表达和转座发生。 [0014] (3) by increasing the temperature of the culture conditions, and inducing transposase expression transposition occurs.

[0015] 本发明的MLE转座子,转座酶由可诱导启动子调控,人为调节转座酶表达,控制转座子跳跃和再跳跃,一方面解决了突变稳定性问题,回复突变也成为可控,可以在植物生长发育的任何时期,对植物的不同组织器官进行有目的的诱导转座突变,真正实现了对诱变在时空量上的全面调控。 [0015] MLE transposon of the present invention, transposase regulated by the inducible promoter, expression of the transposase manual adjustments, and then control jumps transposon jumping, on the one hand solves the problem of stability mutations, has become revertant controllable, can be at any stage of plant growth and development of different tissues and organs of plants carry out the purpose of transposon-induced mutation, truly comprehensive regulation of the amount of mutagenic in time and space. 另外,利用绿色荧光蛋白基因来报告MLE转座子的转座情况,可以简便、有效的对转座发生的植株进行筛选和转座子插入位点进行跟踪。 Further, to report the case of transposition using transposon MLE green fluorescent protein gene, can be simple and effective plants were screened transposition occurs and the transposon insertion site track.

附图说明 BRIEF DESCRIPTION

[0016] 图1:绿色荧光指示转座发生,其中a,b,c为不同转基因株系绿色荧光观察。 [0016] Figure 1: green fluorescence indicates transposition occurs, wherein a, b, c for the green fluorescent observation of different transgenic lines. WT为野生型。 WT is the wild type. 具体实施方式 detailed description

[0017] 下面结合具体实施步骤进一步阐述本发明。 [0017] The following steps with reference to specific embodiments further illustrate the invention. 应理解,这些实例仅用于说明本发明而不用于限制本发明的范围。 It should be understood that these examples are merely to illustrate the invention and not intended to limit the scope of the present invention. 除非特别指出,下列实例中未注明具体条件的实验方法,通常按照常规条件,如Sambrook.等主编的《分子克隆实验指南》中所述条件,或按照试剂盒陈述的步骤进行操作。 Unless otherwise indicated, the following examples no specific conditions of the experimental procedure, generally in accordance with conventional conditions, such as "Molecular Cloning A Laboratory Manual" Sambrook., Eds of the conditions, or follow the steps set forth in the kit.

[0018] 一、毛竹MLE转座子的获得 [0018] First, bamboo is obtained transposon MLE

[0019] 根据MLE转座子两端反向重复序列,利用引物MLE21(引物详情见表1),从毛竹基因组扩增出转座子PpMLE21的全长序列,序列为SEQIDN0.11。 [0019] The ends MLE inverted repeats of the transposon sequences, using primers MLE21 (primer as shown in Table 1), was amplified from genomic Phyllostachys edulis transposon PpMLE21 the full-length sequence, sequence SEQIDN0.11.

[0020] 二、拟南芥转化载体构建 [0020] Second, the transformation vector construct Arabidopsis

[0021] 1.转座子供体载体pBINm-gfp5_ER-miniPpMLE21 的构建 [0021] 1. Construction of transposon donor vector of pBINm-gfp5_ER-miniPpMLE21

[0022] (1)用BseRI (识别的序列为GAGGAG (N) 10 )切除毛竹PpMLE21转座子全长序列中转座酶的大部分序列,再用连接酶将PPMLE21转座子两侧的序列重新连上,命名为miniPpMLE21,(序列为SEQIDN0.2); [0022] (1) with BseRI (recognition sequence GAGGAG (N) 10) cut bamboo PpMLE21 transposon sequences most transit seat full length sequence of the enzyme, ligase and then both sides of the transposon sequences re PPMLE21 connected to the named miniPpMLE21, (sequence SEQIDN0.2);

[0023] (2) Xbal 酶切pBINm-gfp5_ER 载体,将pBINm-gfp5_ER 载体线性化; [0023] (2) Xbal digested pBINm-gfp5_ER vector, the vector was linearized pBINm-gfp5_ER;

[0024] (3)通过in-fusion技术将miniPpMLE21转座子插入到pBINm-gfp5_ER载体(商购可得)的Xbal酶切位点,获得重组载体pBINm-gfp5-ER-miniPpMLE21。 [0024] (3) by in-fusion techniques miniPpMLE21 transposon insertion into the vector pBINm-gfp5_ER (commercially available) of the Xbal cleavage site to obtain a recombinant vector pBINm-gfp5-ER-miniPpMLE21. 当miniPpMLE21转座子转座离开,后面的绿色荧光蛋白基因可以正常表达,可通过绿色荧光蛋白基因表达与否方便分析转座情况。 When miniPpMLE21 Transpositional leave behind the green fluorescent protein gene may be expressed normal or not may facilitate the analysis of transposition case by GFP expression.

