CN112899285A - 水稻耐镉基因OsFWL6的应用 - Google Patents
水稻耐镉基因OsFWL6的应用 Download PDFInfo
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Abstract
本发明属于植物基因工程技术领域，具体涉及了水稻耐镉基因OsFWL6的应用。本发明通过异源表达OsFWL6基因，证明其增强宿主酵母的耐镉能力，减少宿主中的镉积累；通过构建OsFWL6基因超表达水稻植株，证明其种子对镉胁迫的抗性增强。本发明说明OsFWL6基因在水稻耐镉机制中具有重要作用，为镉低积累水稻品种的培育与水稻耐重金属的遗传改良提供理论指导。
Description
技术领域
本发明涉及植物基因工程技术领域，具体涉及水稻耐镉基因OsFWL6的应用。
背景技术
镉是水稻生长的非必需元素，但是镉可以富集在水稻生物体内，通过食物链而危害人畜健康。随着环境污染的加重，大米镉超标情况日益严峻，因此减少水稻对镉的吸收与转运，进而降低稻米中镉的积累具有重要现实意义。研究水稻吸收与转运镉的分子机理将为镉低积累水稻品种的培育与水稻耐重金属的遗传改良提供理论指导。目前已经有一些转运蛋白被证明参与水稻对镉的吸收、转运、隔离等过程。
西红柿的数量性状位点fruit-weight2.2(fw2.2)能够控制最高达到30％的果实重量变异，康奈尔大学的Tanksley课题组通过图位克隆的方法成功克隆到主效基因并命名为FW2.2。FW2.2的同源基因Fruit-Weight2.2-Like(FWL)广泛存在于动植物与真菌中(Guoet al.,2010；Guo and Simmons 2011)。已有研究证明水稻中有8个FWL基因，分别命名为OsFWL1-8，其中OsFWL3和OsFWL5能够分别调节水稻的粒长和株高(Xu et al.,2013)，过量表达OsFWL5能增加水稻的粒重(Song et al.,2015)。RNAi抑制表达OsFWL4能够降低水稻幼苗地上部分的镉含量，减少镉从根到茎的运输(Xiong et al.,2018)，OsFWL4基因敲除植株的分蘖数和产量显著增加(Gao et al.,2020)。虽然已有多个OsFWL基因的功能研究被报道，但是目前OsFWL6基因的功能仍然未被揭示。
发明内容
本发明提供一个耐镉基因OsFWL6(LOC_Os03g61470)的应用。
所述的水稻耐镉基因OsFWL6序列如SEQ ID NO:1所示，用于增强宿主的耐镉能力。
进一步地，增强酵母或者水稻的耐镉能力。
进一步地，增强酵母耐镉能力时，将OsFWL6基因的编码序列连接酵母表达载体，转化宿主酵母，减少宿主酵母中的镉积累，提高宿主酵母对镉的耐受。
进一步地，所述的酵母表达载体pYES2/NTC。
进一步地，增强水稻的耐镉能力时，将OsFWL6基因的编码区序列连接表达载体，转化农杆菌，通过农杆菌介导转化水稻，提高水稻对镉的耐受。
进一步地，提高水稻种子对镉的耐受。
进一步地，转化农杆菌的表达载体为pU1301。
此外，耐镉基因还可以是在SEQ ID NO:1所示的水稻OsFWL6基因核苷酸序列中插入、替换、缺失1个或多个核苷酸得到编码相同蛋白的核苷酸序列。或者是与SEQ ID NO:1所示的核苷酸序列杂交获得具有相同功能的核苷酸序列。或者是与SEQ ID NO:1所示的核苷酸序列具有90％以上相同性的核苷酸序列。
具体而言，水稻耐镉基因OsFWL6(大写斜体表示，下同)来源于水稻品种日本晴(Oryza sativa L cv.Nipponbare)。OsFWL6(大写正体表示，下同)蛋白的氨基酸序列为SEQID NO:2所述；或者在SEQ ID NO:2所述的氨基酸序列中插入、替换、缺失1个或多个氨基酸得到具有相同功能的氨基酸序列。
本发明证明异源表达OsFWL6基因能增强宿主酵母的耐镉能力。具体而言，将OsFWL6基因的编码序列(Coding sequence,CDS)序列连接酵母表达载体pYES2/NTC，转化酵母镉敏感突变株DTY167(ycf1-)。在1/2SG培养基条件下异源表达OsFWL6的酵母菌落生长情况与空载体对照相似。在1/2SG+60μM CdCl2培养基条件下异源表达OsFWL6的酵母菌落生长性能明显优于对照(对照菌落生长受到严重抑制)，进一步试验证明异源表达OsFWL6基因能够减少宿主酵母中39.0％的镉积累。
