WO2024021768A1 - 玉米ZmRAFS基因用于提高作物抗热能力的应用 - Google Patents
玉米ZmRAFS基因用于提高作物抗热能力的应用 Download PDFInfo
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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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- the invention belongs to the technical field of biological genetic engineering, and specifically relates to a key enzyme gene for corn raffinose synthesis ZmRAFS (raffinose synthase/GRMZM2G150906) and its application.
- the inventor's research found that the corn ZmRAFS gene (GRMZM2G150906) was overexpressed in plants, which increased the raffinose content in corn leaves and enhanced the heat resistance of the plants.
- the present invention provides the application of the corn ZmRAFS gene for improving the heat resistance of crops, and the corn ZmRAFS gene sequence is shown in SEQ ID NO: 1. Further, the protein sequence encoded by the gene is shown in SEQ ID NO: 2.
- the corn ZmRAFS gene uses myo-inositol galactoside and sucrose as substrates to synthesize raffinose in the leaves, and improves the plant's heat resistance by regulating the raffinose content in the leaves.
- the crop is preferably corn.
- FIG. 1 Schematic diagram of the corn overexpression vector
- pUbi corn Ubiquitin promoter
- ZmRAFS corn raffinose synthase gene: NOS: terminator
- p35S cauliflower mosaic virus 35S promoter
- Bar herbicide resistance gene
- Tvsp terminator Sub
- HindIII, EcoRV restriction enzyme site.
- Figure 2 The raffinose content in the leaves of the overexpressed ZmRAFS corn line is significantly increased;
- A Genomic level identification of the corn overexpressed line; Marker: DNA molecular weight; Control: PCR water control; Zong31: Genome of the Maize 31 inbred line; Plasmid: plasmid DNA, used as a positive control; pTF101.1-ZmRAFS(1...9): 9 independent transformation events for overexpression of ZmRAFS;
- B Identification of ZmRAFS mRNA levels in leaves of corn overexpression lines;
- C Corn overexpression Identification of ZmRAFS protein levels in leaves of strains.
- Zong31 Maize Zongzi 31 inbred line; ZmRAFS#1, 2, and 3 are three lines overexpressing the ZmRAFS gene (Zong31 background); ZmGAPDH: glyceraldehyde-3-phosphate dehydrogenase gene, used as internal reference; (D ) Determination of raffinose content in leaves of corn overexpression lines.
- FIG. 3 The heat resistance of corn lines overexpressing ZmRAFS gene is significantly improved;
- A Growth phenotypes of corn lines overexpressing ZmRAFS gene and control lines under normal conditions, heat shock treatment and rehydration, the ruler is 5cm;
- B The survival rate of the maize line overexpressing the ZmRAFS gene after heat stress treatment was significantly higher than that of the control line;
- C The electrical conductivity of the maize line overexpressing the ZmRAFS gene after heat stress treatment was significantly lower than that of the control line;
- Zong31 Maize 31 self-bred Line;
- the data represents the mean ⁇ standard error. Each black point represents 1 biological replicate (8 seedlings), and each line has 8 biological replicates. Different lowercase letters represent significant differences (Duncan test, P ⁇ 0.05).
- the coding region of the maize ZmRAFS gene was cloned by PCR, a plant expression vector for the gene was constructed, and maize was transformed to obtain transgenic maize plants.
- the specific methods are as follows:
- the genetically modified positive plants constructed in the above examples were identified by PCR at the genome level and mRNA expression level: the PCR amplification procedure was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. 28 cycles; final extension at 72°C for 8 minutes; the results are shown in Figure 2 (A) and (B).