[0025] 2.转座酶表达载体pCAMBIA1301_hspTp的构建 [0025] 2. Construction of vector pCAMBIA1301_hspTp transposase enzyme expression

[0026] (1)将PART7载体上的基因表达单元通过sacl和PstI从载体切下来;同时利用sad和PstI将pCAMBIA1301载体(商购可得)线性化;通过连接酶将来源于PART7载体的基因表达单元插入到PCAMBIA1301载体sacl和PstI两个酶切位点之间获得重组载体pCAMBIA1301-ex ; [0026] (1) The gene expression unit on a support by sacl PART7 excised from the vector and PstI; sad and PstI while using the pCAMBIA1301 vector (commercially available) linear; by the ligase gene derived from a Vector PART7 the expression unit is inserted into the recombinant vector pCAMBIA1301-ex pCAMBIA1301 between two carrier sacl and PstI restriction sites;

[0027] (2)利用引物MLE21-TP-5和MLE21-TP-3通过PCR技术将PpMLE21转座酶序列(序列为SEQIDN0.3)两端添加Sma I和BamH I酶切位点,PCR扩增后,用Sma I和BamH I双酶切PCR产物,同时利用Smal和BamHI酶切pCAMBIA1301_ex载体,通过连接酶将PpMLE21转座酶的cDNA序列引入pCAMBIA1301-ex载体Smal和BamHI两个酶切位点间,获得重组载体pCAMBIA1301-Tp ; [0027] (2) using primers MLE21-TP-5 MLE21-TP-3, and add both ends was amplified by PCR PpMLE21 transposase sequence (sequence SEQIDN0.3) Sma I and BamH I restriction sites, PCR expansion after the increase, the PCR product was double digested with Sma I and BamH I, while using Smal and BamHI digested pCAMBIA1301_ex vector, the cDNA sequence PpMLE21 introduced transposase vector pCAMBIA1301-ex two Smal and BamHI restriction sites by a ligase Room recombinant vector pCAMBIA1301-Tp;

[0028] (3)通过PCR技术将大豆热激启动子一Gmhspl7.5C启动子(序列SEQIDN0.4)从大豆的基因组扩增出来(引物为Gmhsp-5和Gmhsp-3),并引入sacl和ΚρηΙ两个酶切位点,经测序验证后,将Gmhspl7.5C启动子序列和pCAMBIA1301_Tp载体通过sacl和ΚρηΙ双酶切,用Gmhspl7.5C启动子替换pCAMBIA1301_Tp载体转座酶前的35S启动子,获得重组载体pCAMBIA1301-hspTp。 [0028] (3) a soybean heat shock promoters Gmhspl7.5C promoter (SEQ SEQIDN0.4) was amplified by PCR from genomic techniques of soybean out (primers Gmhsp-5 and Gmhsp-3), and the introduction and sacl ΚρηΙ two restriction sites, after sequencing, the promoter sequence and Gmhspl7.5C pCAMBIA1301_Tp sacl vector by double digestion and ΚρηΙ, before replacing the 35S promoter vector pCAMBIA1301_Tp transposase promoter with Gmhspl7.5C obtain The recombinant vector pCAMBIA1301-hspTp. 转座酶的表达可以通过环境温度来调控。 Transposase expression may be regulated by the ambient temperature.

[0029] 三、拟南芥的转化 [0029] Third, the transformation of Arabidopsis thaliana

[0030] 拟南芥种子在8%NaC10乙醇溶液中消毒6_8min,用无水乙醇洗涤待播种种子5次以上,用无棉纸吸干无水乙醇,在超净工作台上吹干,将种子均匀播撒在1/2MS培养基上,4°C沉积3天,然后放在拟南芥栽培室光下正常培养(栽培室条件:温度23°C,湿度60-70%,光强150umol.s-1.m_2)。 [0030] Arabidopsis seeds were sterilized in 8% NaC10 ethanol 6_8min, washed with ethanol 5 times or more seeds to be sown, blotted dry with lint-free paper ethanol, dried in a clean bench, the seeds uniformly sown in 1 / 2MS medium, 4 ° C deposited 3 days and then placed in the light chamber Arabidopsis cultivated under normal culture (growth chamber conditions: temperature 23 ° C, 60-70% humidity, light intensity 150umol.s -1.m_2). 待一周后长出1对真叶,将拟南芥幼苗移至基质中培养3周左右抽薹开花,去掉主薹,大约一周后,抽出的莲座叶侧枝大量开花,剪掉已经结荚的果荚,准备转化。 After one week grow one pair of true leaves, seedlings Arabidopsis move about 3 weeks culture matrix bolting, flowering, remove the main stalk, about a week later, a large number of collateral extracted rosette leaves flowering, pod has cut the pods ready to convert.

