WO2016029630A1 - miR528的调控位点及其应用 - Google Patents

miR528的调控位点及其应用 Download PDF

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WO2016029630A1
WO2016029630A1 PCT/CN2015/070864 CN2015070864W WO2016029630A1 WO 2016029630 A1 WO2016029630 A1 WO 2016029630A1 CN 2015070864 W CN2015070864 W CN 2015070864W WO 2016029630 A1 WO2016029630 A1 WO 2016029630A1
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mir528
rice
sequence
rna
seq
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French (fr)
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曹晓风
杨荣新
宋显伟
吴建国
李毅
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中国科学院遗传与发育生物学研究所
北京大学
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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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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)

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  • the invention relates to plant breeding, in particular to the application of rice miR528 encoding gene MIR528 and miR528 regulatory sites (Os06g06050, Os06g37150, Os08g04310, Os08g42640 and Os07g38290) in cultivating crop varieties with suitable growth period and high antiviral ability.
  • MIR528 and miR528 regulatory sites Os06g06050, Os06g37150, Os08g04310, Os08g42640 and Os07g38290
  • Flowering time (or heading stage in cereal crops) is the process by which plants change from vegetative growth to reproductive growth. In order to successfully reproduce, plants need to bloom and complete their life history before the arrival of extreme environments (such as high temperature, freezing, etc.). Therefore, the flowering time directly determines whether the plant can complete the life history in its growing area, and is also one of the important agronomic traits that determine crop yield.
  • the cultivation of crop varieties suitable for growth period is also an important factor to be considered in the process of crop breeding. Taking rice as a food crop, rice is a typical short-day plant, that is, short-day sunshine promotes rice heading and long-day sunshine delays heading.
  • cultivated rice and wild rice (O. rufipogon) are thought to have originated from common ancestors.
  • Wild rice is distributed in low latitudes from southern China to eastern India.
  • the limit of distribution latitude is 28° north latitude;
  • the adaptation range of cultivated rice has been greatly expanded, and there is still a distribution of cultivated rice near 45° north latitude. It shows that with the continuous domestication in the process of human breeding, the adaptation area of rice has been greatly expanded to better meet the food needs of human beings. Due to the different latitudes of different regions, the flowering time of crops will also be affected. In order to maximize crop yield, it is particularly important to breed suitable varieties during the growth period. Therefore, it is of great significance to elucidate the molecular mechanism of flowering time of plants, especially food crops, and to isolate genes that control their flowering time, and then apply them to guide molecular breeding and variety improvement.
  • Pests and diseases are important factors affecting the normal growth of plants and the yield of crops. Therefore, the prevention and control of pests and diseases has always been an important part of agricultural production. In the process of prevention and control of pests and diseases, modern agriculture faces enormous contradictions: on the one hand, the use of pesticides plays an important role in ensuring the stable production of crops. On the other hand, excessive use of pesticides also poses great risks to the environment and food safety. It is of great significance to study the plant's own mechanism of resistance to pests and diseases, to explore the genetic resources of plant pests and diseases in nature, and to guide the molecular breeding of crops against pests and diseases, to ensure the stable production of crops and food safety, to protect the ecological environment, and to maintain sustainable agricultural development.
  • Rice stripe virus is an RNA virus transmitted by Laodelphax striatellus.
  • the rice stripe caused by rice is a common rice disease, which can cause severe rice yield reduction in the epidemic year. Plants of rice stripe disease often show dwarfing, reduced tillering, early heart disease curling and drooping, showing "false heart”symptoms; although late onset only on the flag leaf or leaf sheath with fading spots, but Ear dysplasia or malformation, forming "false white spikes.”
  • Rice stripe disease was first discovered in Japan in 1931. It was first reported in China in 1964. The area and area of the disease were obviously enlarged. It was an important viral disease in rice in the 1980s and 1990s.
  • Non-coding small-molecule RNA with regulatory function refers to an RNA molecule with a length of 21-24 nt, which is mostly evolutionarily sequence-conserved. They do not encode proteins, but instead degrade mRNA or inhibit translation at the post-transcriptional level in the form of RNA, and participate in the regulation of the maintenance of eukaryotic chromosome structure, defense against viruses, and regulation of growth and development.
  • the discovery of small-molecule RNA not only reveals the nature of gene silencing that has plagued scientists for many years, but also changes people's traditional understanding of genes. Especially the discovery of miRNA is a major breakthrough in life science research in recent years. Small RNA in 2002 And for two consecutive years in 2003, it was named "Top Ten Scientific and Technological Progress of the Year" by Science magazine.
  • miRNAs recognize target mRNAs by means of sequence complementation, which in turn directs degradation of target mRNA or inhibits translation of its proteins, thereby negatively regulating expression of target sites.
  • the present invention through studying the biological function of miR528, finds that it plays an important regulatory role in rice heading stage and antiviral, and affects rice heading stage and antiviral properties by regulating miR528 expression level. Therefore, the present invention is important for rice crops. The improvement of two important agronomic traits at heading stage and antiviral is of great significance.
  • the present invention demonstrates that miR528 is capable of simultaneously regulating the expression of multiple target mRNAs to exert biological functions.
  • miR528 is capable of simultaneously regulating the expression of multiple target mRNAs to exert biological functions.