- Corn seedling cultivation method Plant ZmRAFS transgenic lines (#1, #2, #3) and wild-type control (Zong 31) in incubators: Seeds from each test group were imbibed and germinated in the dark at 28°C for 72 hours, and their growth was selected. Transplant corns of the same size (root length is about 2-3cm) into pots, with 8 plants in each pot; when transplanting, mix nutrient soil and water at a ratio of 1:1 (m/m), and control the weight of each pot to 170g. After marking, place the corn in an incubator and grow at 28°C with 16 hours of light (10,000 LUX)/8 hours of darkness;
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Abstract
玉米ZmRAFS基因用于提高作物抗热能力的应用，所述玉米ZmRAFS基因的核苷酸序列如SEQ ID NO:1所示，其编码的蛋白序列如SEQ ID NO:2所示。在植物中过表达ZmRAFS基因，通过利用蔗糖和肌醇半乳糖苷合成棉子糖，增加了玉米叶片中棉子糖的含量，提高了玉米的抗热性。
Description
本发明属于生物基因工程技术领域，具体地说，涉及一种玉米棉子糖合成关键酶基因ZmRAFS(棉子糖合成酶/GRMZM2G150906)及其应用。
发明人前期研究成果公开了在拟南芥中过量表达玉米ZmRAFS基因通过水解肌醇半乳糖苷产生肌醇提高植株的耐旱性(Li,T.,et al.,Raffinose synthase enhances drought tolerance through raffinose synthesis or galactinol hydrolysis in maize and Arabidopsis plants.Journal of Biological Chemistry.2020.295:8064-8077.)。耐旱是指植物能够在适应生长温度(如28℃左右)、短期或长期缺水的环境下存活或生长。
发明内容
发明人研究发现玉米ZmRAFS基因(GRMZM2G150906)在植物中过量表达，提高了玉米叶片中棉子糖的含量，增强了植株的抗热性。
基于此，本发明提供了玉米ZmRAFS基因用于提高作物抗热能力的应用，所述玉米ZmRAFS基因序列如SEQ ID NO:1所示。进一步所述基因编码的蛋白序列如SEQ ID NO:2所示。
进一步所述玉米ZmRAFS基因在叶片中以肌醇半乳糖苷和蔗糖为底物合成棉子糖，通过调控叶片中棉子糖含量，提高植株抗热能力。所述作物优选玉米。
图1玉米过表达载体示意图；pUbi:玉米Ubiquitin启动子；ZmRAFS:玉米棉子糖合成酶基因：NOS:终止子；p35S:花椰菜花叶病毒35S启动子；Bar:抗除草剂基因；Tvsp:终止子；HindIII,EcoRV:限制性酶切位点。
图2过表达ZmRAFS玉米株系叶片棉子糖含量显著提高；(A)玉米过表达株系的基因组水平鉴定；Marker:DNA分子量；Control:PCR水对照；Zong31:玉米综31自交系基因组；Plasmid:质粒DNA,作正对照；pTF101.1-ZmRAFS(1…9):过表达ZmRAFS的9个独立转化事件；(B)玉米过表达株系叶片ZmRAFS mRNA水平鉴定；(C)玉米过表达株系叶片ZmRAFS蛋白水平鉴定。Zong31:玉米综31自交系；ZmRAFS#1,2,3为过表达ZmRAFS基因的3个株系(综31背景)；ZmGAPDH:甘油醛-3-磷酸脱氢酶基因，作内参；(D)玉米过表达株系叶片棉子糖(raffinose)含量测定。Zong31:玉米综31自交系；ZmRAFS#1,2,3为过表达ZmRAFS基因的3个株系(综31背景)；数据表示平均值±标准误，n＝3，*表示差异显著(Student’s t-test,*P<0.05，**P<0.01)。
图3过表达ZmRAFS基因玉米株系抗热性显著提高；(A)过表达ZmRAFS基因玉米株系及对照株系在正常、热激处理及复水后生长表型，标尺为5cm；(B)过表达ZmRAFS基因玉米株系热胁迫处理后存活率显著高于对照株系；(C)过表达ZmRAFS基因玉米株系热胁迫处理后电导率显著低于对照株系；Zong31：玉米综31自交系；ZmRAFS(#1,#2,#3)：ZmRAFS转基因株系。数据表示平均值±标准误，每个黑点代表1个生物学重复(8株幼苗)，每个株系8个生物学重复，不同的小写字母代表差异显著(Duncan test，P<0.05)。
除非有特殊说明，本文中的科学与技术术语及方法根据相关领域普通技术人员的认识理解或采用相关领域技术人员公知方法实现。所述抗热性是指植物能够在高温(高于30℃)环境下存活或生长。下面结合附图和具体实施例对本发明的技术方案作进一步详细地说明。如无特殊说明，以下实施例所用材料或试剂均为市售产品。
