CN108148849B - 一种苹果MdPHR1基因及其制备方法和应用 - Google Patents
一种苹果MdPHR1基因及其制备方法和应用 Download PDFInfo
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- CN108148849B CN108148849B CN201810214008.6A CN201810214008A CN108148849B CN 108148849 B CN108148849 B CN 108148849B CN 201810214008 A CN201810214008 A CN 201810214008A CN 108148849 B CN108148849 B CN 108148849B
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Abstract
本发明涉及一种苹果MdPHR1基因及其制备方法和应用,所述MdPHR1基因的核苷酸序列如SEQ.ID.NO.1所示,利用强启动子驱动原理的转基因技术,将MdPHR1基因的超量表达载体转入苹果王林愈伤组织和拟南芥中,获得转基因苹果王林愈伤组织和拟南芥。本发明首次通过植物基因工程技术提高磷酸盐吸收的生产性状,获得了一种从苹果中分离克隆出的促进磷酸盐吸收的基因的完整编码区段的DNA片段,并验证了该基因的功能,利用其功能最终发现采用MdPHR1基因在苹果王林愈伤组织和拟南芥中超量表达之后的转基因材料,磷酸盐的吸收明显提高。
Description
技术领域
本发明一种促进磷酸盐吸收的基因及其应用,具体涉及一种苹果促进磷酸盐吸收的基因MdPHR1及其应用。
背景技术
磷是植物生长发育过程中必需的营养元素,其不仅参与植物体内重要化合物(磷脂、ATP和核酸等)的形成,而且在植物生命活动过程(光合作用、呼吸作用和信号转导等)中发挥着重要作用。然而,多数土壤中磷元素分布不均,并且多以植物难以利用的有机磷形式存在,植物因此常常受低磷胁迫影响。在生产中,人们通过施用磷肥来改善土壤有效磷含量不足的状况提高作物产量,但却导致了一系列环境污染问题。因此,通过分子生物学手段发掘优质基因,提高作物磷吸收能力对减少化肥使用具有重要意义。
AtPHR1是MYB-CC家族转录因子的一员,是拟南芥中磷饥饿反应中一种主要的转录激活因子。AtPHR1主要通过结合基因启动子P1BS顺式元件(GNATATNC),而诱导一些Pi饥饿相关基因的表达。phr1突变体中磷含量显著低于野生型,并且在磷缺乏条件下产生了花青苷积累的表型。相反,AtPHR1过表达株系中磷含量显著增加,并且提高了拟南芥一系列磷饥饿诱导基因的表达。AtPHR1的同源基因在多个物种中已被发现,包括水稻、玉米和大豆等;并且均能够影响植物磷吸收,调控植物的生长发育。
我国是世界上苹果种植面积最大和总产量最高的国家,同时,苹果产业也是我国产量最大的果树产业,苹果产业的发展对于提高农民收入、解决剩余劳动力以及带动下游附加产业的发展起到了关键作用。但是,我国苹果产业与苹果生产先进国家相比,生产水平还存在着较大差距。例如,肥料利用率低,特别是磷肥通过挥发、淋溶和径流等途径损失数量巨大。同时,由于片面追求产量与效益,果园内大量施用磷肥,因此导致了土壤肥力下降、果实品质降低和环境污染严重等后果,这严重制约了苹果产业的可持续发展。
因此,为了减少化肥的施用特别是磷肥的施用,促进磷素的吸收,研究和了解苹果中磷酸盐的吸收和转运相关的基因功能对苹果产业的发展具有重要的意义。
发明内容
本发明所要解决的技术问题是,提供一种苹果促进磷酸盐吸收的MdPHR1基因及其制备方法和应用。
本发明解决其技术问题所采用的技术方案是,一种苹果促进磷酸盐吸收的基因,是从苹果中分离克隆出的磷酸盐吸收相关的基因完整编码区段的DNA片段,并命名为MdPHR1,其核苷酸序列如SEQ.ID.NO.1所示,其所编码的蛋白质氨基酸序列如SEQ.ID.NO.2所示。
本发明进一步解决其技术问题所采用的技术方案是,一种苹果MdPHR1基因的制备方法,包括以下步骤:
(1)提取皇家嘎啦苹果组培叶片中的RNA及反转录;