  • rice heading time was advanced, and it was hypersensitive to rice stripe virus RSV; while rice plants with down-regulated miR528 expression showed delayed heading time and strong anti-RSV virus ability.
  • miR528 has important regulatory functions in rice heading and antiviral.
  • the present invention analyzes the polymorphism of the MIR528 gene locus in different rice cultivars, and finds that there are two distinct insertion (or deletion) sequences in the regulatory region of the upstream promoter of MIR528, and the expression level of the mature miR528 is Important regulatory role. Therefore, the polymorphism of MIR528 gene locus in different rice varieties can be used as a molecular marker in the genetic breeding process, and it can be applied to the screening of two important agronomic shapes of rice heading stage and antiviral.
  • the invention detects the conserved presence of miR528 in important grass crops such as corn, sorghum and sugarcane, indicating that miR528 has extensive regulation on flowering time and antiviral ability of gramineous plants. Therefore, the MIR528 gene can be used as a molecular marker in the genetic breeding process of several crops mentioned above.
  • the rice MIR528 gene and its upstream regulatory sequences provided by the invention have the following characteristics:
  • SEQ ID NO: 1 in the Sequence Listing consists of a total of 3154 deoxyribonucleotides from the rice MIR528 gene and its upstream and downstream regulatory sequences.
  • the deoxyribonucleotide from position 2149 to position 2800 in sequence 1 is the MIR528 gene transcription sequence (wherein the mature miR528 gene corresponds to the 2231 bp to 2251 bp transcript of SEQ ID NO: 1, for a total of 21 nt, ie SEQ ID NO: 2), and the rest Part of the upstream and downstream regulatory sequences.
  • MIR528 gene sequence and the target sequence regulated by mature miR528 in the present invention are all protected in molecular breeding markers, transgenic lines, and transgenic new varieties.
  • this content relates to the following scenarios:
  • the upstream regulatory region of MIR528 gene (Os03g03724) derived from different rice varieties has sequence polymorphism (as shown in Figure 6), which can be involved in the regulation of mature miR528 expression level, thereby optimizing rice heading stage by hybridization or transgenic method. And rice stripe disease resistance.
  • Rice MIR528 has one or more deoxynucleotide mutations (including nucleotide sequence mutations caused by physical or chemical methods) at position 1 and Figure 1 corresponding regulatory sequences.
  • Target genes Os06g06050, Os06g37150, Os08g04310, Os08g42640, Os07g38290 regulated by miR528 have one or more deoxynucleotide mutations (including nucleotide sequence mutations caused by physical or chemical methods) at the position of the corresponding recognition sequence in Figure 1.
  • MIR528 and its recognition target genes Os06g06050, Os06g37150, Os08g04310, Os08g42640, Os07g38290 were constructed on an expression vector to transform plant cells, tissues or organs of interest, and transgenic lines with optimized rice heading stage and rice stripe disease resistance were obtained.
  • the plant of interest is any monocotyl or dicotyledonous plant. More preferably, the monocot plants are rice, corn, wheat, sorghum, millet, barley, oats, rye, and the dicots are cotton, soybean, tobacco, canola, tomato.
  • FIG. 1 shows the regulatory site of miR528 in rice.
  • miR528 has five target mRNAs in rice, which are transcribed from partial sequences of genes Os06g06050, Os06g37150, Os08g04310, Os08g46240, Os07g38290 (ie, SEQ ID NOs: 14, 15, 16, 17 and 18, respectively).
  • the recognition site for miR528 is a nucleotide sequence of approximately 21 nt in length, showing the reverse complement of the sequence in which miR528 and the target mRNA are present at the corresponding recognition site.
  • FIG. 2 shows the miR528 overexpression vector map.
  • the MIR528 gene sequence was ligated into the binary expression vector pCAMBIA2300, driven by the constitutive promoter ACTIN1 promoter.
  • the vector was used to transform wild type rice, thereby overexpressing miR528 in rice.
  • Figure 3 shows the expression of mature miR528 in miR528 overexpression and knockout rice lines.
  • A shows that small RNA Northem hybridization shows the expression level of miR528 in the mir528 mutant and its wild type control Dongjin, U6 and 5s RNA as internal parameters.
  • B shown Northern RNA hybridization revealed expression levels of miR528 in three independent miR528 overexpressing transgenic lines and their wild type controls.
  • Figure 4 shows the overexpression of miR528 in Beijing field and the phenotype of the heading stage of knockout rice lines. The results showed that the mir528 mutant showed late flowering compared to its wild type control, while the miR528 overexpressing transgenic plants showed early flowering.
  • Figure 5 shows miR528 overexpressing the phenotype of rice lines (miR528OE) and mir528 mutants after RSV inoculation.
  • Mock is a control plant that is not vaccinated.
  • RSV indicates plants inoculated with RSV.
  • Figure 6 shows the sequence insertion (or deletion) of the upstream regulatory region of the MIR528 gene in different varieties.
  • FIG. 7 miR528 expression levels differ in different rice varieties.
  • the expression level of miR528 in rice varieties containing MIR528-A1 allele was higher than that of rice varieties containing MIR528-A2 allele.
  • miR156, miR172, U6 and 5s RNA were used as control and internal reference.