实施例：
该实施例通过PCR方法克隆了玉米ZmRAFS基因的编码区，构建了该基因植物表达载体，转化玉米，获得了玉米转基因植株。具体方法如下：
(1)从B73三叶期玉米幼苗的叶片提取RNA并将其反转录成cDNA；
(2)以cDNA为模板，用上游引物5’-CGCGGATCCATGGCTCCCAACCTCAGCAAGAAG-3’和下游引物5’-TGCTCTAGAGGTAGACGTACTGGACGCGACACAG-3’对SEQ ID NO:1所示的ZmRAFS的编码区进行扩增；扩增程序为：95℃预变性5min；95℃变性30s，60℃退火30s，72℃延伸1min20s，35个循环；72℃终延伸10min；
(3)将扩增产物切胶回收，连接到玉米表达载体pTF101上，之后将载体转化到农杆菌AGL1菌株中，参见图1所示；
(4)所得农杆菌AGL1菌株用于玉米的遗传转化，玉米转化受体为综31自交系。通过侵染综31授粉后10天的幼胚，经过恢复、筛选、再生等培养阶段，获得9株转基因阳性植株，结果如图2(A)。
进一步对上述其中的3个株系(#1、#2和#3)转基因植株进行了分子鉴定，发现转基因植株叶片中ZmRAFS基因的mRNA和蛋白过量表达，棉子糖含量显著高于对照，具体方法如下：
(1)通过PCR对上述实施例构建的转基因阳性植株进行基因组水平和mRNA表达水平鉴定：PCR扩增程序为：95℃预变性5min；95℃变性30s，60℃退火30s，72℃延伸30s，28个循环；72℃终延伸8min；结果如图2(A)和(B)所示。
(2)通过Western blot对上述实施例构建的转基因阳性植株蛋白水平进行鉴定；Western blot步骤参照文献：Gu,L.,et al.,ZmGOLS2,a target of transcription factor ZmDREB2A,oferssimilar protection against abiotic stress as ZmDREB2A.Plant Mol Biol,2016.90(1-2):p.157-70.中公开的方法，方法中所用ZmRAFS一抗为参照文献Li,T.,et al.,Regulation of seed vigor by manipulation of raffinose family oligosaccharides(RFOs)in maize and Arabidopsis.Molecular Plant.2017.10(12):1540-1555.中公开的方法制备，使用稀释倍数为1：5000；二抗(羊抗兔)购于康为公司，使用稀释倍数为1：10000；结果如图2(C)所示。
(3)通过HPLC对上述#1、#2、#3株系及野生型对照植株叶片的棉子糖含量进行了鉴定，方法参照文献(Li,T.,et al.,Raffinose synthase enhances drought tolerance through raffinose ynthesis or galactinol hydrolysis in maize and Arabidopsis plants.Journal of Biological Chemistry.020.295:8064-8077)；结果如图2(D)所示。
对实施例所得转基因阳性植株的抗热性鉴定具体方法如下：
(1)玉米幼苗培养方法：培养箱种植ZmRAFS转基因株系(#1、#2、#3)及野生型对照(综31)：将各试验组的种子28℃黑暗吸胀萌发72h，挑选长势一致(根长2-3cm左右)的玉米移栽到盆中，每个盆种8棵；移栽时，营养土和水按照1:1(m/m)比例混匀，每盆重量控制到170g，做好标记后将玉米放培养箱，28℃16h光照(10,000LUX)/8h黑暗生长；
(2)幼苗热激处理及存活率统计：待幼苗长到3叶期时进行42℃热激处理，当对照组叶片不可逆全卷后(次日早上卷叶不可恢复)，拍照记录，28℃恢复培养观察表型并拍照，结果如图3(A)所示；统计存活率并测定电导率；存活率＝存活株数/总株数×100％；结果如图3(B)所示。
(3)电导率测定：取热激恢复后的ZmRAFS转基因玉米株系及对照株系叶片进行电导率测定，取同一叶位叶片，浸泡于15ml去离子水中，抽真空30min后，25℃、120rpm处理1h测定电导率记录为C1；叶片经沸水浴30min后，冷却至室温后测定电导率记录为C2；C1/C2×100％即为叶片相对电导率。电导率仪型号为雷磁DDS-307；结果如图3(C)所示。
以上所述，仅为本发明较佳的具体实施方式，本发明的保护范围不限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，可显而易见地得到技术方案的简单变化或等效替换均属于本发明的保护范围内。
Claims (4)
- 玉米ZmRAFS基因用于提高作物抗热能力的应用，所述玉米ZmRAFS基因序列如SEQ ID NO:1所示。
- 如权利要求1所述的应用，其特征在于，所述玉米ZmRAFS基因编码的蛋白序列如SEQ ID NO:2所示。
- 如权利要求1所述的应用，其特征在于，所述玉米ZmRAFS基因通过调控作物叶片中棉子糖的含量提高作物抗热能力。
- 如权利要求1或2所述应用，其特征在于，所述作物为玉米。
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