(2)cDNA全长序列的获得:根据NCBI查到的拟南芥中AtPHR1基因保守氨基酸序列并进行同源序列比对获得苹果中MdPHR1基因的核苷酸序列,设计引物MdPHR1-F,MdPHR1-R,然后以反转录合成的嘎啦基因组cDNA为模板进行PCR扩增,得到cDNA全长序列;其中,
MdPHR1-F序列如SEQ.ID.NO.3所示,
MdPHR1-R序列如SEQ.ID.NO.4所示;
(3)PCR产物进行凝胶回收、载体连接、大肠杆菌转化,测序得到MdPHR1基因,MdPHR1基因的开放阅读框(open reading frame,ORF)为1458bp,编码485个氨基酸。进一步,步骤(2)中,所述PCR扩增反应体系为:
本发明进一步解决其技术问题所采用的技术方案是,一种苹果MdPHR1基因在转基因苹果愈伤组织和拟南芥中的应用,利用强启动子(花椰菜花叶病毒35S启动子)驱动原理的转基因技术,将MdPHR1基因的超量表达载体转入苹果愈伤组织和拟南芥中,从而获得转基因材料。实验证明,超量表达MdPHR1基因的转基因苹果愈伤组织和拟南芥在低浓度的磷酸盐的处理条件下,磷酸盐的吸收相比于对照明显提高,沉默表达MdPHR1基因的苹果愈伤组织磷吸收明显受到抑制,说明MdPHR1基因在低浓度的磷酸盐的条件下植株磷酸盐的吸收过程中起着重要作用。
综上,本发明首次通过植物基因工程技术提高植物磷酸盐吸收的生产性状,从皇家嘎啦苹果组培苗中分离克隆出的磷酸盐吸收相关基因的完整编码区段的DNA片段,并验证了该基因的功能,利用其功能最终发现采用超量表达之后,转基因材料磷酸盐的吸收能力明显提高。
附图说明
图1为PCR扩增产物电泳图谱
图2为MdPHR1转基因苹果王林愈伤组织中MdPHR1基因的表达分析图。
图3为MdPHR1转基因苹果王林愈伤组织在不同浓度的磷酸盐的条件下的生长状态。
图4为MdPHR1转基因苹果王林愈伤组织在不同浓度的磷酸盐的条件下的鲜重。
图5为MdPHR1转基因苹果王林愈伤组织在不同浓度的磷酸盐的条件下磷酸盐的含量。
图6为MdPHR1转基因苹果王林愈伤组织在不同浓度的磷酸盐的条件下总磷含量。
图7为MdPHR1转基因拟南芥(MdPHR1-OX-1/2/3)中MdPHR1基因的表达分析。
图8为MdPHR1转基因拟南芥(MdPHR1-OX-1/2/3)在不同浓度的磷酸盐的条件下的生长状态。
图9为MdPHR1转基因拟南芥(MdPHR1-OX-1/2/3)在不同浓度的磷酸盐的条件下的鲜重。
图10为MdPHR1转基因拟南芥(MdPHR1-OX-1/2/3)在不同浓度的磷酸盐的条件下磷酸盐的含量。
图11为MdPHR1转基因拟南芥(MdPHR1-OX-1/2/3)在不同浓度的磷酸盐的条件下总磷的含量。
具体实施方式
下面结合附图和实施例对本发明进一步加以说明。
实施例1:苹果MdPHR1基因的克隆
一、嘎啦组培叶片RNA提取及反转录
1、嘎啦组培叶片RNA提取和含量检测
利用RNAplant Plus植物总RNA提取试剂(天根生化科技(北京)有限公司)小规模提取嘎啦组培叶片的总RNA,操作步骤按提取试剂说明书进行。吸取1μl总RNA溶液用Nanodrop超微量分光光度计进行测定,以无RNase的水为空白对照,测定溶液中RNA浓度(μg/ml)。
2、cDNA模板的合成
利用宝生物cDNA反转录试剂盒(PrimeScriptTMRT reagent Kit with gDNAEraser)操作步骤的第一步去除基因组DNA反应去除DNA。然后利用宝生物试剂盒(PrimeScriptTMII 1st Strand cDNA Synthesis Kit)操作步骤合成cDNA模板。操作步骤按试剂盒说明书进行。
二、cDNA全长序列的获得
根据NCBI查到的拟南芥中AtPHR1基因保守氨基酸序列并进行同源序列比对获得苹果中MdPHR1基因的核苷酸序列,设计引物MdPHR1-F,MdPHR1-R,然后以反转录合成的嘎啦基因组cDNA为模板进行PCR扩增。
SEQ.ID.NO.3MdPHR1-F:5’-GTCGACATGGAGGCACGCCCTGCTAT-3’;
SEQ.ID.NO.4MdPHR1-R:5’-GAATTCTTCTTTGATTTTGGCAC-3’;
其中,PCR扩增体如下:
PCR反应结束后,进行2.0%琼脂糖凝胶电泳以检测是否有适当大小的条带,并将PCR产物回收(回收操作步骤根据Takara公司的Agarose Gel DNA Extraction Kit试剂盒的说明书进行)、载体连接(取3.0μl PCR回收产物与pMD18-T载体连接,操作步骤按pMD18-TVector说明书进行)、大肠杆菌转化(连接产物转化大肠杆菌感受态细胞DH5α,操作步骤按全式金生物科技公司的Trans5αChemically Competent Cell感受态的说明书进行,在含有氨苄青霉素的LB平板培养基上,37℃倒置培养12-20小时;PCR检测阳性克隆,挑取阳性克隆,在LB液体培养基中培养过夜)、序列测定(将摇好的菌取1ml放到1.5ml离心管中,密封,在北京六合华大基因科技股份有限公司进行序列测定)。
测序后得到MdPHR1基因,其核苷酸序列如SEQ.ID.NO.1所示;其氨基酸序列如SEQ.ID.NO.2所示。
实施例2:转基因愈伤组织的获得
1、准备苹果王林的愈伤组织用于侵染,每隔2-3周在王林愈伤组织的固体培养基(MS基本培养基加入1.5mg/L 2.4-D和0.4mg/L 6-BA)上进行继代一次,在23℃-25℃的暗室培养。
2、将扩增获得的MdCEPR1基因连接中间载体pMD18-T,获得MdCEPR1-OX-pMD18载体,然后通过限制性内切酶进行酶切反应,将含有目的基因片段的酶切产物进行回收。对pBI121表达载体用相同的限制性内切酶进行酶切反应,同样将酶切的载体产物进行回收,然后将回收的目的基因片段和pBI121混合,在16℃进行连接反应,连接过夜后转化大肠杆菌感受态细胞。筛选阳性克隆,获得MdCEPR1-OX-pBI121植物表达载体。将构建的表达载体转化农杆菌LBA4404。挑取农杆菌单克隆菌落接种于10mL YEP液体培养基(含50mg/L卡那霉素以及50mg/L利福平)中,28℃、200rpm,振荡培养至OD600为0.6-0.8(约48h);取其中l mL菌液加入20mL YEP液体培养基(含50mg/L卡那霉素、50mg/L利福平以及100μmol/L乙酰丁香酮)内,28℃、200rpm,振荡培养至OD600为0.6-0.8(约5h);然后离心收集菌体,用侵染液(含0.05g/ml蔗糖、0.03-0.05%Silweet)悬浮菌体,备用。
3、将侵染用的农杆菌的菌体悬浮在无菌水中,使其终浓度OD600为0.5-0.6,将苹果王林的愈伤组织移入上述侵染液中,在摇床上慢慢摇15min左右,用无菌的滤纸吸除表面的侵染液,转移到不含抗性的王林愈伤组织的固体培养基玻璃平板上,室温暗处培养2天左右。然后将共培养的王林愈伤组织加入头孢的灭菌水洗3-5次,洗去农杆菌。最后将上述获得的王林愈伤组织均匀的铺到的固体筛选培养基(含250mg/L的头孢霉素和30mg/L的潮霉素)玻璃平板上;大约培养30天左右,将新长出的王林愈伤组织移到新的筛选培养基(含250mg/L的头孢霉素和30mg/L的潮霉素)上。
4、提取筛选出来的抗性王林愈伤组织的DNA和RNA,PCR和RT-PCR鉴定是否为转基因的王林愈伤组织。将确定为转基因的王林愈伤组织每隔2-3周进行继代一次,进行表型分析。
实施例3:转基因愈伤组织的相关生理指标检测
选择继代2周、长势良好并且基本一致的对照(WT)和转基因苹果王林愈伤组织(MdPHR1-L1和MdPHR1-L2),在不同磷酸盐浓度的王林愈伤组织培养基(磷饥饿处理)上暗培养1周左右,然后转移到不同浓度的磷酸盐(50μM的PO4 3+,-Pi和1.25mM的PO4 3+,+Pi)的王林愈伤组织培养基上暗培养15天左右,检测其鲜重、磷酸盐的含量和总磷含量。结果表明,在低浓度的磷酸盐(50μM的PO4 3+,-Pi)的条件下,MdPHR1过表达转基因愈伤组织与野生型愈伤组织相比具有较高的鲜重、磷酸盐的含量以及总磷含量,而在正常浓度的磷酸盐(1.25mM的PO4 3+,+Pi)条件下则没有明显差异,证明MdPHR1在低浓度磷酸盐((50μM的PO4 3+,-Pi)而不是高浓度磷酸盐(1.25mM的PO4 3+,+Pi)的条件下在调控磷酸盐的吸收。
实施例4:转基因拟南芥的获得
1、将获得的拟南芥种子,分别用70%酒精消毒2min,4%次氯酸钠消毒10min(期间多次摇晃),灭菌水冲洗5次,将种子均匀播种到MS培养基上。首先在4℃层积培养2-4天,然后在长日照的条件下进行培养2周(19-25℃,16h/8h长日照),至成长为小苗,移栽到基质培养到开花。