  • FIG. 8 conserves of the rice MIR528 gene in monocots.
  • the plant transcriptional assembly database http://planttajcvi.org/
  • the plant transcriptional assembly database http://planttajcvi.org/
  • the alignment showed that the miR528 precursor was conserved in grass, sorghum, sugarcane and other grass crops, especially in the mature miR528 and its asterisk chain (miR528*) region, indicating that miR528 is conserved in these species.
  • Table 1 shows the allelic distribution of the upstream regulatory region of MIR528 gene in wild rice and some common rice varieties. It contains the identified MIR528 allelic forms of wild rice rufipogon, 9 indica varieties and 17 indica varieties, their origin and distribution.
  • the recognition site of miR528 is a nucleotide sequence of about 21 nt in length, and miR528 has a good sequence match with the target mRNA at the corresponding recognition site, as shown in FIG.
  • the corresponding 21 nt miR528 is referred to as the mature miR528 gene, and its transcribed sequence corresponds to 2231 bp to 2251 bp (SEQ ID NO: 2) of SEQ ID NO: 1.
  • This example is a general description of the construction of the pCAMBIA2300-pACTIN1-MIR528 vector.
  • cx353 (5'-CTGCAGGCAGCAGCCACAGCAAAATTTGGTTTGGGA-3', SEQ ID NO: 3) was used as a forward primer to cx352 (5'-CTGCAGTTCTTTCGCATCTAATCCCCGATCTGA-3', SEQ ID NO: 4).
  • the reverse primer (where the 5' end of the forward and reverse primers respectively contain the PstI restriction site sequence), the MIR528 gene sequence is amplified by high-fidelity DNA polymerase PCR (ie, 2149 bp to 2800 bp of SEQ ID NO: 1). Deoxyribonucleotide sequence).
  • the PCR product was electrophoresed and ligated to the cloning vector P-EASY blunt, transformed into E. coli DH5 ⁇ , and plated to obtain a monoclonal.
  • the correct cloned plasmid was selected and sequenced, and the MIR528 fragment was digested with PstI.
  • the vector pCAMBIA2300 was digested with PstI overnight, and the terminal was dephosphorized and recovered.
  • the recovered MIR528 fragment was ligated to the vector fragment in a molar ratio of about 3:1 (T 4 Ligase )overnight.
  • the ligated product was transformed into E.
  • RNA hybridization was affected by small RNA hybridization.
  • the specific method was as follows: We extracted the mutant mir528 and its wild type control Dongjin (http://signal.salk.edu/cgi) according to the conventional method. Total RNA of -bin/RiceGE), 15 ug of total RNA was quantified, and an equal volume of 8 M LiCl was added to precipitate at 4 ° C for 2 hours, centrifuged at 12,000 rpm for 5 minutes, and the supernatant was added to add 1/10 volume of 3 M sodium acetate and 3 volumes.
  • RNA loading buffer was added, and the mixture was denatured by heating at 90 ° C for 3 minutes, and then inserted into ice.
  • the small RNA was electrophoresed through 15% PAGE gel containing 8 M urea for 2 hours, and then EB-containing.
  • 0.5x TBE was stained with PAGE and photographed to reveal a clear 5sRNA band. After washing the PAGE gel with 0.5xTBE, the film was transferred by an electric film transfer machine and UV-crosslinked.
  • the expression level of mature miR528 showed that the expression level of MiR528 was up-regulated in the transgenic lines (miR528-OE1, miR528-OE2 and miR528-OE3) compared with wild-type Nipponbare, which achieved the purpose of over-expression of miR528 in rice.
  • Figure 3B The expression level of mature miR528 showed that the expression level of MiR528 was up-regulated in the transgenic lines (miR528-OE1, miR528-OE2 and miR528-OE3) compared with wild-type Nipponbare, which achieved the purpose of over-expression of miR528 in rice.
  • Figure 3B The expression level of mature miR528 showed that the expression level of MiR528 was up-regulated in the transgenic lines (miR528-OE1, miR528-OE2 and miR528-OE3) compared with wild-type Nipponbare, which achieved the purpose of over-expression of miR528 in rice.
  • Example 4 Phenotypic analysis of heading stage of miR528 knockout and overexpressing rice lines
  • the statistical analysis of the heading stage revealed that the miR528 knockout mutant mir528 strain showed a slight delay (about 3.5 days) when the heading time (1 cm of rice main spike) was taken out compared with the wild type control Dongjin.
  • miR528 overexpressed rice lines (miR528-OE1, miR528-OE2 and miR528-OE3) had an early heading period, see Figures 4A and B.
  • the above results indicate that miR528 has a positive regulatory effect on rice heading.
  • RSV showed a strong sensitive phenotype (slow plant, yellowing of leaves); while showing moderate susceptibility (partial leaf yellowing) relative to wild type control (Dongjin-RSV), miR528 deletion mutant mir528 (mir528-RSV) ) showed a strong anti-RSV phenotype (the plants grew well and no obvious susceptible plants were found). This result indicates that miR528 has a negative regulatory effect on rice resistance to rice stripe virus (RSV).