[0031] 取5mL农杆菌(EHA105)加入100mL含50 μ g/mL利福平(Rif )+50 μ g/mL卡纳青霉素(Kan )液体YEP (配方:酵母提取物,10g/L ;蛋白胨,10g/L ;NaCl,5g/L)中,28 °C,180rpm,摇菌过夜使其0D600达到L 0-1.2,4000rpm,离心15min,弃上清,加入lOOmL浸润法培养基(l/2MS+5%蔗糖),并加入表面活性剂Silwet-77,使其终浓度达0.02%。 [0031] Take 5mL Agrobacterium (EHA105) was added 100mL containing 50 μ g / mL rifampicin (Rif) +50 μ g / mL penicillin Fabio (Kan) liquid YEP (formulation: yeast extract, 10g / L; Peptone , 10g / L; NaCl, 5g / L) of, 28 ° C, 180rpm, bacteria overnight shaking it reaches 0D600 L 0-1.2,4000rpm, centrifugal 15min, supernatant was discarded, the medium was added lOOmL infiltration method (l / 2MS + 5% sucrose), and adding a surfactant Silwet-77, at a final concentration of 0.02%. 将重悬的农杆菌菌液置于培养皿中,将拟南芥花序完全浸没在溶液中,保持30s-60s。 The resuspended Agrobacterium cell suspension was placed in a petri dish, Arabidopsis inflorescence completely immersed in the solution, maintained 30s-60s. 用保鲜膜保湿,向周转柜中加水,暗室培养24h后,光下正常培养。 Moisturizing with plastic wrap, adding water to the working cabinet darkroom after 24h culture, culture under normal light. 隔5天左右可对已浸染植株再次浸染,提高转化率。 About every 5 days dip again been disseminated plants, improve the conversion rate. 种子成熟后适当减少浇水次数,待种荚发黄开裂时收获种子,用于下一代的筛选。 Appropriate to reduce watering frequency after seed maturation, seed pods to be made when harvesting seed yellow cracking, screening for the next generation.

[0032] 四、转基因阳性植株筛选和鉴定 [0032] Fourth, positive transgenic plants Screening and Identification

[0033] 导入转座子供体载体pBINm-gfp5_ER-miniPpMLE21的拟南芥种子播撒到含50mg/L卡那霉素的1/2MS培养基中筛选,抗性植株通过PCR验证(引物为MLE21),能扩增出1.2kb左右的目的条带为阳性植株 [0033] introducing the transposon donor vector pBINm-gfp5_ER-miniPpMLE21 to sow the seeds of Arabidopsis thaliana containing 50mg / 1 / 2MS medium L kanamycin screened, resistant plants was verified by PCR (primers MLE21), Article object could be amplified band of about 1.2kb positive plants

[0034] 以上述阳性拟南芥植株为受体,继续转化转座酶表达载体pCAMBIA1301_hspTp,将收获的拟南芥种子播撒到含50mg/L卡那霉素+50mg/L潮霉素的1/2MS培养基中筛选,转基因抗性植株通过PCR验证(引物为MLETP21-5和MLE21TP-3),能扩增出1.5kb左右的目的条带为阳性植株,该阳性转基因植株命名为双阳性植株。 [0034] In the above-described receptor positive Arabidopsis plants is continued conversion transposase expression vector pCAMBIA1301_hspTp, Arabidopsis thaliana sown seeds harvested to kanamycin + 50mg / L hygromycin containing 50mg / L card 1 / 2MS medium screening, transgenic resistant plants were confirmed by PCR (primers MLETP21-5 and MLE21TP-3), can be amplified with the purpose of article about 1.5kb positive plants, the plants were designated as positive transgenic plants were double positive.

[0035] 五、转座诱导和转座事件的鉴定 [0035] Fifth, the induction and identification of transposition transposition events

[0036] ( 1)双阳性植株的转座诱导 [0036] (a) inducing a double-positive plants transposition of

[0037] 42°C连续热激两次双阳性植株的幼苗,每次2小时可以诱导最高的转座活性。 [0037] 42 ° C heat shock two consecutive double-positive plant seedlings, each 2 hours to induce the highest transposition activity. 然后放在21°C恢复培养,同时增加培养环境的湿度,两天后在突光显微镜下观察突光。 21 ° C and then placed in recovery culture, while increasing the humidity in the culture environment, was observed two days after the light projecting projection light microscope.