本发明证明超量表达OsFWL6基因能增强水稻的耐镉能力。具体而言，构建OsFWL6超量表达水稻株系，将OsFWL6基因的编码区(CDS)序列连接超量表达载体pU1301，转化根癌农杆菌，通过农杆菌介导转化水稻，经过筛选以及验证获得pU1301-OsFWL6超量表达水稻植株。将pU1301-OsFWL6与pU1301空载体水稻种子进行发芽试验，在非镉胁迫培养条件下pU1301-OsFWL6与pU1301的种子均能正常发芽，生长无明显差异；在200μM CdCl2条件下，pU1301-OsFWL6水稻种子生长明显优于pU1301。
本发明说明OsFWL6基因在水稻耐镉机制中具有重要作用，为镉低积累水稻品种的培育与水稻耐重金属的遗传改良提供理论指导。
附图说明
图1为异源表达OsFWL6对宿主酵母耐镉性能的影响；
图2为遗传转化T0代植株OsFWL6表达量检测；
图3为超量表达OsFWL6对水稻种子镉胁迫的影响。
具体实施方式
以下将结合实施例对本发明做进一步说明，而不会形成对本发明的限制。以下实施例中的试验方法，如无特殊说明，均为常规方法。
实施例1
利用镉敏感酵母突变体分析OsFWL6基因的耐镉功能
步骤1酵母异源表达载体的构建
以水稻品种日本晴cDNA为模板，利用引物序列(SEQ ID NO:3/NO:4)扩增OsFWL6基因的编码序列，通过BamHI、EcoRI双酶切CDS片段和酵母诱导表达载体pYES2/NTC(购自赛默飞世尔公司)，回收纯化两个线性片段后用T4连接酶进行连接反应，转化大肠杆菌感受态菌株DH10B，在含Amp的LB平板上筛选培养，挑选单克隆经过双酶切和测序验证为重组载体pYES2/NTC-OsFWL6。
步骤2醋酸锂介导的酵母转化
本试验用到的酵母菌株是镉敏感ycf1突变菌株DTY167(MATa ura3 leu2 his3trp3 lys2 suc2 ycf::hisG)。首先将菌株DTY167在YPD培养基上划线培养3～5d，挑取单克隆接种于5mL YPD液体培养基中，30℃，200rpm培养1d，菌液离心后弃上清，使用1mL dd H2O重悬菌体，用0.1M的LiAc洗涤菌体3次，分装成每管20μL的感受态细胞，每管加入5μL SSDNA混匀，分别加入5μL重组载体质粒pYES2/NTC-OsFWL6和空载体质粒pYES2/NTC混匀，然后加入345μL Mixture(35μL LiAc，35μL 10×TE，275μL PEG3350)混匀，42℃水浴20min后转入冰上静置3min，离心去上清，加入100μL dd H2O重悬菌体。在SD/-Ura固体培养基上筛选培养2～3d。
步骤3镉胁迫平板对比培养
挑取单菌落在SG/-Ura液体培养基中培养2～3d，离心收集菌体，用dd H2O洗涤2次后将菌液浓度调整至OD600＝1.0，进一步将菌液进行10倍梯度稀释(OD600＝0.1、0.01、0.001)，取各浓度菌液5μL点接于1/2SG/-Ura和1/2SG/-Ura+60μM CdCl2培养基上培养5d。结果如图1所示在无镉胁迫条件下pYES2/NTC和pYES2/NTC-OsFWL6酵母转化菌株的生长情况基本相同，在60μM镉胁迫条件下pYES2/NTC菌株不能够生长，pYES2/NTC-OsFWL6菌株仍然可以生长，说明异源表达OsFWL6基因能显著提高宿主酵母的耐镉能力。
步骤4转化酵母菌体镉含量对比
挑取转化pYES2/NTC和pYES2/NTC-OsFWL6的酵母单菌落接种于含40μM CdCl2的SG/-Ura液体培养基中，培养60h后离心去上清，用dd H2O洗涤3次，菌体经过烘干粉碎后过100目筛，选用硝酸和高氯酸(V/V＝7:3)混合液消解，采用电感耦合等离子体质谱仪(ICP-MS)测定Cd含量，结果显示转化pYES2/NTC-OsFWL6的酵母菌镉含量为392.4±31.7mg/kg，比pYES2/NTC空载体对照(643.1±46.8mg/kg)减少39.0％，本试验证明异源表达OsFWL6基因能够显著降低镉在宿主酵母中的积累。
实施例2利用水稻超表达植株分析OsFWL6基因的耐镉功能
步骤1水稻超表达载体的构建