  • RSV rice stripe virus
  • MIR528-A1 and MIR528-A2 allelic forms of the upstream regulatory region of MIR528 from different varieties (ie MIR528-A1 and MIR528-A2, of which MIR528-A1 It indicates that a base sequence exists at -590 bp and -837 bp upstream of the transcription start site, and MIR528-A2 indicates that the corresponding base sequence is deleted at -590 bp and -837 bp upstream of the transcription start site).
  • MIR528-A2 indicates that the corresponding base sequence is deleted at -590 bp and -837 bp upstream of the transcription start site.
  • the corresponding allelic form is MIR528-A1
  • the identified japonica varieties Nipponbare, Zhonghua 11, Dongjin, etc.
  • There are 30 bp and 38 bp sequence deletions at the -590 bp and -837 bp sites upstream of the transcription start site the corresponding allelic form is MIR528-A2
  • the labeled DNA sequence 5'-GTGCTCACTCTCTTCTGTCA-3' is a hybridization probe, SEQ ID NO: 11) and miR172 (a 32 P isotopically labeled DNA sequence 5'-GATGCAGCATCATCAAGATTCT-3' is a hybridization probe, SEQ ID NO: 12
  • U6 and 5s RNA were used as hybridization and loading internal reference, respectively, see Figure 7.
  • Example 7 MIR528 is conserved in gramineous plants
  • the present invention investigates the conservation of MIR528 in plants.
  • the alignment showed that the miR528 precursor was conserved in grass, sorghum, sugarcane and other grass crops, especially in the mature miR528 (ie 21nt miR528) and its asterisk chain (miR528*) region, see Figure 8.

Abstract

提供了水稻miR528编码基因MIR528及miR528调控位点（Os06g06050、Os06g37150、Os08g04310、Os08g42640和Os07g38290）在控制水稻生育期和病毒抗性方面的用途。通过miR528过量表达和敲除水稻株系的研究，发现miR528能够影响水稻抽穗期和抗水稻条纹叶枯病毒（RSV）能力，过量表达miR528会导致抽穗期提前和对RSV病毒敏感，而敲除miR528则会导致抽穗期延迟和抗RSV病毒。MIR528在水稻不同品种中的存在直接影响miR528表达水平的调控序列多态性，可用于水稻杂交育种及分子辅助育种。

Description

miR528的调控位点及其应用 技术领域：
本发明涉及植物育种，具体涉及水稻miR528编码基因MIR528及miR528调控位点(Os06g06050、Os06g37150、Os08g04310、Os08g42640和Os07g38290)在培育生育期适宜和高抗病毒能力的作物品种中的应用。
技术背景：
开花时间(或谷类作物中的抽穗期)是植物从营养生长到生殖生长转变的过程，为了成功繁衍，植物需要在极端环境(如高温、冷冻等)到来之前开花并完成其生活史。因此，开花时间直接决定着植物能否在其生长地域完成生活史，同时也是决定作物产量的重要农艺性状之一。培育生育期适宜的作物品种，也是作物育种过程中需要考虑的重要因素。以粮食作物水稻为例，水稻是典型的短日照植物，即短日照促进水稻抽穗而长日照延迟其抽穗。现在栽培稻和野生稻(O.rufipogon)被认为起源于共同的祖先，野生稻分布在中国南部到印度东部等低纬度地区，其分布纬度的极限是北纬28°；而与其相对应的是，现在栽培稻的适应范围已经大大扩展，目前在北纬45°附近仍有栽培稻的分布。说明随着人类育种过程中的不断驯化，水稻的适应地域被大大扩展，从而更好地满足人类的粮食需求。由于不同地域的纬度不同，作物开花时间亦会受到影响，为了实现作物产量最大化，选育生育期适宜的品种显得尤为重要。因此，阐明植物尤其是粮食作物开花时间的分子机制，分离控制其开花时间的基因，进而应用于指导分子育种和品种改良具有重要意义。