[0038] (2)绿色荧光观察 [0038] (2) green fluorescent observation

[0039] 取拟南芥新鲜的叶片,手工撕成0.1-1.0mm薄片,将荧光显微镜的光源波长调到477nm处,观察绿色荧光的位置。 [0039] Arabidopsis taken fresh leaves, 0.1-1.0mm manually torn sheet will be transferred to the light source wavelength fluorescence microscopy at 477nm, green fluorescent observation position. 有绿色荧光点的细胞表明有转座发生。 There are cell green fluorescent dots indicates a transposition occurs. 从图1中可以看出和野生型相比,经过高温诱导后,可看到大量发生转座的细胞(绿色荧光的部分)。 As can be seen from Figure 1 and compared to wild type after high temperature induction, the cells can be seen transposition (part of the green fluorescence) high incidence. 这说明,构建的Mariner-LikeElement (MLE)转座子试剂盒可以在人工诱导下转座,并保持较高的转座效率。 This description, constructed Mariner-LikeElement (MLE) transposon transposition kit may at artificially induced, and maintain high efficiency of transposition.

[0040] 表1引物表 [0040] Table 1 Primer table

[0041] [0041]

Figure CN103710342AD00071

[0042] 以上所述是本发明的优选实施例,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明所述原理的前提下,还可以作出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。 [0042] The above is a preferred embodiment of the present invention, it should be noted that those of ordinary skill in the art, in the present invention without departing from the principles of the premise, further improvements and modifications may be made, these improvements and modifications should also be regarded as the protection scope of the present invention.

[0043] [0043]

Figure CN103710342AD00081
Figure CN103710342AD00091
Figure CN103710342AD00101
Figure CN103710342AD00111

Claims (5)

  1. 1.一种MLE转座子试剂盒,其特征在于包括含有核苷酸序列为SEQIDN0.2的miniPpMLE21的载体以及含有核苷酸序列为SEQIDN0.3的转座酶序列的载体。 A kit MLE transposons, comprising the nucleotide sequence comprising the SEQIDN0.2 miniPpMLE21 carrier and vectors comprising nucleotide sequences of the transposase sequence of SEQIDN0.3.
  2. 2.根据权利要求1所述的试剂盒,其中含有核苷酸序列为SEQIDN0.2的miniPpMLE21的载体是将miniPpMLE21转座子构建到pBINm-gfp5_ER载体上,获得转座子供体载体pBINm-gfp5_ER-miniPpMLE21ο The kit of claim 1, wherein the carrier comprising the nucleotide sequence of SEQIDN0.2 miniPpMLE21 is miniPpMLE21 transposon constructs to pBINm-gfp5_ER vector, transposon donor vector is obtained pBINm-gfp5_ER- miniPpMLE21ο
  3. 3.根据权利要求1所述的试剂盒,所述的含有核苷酸序列为SEQIDN0.3的转座酶序列的载体是转座酶序列构建到PCAMBIA1301载体上。 3. The kit of claim 1, said vector containing a nucleotide sequence of a transposase sequences SEQIDN0.3 transposase sequence is built onto PCAMBIA1301 carrier.
  4. 4.上述MLE转座子试剂盒在诱变转基因植物中的应用。 4. Application of the above MLE transposon mutagenesis kit in transgenic plants.
  5. 5.根据权利要求1所述的应用,所述的应用包括如下步骤:(1)将miniPpMLE21转座子构建到pBINm-gfp5_ER载体上,获得转座子供体载体pBINm-gfp5-ER-miniPpMLE21,转化拟南芥,通过卡那霉素筛选阳性植株;(2)转座酶序列(SEQIDN0.3)构建到pCAMBIA1301载体,获得转座酶表达载体pCAMBIA1301-Tp,转化携带有PpMLE21转座子的拟南芥,通过卡那霉素和潮霉素共同筛选阳性植株;(3 )通过提高培养环境的温·度,诱导转座酶表达和转座发生。 5. The use according to claim 1, said application comprising the steps of: (1) Construction of a transposon to miniPpMLE21 pBINm-gfp5_ER vector, transposon donor vector is obtained pBINm-gfp5-ER-miniPpMLE21, conversion Arabidopsis plants are screened by kanamycin positive; (2) transposase sequence (SEQIDN0.3) Construction of vector pCAMBIA1301 to obtain a transposase expression vector pCAMBIA1301-Tp, carrying PpMLE21 transformed Arabidopsis transposon mustard, by kanamycin and hygromycin common positive screening plants; (3) by raising the temperature-culture of the environment, inducing transposase expression and transposition occurs.
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