利用引物序列(SEQ ID NO:5/NO:6)扩增OsFWL6基因的编码序列，通过KpnI、BamHI双酶切OsFWL6基因的CDS片段和水稻超表达载体pU1301(该载体整个编码框由玉米Ubiquitin强启动子驱动，pU1301载体由pCAMBIA1301载体(购自上海柯雷生物科技有限公司)改造，即用Ubiquitin启动子替代35S启动子，回收纯化两个线性片段后用T4连接酶连接转化大肠杆菌感受态菌株DH10B，在含Kan的LB平板上筛选培养，挑选单克隆经过双酶切和测序验证为重组载体pU1301-OsFWL6。
步骤2农杆菌介导的水稻遗传转化
将空载体质粒pU1301和重组质粒pU1301-OsFWL6分别转化根癌农杆菌EHA105，通过菌落PCR验证阳性菌株，采用农杆菌介导的水稻遗传转化方法将菌株EHA105-pU1301和EHA105-pU1301-OsFWL6侵染水稻Nipponbare愈伤组织，经过共培养、筛选培养、分化培养，生根培养以及阳性苗验证，得到成功的水稻转化苗(具体遗传转化方法参考Lin andZhang,2005)。共获得空载体pU1301转化苗15株和pU1301-OsFWL6转化苗24株，在2组转基因家系中随机各挑选5株，通过Realtime PCR分析其OsFWL6的相对表达量，结果显示在pU1301转化苗中OsFWL6表达量无明显变化，而在pU1301-OsFWL6转化苗中其表达量升高141-393倍，结果见图2。将这10份T0家系的下一代植株进行大田种植，收集各个转基因株系的水稻种子，分别以自主分离的方式筛选纯合转化植株。
步骤3转基因水稻种子发芽试验
将筛选出来的pU1301和pU1301-OsFWL6转基因T2代水稻种子去壳，用0.15％氯化汞消毒15min，无菌水清洗干净后将种子分别接种于1/2MS培养基和含200μM CdCl2的1/2MS培养基，温度控制在25-28℃，光照时间为14h/d，培养10-15d。结果显示在无镉胁迫条件下两个转基因水稻的种子均能正常发芽，生长无显著性差异；在镉胁迫条件下pU1301转基因水稻种子生长受到严重抑制，pU1301-OsFWL6转基因水稻种子的生长显著优于pU1301转基因水稻种子，结果见图3。说明OsFWL6能显著提高水稻的耐镉性能。
序列表
<110> 湖南省微生物研究院
江西省农科院水稻研究所
<120> 水稻耐镉基因OsFWL6的应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 679
<212> DNA
<213> 水稻(Oryza sativa L.)
<400> 1
atggccaagc caagcgccgc tccggtgacc ggcgtccccg tcggctccgc cgcctggtcc 60
accggcctct gcgactgctt cgacgactgc ggcctatgta cgttctcgct cttttttata 120
agcttcagct ctttattttg ctttttctcc cttcttctga cataagttgg tctggctgat 180
tccattgtgg tatattgata tgtaatcatt ttacgtgttc acgaaaaaaa cgttctcgct 240
ctcgccgccg gccaccgtgc catggctaga tcatatcatg aacatgatct gtaatttact 300
aattaattca cattaatctg caggctgctt gacgtgctgg tgcccgtgca tcacgttcgg 360
gagggtggcg gagatggtgg acaggggctc gacgtcgtgc ggcaccggcg gcgcgctgta 420
cgggctgctg tgcgcgttca ccgggtgcca gtggatctac tcctgcacct accggggcaa 480
gatgcgcact cagtacgggc tcgccgaagc cggctgcgcc gactgctgcg tccacttctg 540
ctgcgagcca tgcgcgctgt gccaggagta ccgcgagctc gtcgcccgcg gctacgaccc 600
caagctcggc tggcacctca acgccgaccg cgccgccgcc gccggcgccg ctcccgccgt 660
gcagtacatg ggccgctaa 679
<210> 2
<211> 150
<212> PRT
<213> 水稻(Oryza sativa L.)