病虫害是影响植物正常生长和农作物产量的重要因素，因此，病虫害的防治一直是农业生产的重要环节。在病虫害的防治过程中，现代农业面临着巨大的矛盾：一方面，农药使用在保障农作物稳产中功不可没，另一方面，过度使用农药也给环境及食品安全带来极大风险。研究植物自身抗病虫害机制，发掘自然界中植物抗病虫害基因资源，进而指导作物抗病虫害的分子育种，对于保障农作物稳产和食品安全、保护生态环境、维持农 业可持续发展有着重要意义。水稻条纹叶枯病毒(RSV)是一种由灰飞虱传播的RNA病毒，由其引起的水稻条纹叶枯病(rice stripe)是一种常见水稻病害，在流行年份能造成水稻的严重减产。水稻条纹叶枯病的发病植物往往表现出矮化，分蘖减少，发病早的心叶卷曲下垂，呈“假枯心”症状；发病晚的虽只在剑叶或叶鞘上有退色斑，但穗发育不良或畸形，形成“假白穗”。水稻条纹叶枯病最早于1931年在日本发现，我国最早于1964年报道，随后发病地区和面积明显扩大，在80-90年代一直是水稻重要病毒病害。自本世纪初以来日趋严重，2004年仅江苏省发病面积就达到133万hm2，随后在全国范围内大面积流行。根据全国农技推广中心的病害测报，该病发病面积连续5年一直在不断扩大，2008年达到200万hm2。
miRNA和siRNA的研究是近年来国际生命科学研究的热点之一。具有调节功能的非编码小分子RNA是指长度为21-24nt左右，大多在进化上具有序列保守性的RNA分子。它们不编码蛋白质，而是以RNA的形式在转录后水平上通过降解mRNA或抑制其翻译，参与调节真核生物染色体结构的维持、防御病毒及调控生长发育等过程。小分子RNA的发现不仅揭示了困扰科学家们多年的基因沉默现象的本质，还改变了人们对基因的传统认识，特别是miRNA的发现是近年来生命科学研究的一个重大突破，小分子RNA在2002和2003连续两年被《Science》杂志评为“年度十大科技进展”。
大量的实验证据表明miRNA在动植物的生长、发育、环境适应等各个生物学过程中具有重要的调控作用。在植物体内，miRNA通过序列互补的方式识别靶mRNA，进而指导靶mRNA的降解或抑制其蛋白质的翻译，从而负调控靶位点的表达。虽然小分子RNA在理论上的研究已经取得了巨大的进步，但是如何在生产上利用小分子RNA，为现代农业服务，产生经济价值是现代生物领域的一大难题。本发明通过对miR528生物学功能的研究，发现其在水稻抽穗期和抗病毒上具有重要调控作用，并通过调节miR528表达水平影响水稻抽穗期及抗病毒特性，因此，本发明对于重要粮食作物水稻在抽穗期和抗病毒两个重要农艺性状的改良具有重要的意义。
发明内容：
本发明证实miR528能够同时调控多个靶mRNA表达而发挥生物学功能。通过在水稻中过量表达miR528使水稻抽穗时间提前，并且对水稻条纹病毒RSV表现出超敏感；而miR528表达被下调的水稻植株则会表现出抽穗时间延迟和强抗RSV病毒能力。通过系统研究，我们发现miR528在水稻抽穗期和抗病毒方面有着重要调控功能。
本发明分析了MIR528基因位点在不同水稻品种中的多态性，发现MIR528上游启动子调控区域存在两段明显的插入(或缺失)序列，而且这种多态性对于成熟miR528的表达量有重要调控作用。因此MIR528基因位点在不同水稻品种中的多态性可以在遗传育种过程中作为分子标记，应用于水稻抽穗期和抗病毒两个重要农艺形状的筛选。
本发明在玉米、高粱、甘蔗等重要禾本科作物中检测到miR528保守存在，说明miR528对禾本科植物开花时间和抗病毒能力的调控广泛存在。因此，MIR528基因可以作为分子标记应用于上述几种农作物的遗传育种过程中。
本发明所提供的水稻MIR528基因及其上游调控序列具有以下特性：
1、序列表中的SEQ ID NO：1
序列表中的SEQ ID NO：1由水稻MIR528基因及其上下游调控序列共3154个脱氧核糖核苷酸组成。序列1中自2149位至2800位脱氧核糖核苷酸为MIR528基因转录序列(其中成熟miR528基因对应SEQ ID NO：1的2231bp-2251bp的转录序列，共计21nt，即SEQ ID NO：2)，其余部分为上下游调控序列。
2、本发明涉及的MIR528基因序列以及受到成熟miR528调控的靶位点序列在分子育种标记、转基因株系、转基因新品种中的应用均属于保护内容。
具体地，本内容涉及以下方案：
来源于水稻MIR528基因(Os03g03724)，具有调控水稻抽穗期和水稻条纹叶枯病抗性的功能，其特征在于其脱氧核苷酸序列是序列列表中的序列1。
来源于水稻Os06g06050、Os06g37150、Os08g04310、Os08g42640、Os07g38290基因，具有调控水稻抽穗期和水稻条纹叶枯病抗性的功能， 其特征在于其脱氧核苷酸序列是可转录成含有图1对应的核糖核酸序列，该序列的脱氧核苷酸序列能够被miR528特异性识别。
来源于不同水稻品种的MIR528基因(Os03g03724)上游调控区具有序列多态性(如图6所示)，该多态性能参与成熟miR528表达水平的调控，从而通过杂交或转基因的方式优化水稻抽穗期和水稻条纹叶枯病抗性。
水稻MIR528在序列1和图1对应调控序列位置发生了一个或一个以上脱氧核苷酸突变(包括用物理或化学的方法导致的核苷酸序列突变)。
受miR528调控的靶基因Os06g06050、Os06g37150、Os08g04310、Os08g42640、Os07g38290在图1对应识别序列位置发生了一个或一个以上脱氧核苷酸突变(包括用物理或化学的方法导致的核苷酸序列突变)。将MIR528及其识别靶基因Os06g06050、Os06g37150、Os08g04310、Os08g42640、Os07g38290构建在表达载体上转化目的植物细胞、组织或器官，能够获得具有优化水稻抽穗期和水稻条纹叶枯病抗性的转基因株系。优选地，目的植物为任何单子叶或双子叶的植物。更优选地，单子叶植物为水稻、玉米、小麦、高粱、谷子、大麦、燕麦、黑麦，双子叶植物为棉花、大豆、烟草、油菜、番茄。
附图说明
图1所示是miR528在水稻中的调控位点。miR528在水稻有5个靶mRNA，分别由基因Os06g06050、Os06g37150、Os08g04310、Os08g46240、Os07g38290的部分序列转录而成(即，分别是SEQ ID NO：14，15，16，17和18)。miR528的识别位点是长度约为21nt的核苷酸序列，图中显示了miR528与靶mRNA在对应的识别位点存在序列的反向互补。
图2所示的是miR528过量表达载体图谱。MIR528基因序列在组成型启动子ACTIN1启动子的驱动下，连入到双元表达载体pCAMBIA2300。利用该载体转化野生型水稻，进而在水稻中过量表达miR528。
图3所示的是miR528过量表达和敲除水稻株系中成熟miR528表达量鉴定。A所示的是小RNA Northem杂交显示mir528突变体及其野生型对照Dongjin中miR528的表达水平，U6和5s RNA作为内参。B所示的 是小RNA Northern杂交显示3个独立的miR528过表达转基因系和其野生型对照中miR528的表达水平。
图4所示的是北京田间miR528过量表达和敲除水稻株系抽穗期表型。结果显示相对也其野生型对照，mir528突变体表现出晚花，而miR528过表达转基因植株则表现出早花。
图5所示miR528过量表达水稻株系(miR528OE)和mir528突变体接种RSV后的表型。Mock为未接种病毒的对照植株。RSV表示接种RSV的植株。
图6所示的是MIR528基因上游调控区在不同品种中存在的序列插入(或缺失)。通过对来自不同水稻品种的MIR528基因及其顺式调控序列分析，我们发现来自不同品种的MIR528上游调控区域序列存在两种不同的等位形式(MIR528-A1和MIR528-A2)。其中相对于MIR528-A1序列来说，MIR528-A2在转录起始位点上游-590bp和-837bp位点分别存在30bp和38bp的序列缺失。
图7miR528表达水平在不同水稻品种中存在差异。含有MIR528-A1等位基因的水稻品种中miR528的表达水平整体高于含有MIR528-A2等位基因的水稻品种。其中miR156、miR172、U6和5s RNA作为对照和内参。
图8水稻MIR528基因在单子叶植物中的保守性。利用水稻miR528前体序列(形成miR528前体茎-环结构的序列)通过序列比对的方式搜索了植物转录组装数据库(http：//planttajcvi.org/)。比对结果显示，miR528前体在玉米、高粱、甘蔗等禾本科作物中保守存在，尤其是在成熟miR528及其星号链(miR528*)区域高度保守，说明miR528在这些物种保守存在。
表1野生稻及部分常见水稻品种中MIR528基因上游调控区等位特征分布。其中包含已鉴定的野生稻rufipogon、9个籼稻品种和17个粳稻品种名称、原产地及分布所属的MIR528等位形式。
具体实施方式
下面结合具体的实施例进一步阐述本发明。需要说明的是，这些实施例仅仅是为了说明本发明，而不能以任何方式构成对本发明权利要求范围 的限制。
下述实施例中所用方法如无特别说明均为常规方法，具体步骤可参见：《Molecular Cloning：A Laboratory Manual》(Sambrook，J.，Russell，David W.，Molecular Cloning：A Laboratory Manual，3rd edition，2001，NY，Cold Spring Harbor)或相关产品。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1、miR528识别位点的获得
利用植物miRNA与靶mRNA存在序列互补的原理，通过水稻全基因组扫描，我们获得了水稻中miR528的识别位点。在水稻中miR528的靶mRNA有5个，分别由Os06g06050、Os06g37150、Os08g04310、Os08g46240、Os07g38290基因转录而成。miR528的识别位点是长度约为21nt的核苷酸序列，miR528与靶mRNA在对应的识别位点存在很好的序列匹配，见附图1。将对应的21nt的miR528称为成熟miR528基因，其转录序列对应SEQ ID NO：1的2231bp-2251bp(SEQ ID NO：2)。
实施例2、MIR528基因过量表达载体的构建
本实施例是关于pCAMBIA2300-pACTIN1-MIR528载体构建一个通用说明。