<400> 2
Met Ala Lys Pro Ser Ala Ala Pro Val Thr Gly Val Pro Val Gly Ser
1 5 10 15
Ala Ala Trp Ser Thr Gly Leu Cys Asp Cys Phe Asp Asp Cys Gly Leu
20 25 30
Cys Cys Leu Thr Cys Trp Cys Pro Cys Ile Thr Phe Gly Arg Val Ala
35 40 45
Glu Met Val Asp Arg Gly Ser Thr Ser Cys Gly Thr Gly Gly Ala Leu
50 55 60
Tyr Gly Leu Leu Cys Ala Phe Thr Gly Cys Gln Trp Ile Tyr Ser Cys
65 70 75 80
Thr Tyr Arg Gly Lys Met Arg Thr Gln Tyr Gly Leu Ala Glu Ala Gly
85 90 95
Cys Ala Asp Cys Cys Val His Phe Cys Cys Glu Pro Cys Ala Leu Cys
100 105 110
Gln Glu Tyr Arg Glu Leu Val Ala Arg Gly Tyr Asp Pro Lys Leu Gly
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Trp His Leu Asn Ala Asp Arg Ala Ala Ala Ala Gly Ala Ala Pro Ala
130 135 140
Val Gln Tyr Met Gly Arg
145 150
<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tgggatccat ggccaagcca agc 23
<210> 4
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cggaattcgc ggcccatgta ctg 23
<210> 5
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ggggtaccat ggccaagcca agc 23
<210> 6
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tgggatccgc ggcccatgta ctg 23
Claims (10)
1.水稻耐镉基因OsFWL6的应用，所述的水稻耐镉基因OsFWL6序列如SEQ ID NO:1所示，用于增强宿主的耐镉能力。
2.根据权利要求1所述的应用，其特征在于，增强酵母或者水稻的耐镉能力。
3.根据权利要求2所述的应用，其特征在于，将OsFWL6基因的编码序列连接酵母表达载体，转化宿主酵母，减少宿主酵母中的镉积累，提高宿主酵母对镉的耐受。
4.根据权利要求3所述的应用，其特征在于，所述的酵母表达载体pYES2/NTC。
5.根据权利要求2所述的应用，其特征在于，将OsFWL6基因的编码区序列连接表达载体，转化农杆菌，通过农杆菌介导转化水稻，提高水稻对镉的耐受。
6.根据权利要求5所述的应用，其特征在于，提高水稻种子对镉的耐受。
7.根据权利要求5所述的应用，其特征在于，所述的表达载体为pU1301。
8.根据权利要求1-7任一项所述的应用，其特征在于，所述的水稻耐镉基因OsFWL6替换为水稻OsFWL6基因核苷酸序列中插入、替换、缺失1个或多个核苷酸得到编码相同蛋白的核苷酸序列。
9.根据权利要求1-7任一项所述的应用，其特征在于，所述的水稻耐镉基因OsFWL6替换为与水稻耐镉基因OsFWL6核苷酸序列杂交获得具有相同功能的核苷酸序列。
10.根据权利要求1-7任一项所述的应用，其特征在于，所述的水稻耐镉基因OsFWL6替换为与水稻耐镉基因OsFWL6核苷酸序列具有90％以上相同性的核苷酸序列。
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| CN114410658A (zh) * | 2022-03-11 | 2022-04-29 | 四川农业大学 | 一种降低水稻糙米镉含量的基因OsWNK9及其编码蛋白和应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113897372A (zh) * | 2021-10-08 | 2022-01-07 | 淮阴师范学院 | OsFWL7基因在增加水稻籽粒金属微量元素含量中的应用 |
| CN113897372B (zh) * | 2021-10-08 | 2022-03-08 | 淮阴师范学院 | OsFWL7基因在增加水稻籽粒金属微量元素含量中的应用 |
| CN114410658A (zh) * | 2022-03-11 | 2022-04-29 | 四川农业大学 | 一种降低水稻糙米镉含量的基因OsWNK9及其编码蛋白和应用 |
| CN114410658B (zh) * | 2022-03-11 | 2023-04-25 | 四川农业大学 | 一种降低水稻糙米镉含量的基因OsWNK9及其编码蛋白和应用 |
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