首先以日本晴水稻(Nipponbare)DNA为模板，以cx353(5’-CTGCAGGCAGCAGCCACAGCAAAATTTGGTTTGGGA-3’，SEQ ID NO：3)为正向引物，以cx352(5’-CTGCAGTTCTTTCGCATCTAATCCCCGATCTGA-3’，SEQ ID NO：4)为反向引物(此处正反向引物的5’端分别包含有PstI酶切位点序列)，利用高保真DNA聚合酶PCR扩增MIR528基因序列(即SEQ ID NO：1的2149bp-2800bp的脱氧核糖核苷酸序列)。电泳回收PCR产物，并连接到克隆载体P-EASY blunt上，转化大肠杆菌DH5α，涂板获取单克隆。选择测序正确的克隆提取质粒，用PstI酶切，回收MIR528片段；同时，将载体pCAMBIA2300用PstI酶切过夜，末端脱磷并回收。将回收的MIR528 片段与载体片段以摩尔比约3∶1的量进行连接(T4Ligase
Figure PCTCN2015070864-appb-000001
)过夜。第二天将连接产物转化大肠杆菌DH5α，并于37℃培养过夜，可获得单克隆。选取多个单克隆进行液体培养基培养，同时利用引物cx245(5’-GACAAATGCAGCCTCGTGCGGA-3’，SEQ ID NO：5)和cx352(5’-CTGCAGTTCTTTCGCATCTAATCCCCGATCTGA-3’，SEQ ID NO：6)对其进行PCR验证，对于PCR阳性的克隆送测序，最后提取测序正确的克隆质粒，从而获得目的载体用作转基因水稻的转化。图谱见附图2。
实施例3、miR528敲除和过量表达水稻株系的鉴定
通过搜索水稻数据库RiceGE(http：//signal.salk.edu/cgi-bin/RiceGE)，我们从中找到一个T-DNA插入位点位于miR528前体(pre-miR528)序列内的水稻突变体株系(PFG_3A-11864.R)，我们将其命名为mir528。
我们通过小RNA杂交的方法鉴定了其中成熟miR528的表达水平是否受到影响，具体方法如下：我们按常规方法提取了突变体mir528和其野生型对照Dongjin(http：//signal.salk.edu/cgi-bin/RiceGE)的总RNA，定量后取15ug总RNA，加入等体积8M LiCl在4℃下沉淀2小时，12000rpm离心5分钟，取上清加入1/10体积的3M醋酸钠和3倍体积的乙醇于-80℃下沉淀4小时，12000rpm离心10分钟所得沉淀即为富集的小RNA。小RNA溶于去离子甲酰胺后，加入RNA上样缓冲液，90℃加热变性3分钟后立即插到冰上，小RNA通过含8M尿素的15％PAGE胶电泳2小时后，用含有EB的0.5xTBE染PAGE胶并照相，能看到清晰的5sRNA条带。0.5xTBE清洗PAGE胶后，利用电转膜仪转膜并紫外交联。我们利用磷酸激酶将DNA序列(5’-GCTCCTCTGCATGCCCCTTCCA-3’，SEQ ID NO：7)末端标记上32P同位素信号，以此作为探针与miR528杂交，同时用上述方法32P同位素标记DNA序列(5’-TGTATCGTTCCAATTTTATCGGATGT-3’，SEQ ID NO：8)与水稻内源的U6RNA杂交，并以U6作为杂交内参，最后用磷屏记录放射信号。小RNA Northem杂交结果显示，mir528株系中miR528被完全敲除，见附图3A。另一方面，我们将实施例2构建好的含有水稻內源强启动子ACTIN1驱动MIR528序列的pCAMBIA2300载体 (pCAMBIA2300-pACTIN1-MIR528)转入野生型日本晴水稻Nipponbare，并利用引物cx245(序列同上cx245)和cx352(序列同上cx352)对获得的转基因株系进行PCR验证，鉴定获得了多个独立的转基因阳性株系，进而我们利用前面提到的小RNA Northem杂交的方法，鉴定了转基因阳性株系中成熟miR528的表达水平，结果显示与野生型日本晴相比，转基因株系(miR528-OE1，miR528-OE2和miR528-OE3)中MiR528表达水平被上调，实现了在水稻中过量表达miR528的目的，见附图3B。
实施例4、miR528敲除和过量表达水稻株系抽穗期表型分析
我们对大田种植miR528敲除突变体mir528和过表达水稻株系(miR528-OE1，miR528-OE2和miR528-OE3)的表型进行了分析，具体操作如下：所用地块为北京昌平区农场，水稻播种期为5月上旬，育苗45天后插秧，大田常规水肥管理。田间结果显示，miR528敲除和过量表达水稻株系在整个苗期和营养生长期内，与野生型相比，没有明显差异。而抽穗期的统计分析发现，与野生型对照Dongjin相比，miR528敲除突变体mir528株系表现出抽穗时间(以水稻主穗抽出1cm的时间)稍有延迟(约3.5天)。而与野生型对照Nipponbare相比，miR528过量表达的水稻株系(miR528-OE1，miR528-OE2和miR528-OE3)抽穗期会有所提前，见附图4A和B。以上结果表明，miR528对于水稻抽穗具有正调控作用。
实施例5、miR528敲除和过量表达水稻株系抗水稻条纹叶枯病毒(RSV)功能分析
为了验证miR528在抗水稻条纹叶枯病毒中的功能，我们对分蘖期的miR528敲除突变体mir528和过量表达水稻株系分别做了RSV接病毒实验，具体操作如下：水稻种子催芽3天后播种，经过14天育苗，释放携带有RSV病毒的飞虱进行病毒接种，3天后接种完成，然后移到正常条件下生长，水稻苗再经过两周的生长可表型观察和统计。结果显示，miR528敲除突变体mir528株系具有更强的抗病毒能力；而与野生型对照相比，两个独立的miR528过量表达水稻株系(miR528OE1和miR528OE2)则对病毒表现出更加敏感，表现出更高的发病率，见附图5，其中Mock 为未接种病毒的对照植株，RSV表示接种RSV的植株。从图5可见，不同株系水稻接种RSV后，相对于日本晴(Nip)对照(Nip-Mock和Nip-RSV)，两个独立的miR528过量表达水稻株系(miR528OE1-RSV和miR528OE2-RSV)对RSV表现出强敏感表型(植株变矮，叶片黄化)；而相对于野生型对照(Dongjin-RSV)表现出中度感病(部分叶片黄化)，miR528缺失突变体mir528(mir528-RSV)则表现出强的抗RSV表型(植株生长良好，未发现明显感病植株)。这一结果表明，miR528对于水稻抗水稻条纹叶枯病毒(RSV)具有负调控作用。
实施例6、水稻MIR528基因在不同品种中的差异
我们利用引物cx9125(5’-GCATCTGGTGGCGAGCAAGTGG-3’，SEQ ID NO：9)和cx9126(5’-CCGGGAGTGACCGGCATTCTGT-3’，SEQID NO：10)，通过PCR克隆了来自不同水稻品种(见表1)的MIR528基因及其上游启动子区序列(即SEQ ID N0：1)。
通过对来自不同水稻品种的MIR528基因及其顺式调控序列分析，我们发现来自不同品种的MIR528上游调控区域序列存在两种不同的等位形式(即MIR528-A1和MIR528-A2，其中MIR528-A1表示在转录起始位点上游-590bp和-837bp处存在碱基序列，MIR528-A2表示在转录起始位点上游-590bp和-837bp处缺失相应的碱基序列)。其中相对于已鉴定到的野生稻(rufipogon)和常见籼稻品种93-11、明恢63、kasalath等(其对应的的等位基因形式为MIR528-A1)来说，已鉴定到的粳稻品种(日本晴、中花11、东粳即Dongjin等)在转录起始位点上游-590bp和-837bp位点分别存在30bp和38bp的序列缺失(其对应的的等位基因形式为MIR528-A2)，见附图6和表1。
为了研究这种序列缺失对于成熟miR528表达水平的影响，我们进一步对不同品种叶片中成熟miR528的表达水平做了鉴定，按照实施例3的方法进行小RNA杂交，结果显示，MIR528上游调控区域存在序列缺失的粳稻品种(其对应的的等位基因形式为MIR528-A2)成熟miR528(即21nt的miR528基因)表达水平要整体较籼稻品种低，而另外两个植物中保守的miR156(以32P同位素标记的DNA序列5’-GTGCTCACTCTCTTCTGTCA-3’ 为杂交探针，SEQ ID NO：11)和miR172(以32P同位素标记的DNA序列5’-GATGCAGCATCATCAAGATTCT-3’为杂交探针，SEQ ID NO：12)则未见类似趋势，其中U6和5s RNA分别作为杂交和上样内参，见附图7。以上结果表明，来自不同水稻品种的MIR528上游调控序列可以直接影响成熟miR528的表达水平，因此可以作为育种中的分子标记，通过杂交的方式优化水稻体内成熟miR528的表达水平。
实施例7、MIR528在禾本科植物中保守存在
本发明研究了MIR528在植物中的保守性。首先，我们分析了miR528前体在植物中的保守性，利用水稻miR528前体序列(AGUGGAAGGGGCAUGCAGAGGAGCAGGAGAUUCAGUUUGAAGCUGGACUUCACUUUUGCCUCUCUCUCCUGUGCUUGCCUCUUCCAUU，SEQ ID NO：13)搜索了植物转录组装数据库(http：//planttajcvi.org/)。比对结果显示，miR528前体在玉米、高粱、甘蔗等禾本科作物中保守存在，尤其是在成熟miR528(即21nt miR528)及其星号链(miR528*)区域高度保守，见附图8。进一步利用在线网站RNAfold(http：//rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi)对这些转录本的二级结构进行预测分析，结果显示，它们都能形成典型的miRNA前体的茎-环结构，说明miR528在这些物种保守存在。
表1
Figure PCTCN2015070864-appb-000002

Claims (11)

  1. DNA序列，其序列如SEQ ID NO：1所示及其变体。
  2. 权利要求1的DNA序列，其中的2231位-2251位脱氧核糖核苷酸序列转录成成熟RNA序列及其变体。
  3. RNA序列，其中序列如SEQ ID NO：2所示及其变体。
  4. 权利要求3的RNA序列，其中识别的mRNA序列选自由SEQ ID NO：14，15，16，17和18及其变体组成的组。
  5. 权利要求1或2所述的DNA序列或权利要求3或4所述的RNA序列在调控水稻抽穗期和水稻条纹叶枯病抗性中的应用。
  6. 筛选调控水稻抽穗期和水稻条纹叶枯病抗性基因的方法，包括利用碱基互补配对寻找权利要求1-2任一项所述的DNA序列或权利要求3-4任一项所述的RNA序列所互补配对的靶基因。
  7. 表达载体，其包含权利要求1或2的DNA序列。
  8. 宿主细胞，其包含权利要求7的表达载体。
  9. 权利要求8的宿主细胞，其为植物细胞。
  10. 权利要求9的宿主细胞，其中所述植物是单子叶或双子叶的植物。
  11. 权利要求10的宿主细胞，其中所述单子叶植物为水稻、玉米、小麦、高粱、谷子、大麦、燕麦或黑麦，所述双子叶植物为棉花、大豆、烟草、油菜或番茄。
PCT/CN2015/070864 2014-08-26 2015-01-16 miR528的调控位点及其应用 WO2016029630A1 (zh)

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