2、农杆菌的活化按照实施例2中的步骤2的方法进行操作。
3、将拟南芥花序浸到侵染液中15~20s,收集果荚,播种到MS筛选培养基(30mg/L的潮霉素)上筛选,PCR和RT-PCR检测得到阳性转基因植株,经过连续3代筛选得到T3代纯合体,收取种子,进行表型分析。
实施例5:转基因拟南芥的相关生理指标检测
选取萌发2周左右、长势良好并且一致的野生型拟南芥(col)和转基因拟南芥(MdPHR1-OX-1/2/3),转移到不同浓度的磷酸盐(50μM的PO4 3+,-Pi和1.25mM的PO4 3+,+Pi)营养液处理的营养钵中长日照培养3周左右,检测其鲜重、磷酸盐的含量以及总磷含量。结果表明,在低浓度的磷酸盐(50μM的PO4 3+,-Pi)的条件下,MdPHR1过表达转基因拟南芥与对照相比具有较高鲜重、磷酸盐的含量以及总磷含量,而在正常浓度的磷酸盐(1.25mM的PO4 3+,+Pi)条件下则没有明显差异,同样证明MdPHR1在低浓度磷酸盐(50μM的PO4 3+,-Pi)而不是高浓度磷酸盐(1.25mM的PO4 3+,+Pi)的条件下在调控磷酸盐的吸收。上述检测鲜重、磷酸盐的含量以及总磷含量的方法均为实验室常规检测方法。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 山东农业大学
<120> 一种苹果MdPHR1基因及其制备方法和应用
<130> PCNGS
<141> 2018-03-15
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1458
<212> DNA
<213> MdPHR1
<400> 1
atggaggcac gccctgctat gtccatccgg agatcggctg caaatcagct tgctcatatg 60
ggggtccctg cagcaatgtc ttcatcctta ccagtccttc caacttcttt ggaagagaca 120
catcccaatt taccagactc ccaacaggtt tccatggaaa gagaacttat gacaaggcct 180
gttgtgcatg ctggtcactt aacctccaac agtggagtag ttggtcacat attttcatca 240
tcgtcgggat tttcaacaga tcttcactac tcaactcatt cacctcatga aaaacagcaa 300
aaaaactctc ctttcatttc tcagtctcct cattcgggat ttcttcagtc aacagaatct 360
tgtccttatc ccaaagaaaa cagtggttcc tggtgtacag atccactgcc aggtttcctt 420
gattttcctg taaataacca tatcgagaat agtcaaatag agagcagtag ttgtagcggc 480
ataatggctg ctgatgaatt tgctaagcga catgattggc aggaatgggc agatcagcta 540
attactgatg acgatgcttt aacttctaac tggaatgagc ttcttgttga caacgttaca 600
gatctggaac aaaagatgaa ataccaggct cccaaaccgt ctccaaattt ttcggtccag 660
cagtcccaag ttcatcagca acaacctgct tcatctgggg aaatcattcc tgctccatct 720
agggaaatca tttctgttac tgctccttct tcagctaata gtgccactgc caaggcacgc 780
atgcgttgga cgcctgaact tcatgagtcc tttgtggagg ctgttaacca acttggcggt 840
agtgaaagag caactcctaa gggtgtgcta aagctcatga aagttgaaca cttgactatc 900
tatcatgtga aaagtcactt gcagaaatat aggactgcta gatacagacc agaatcatcc 960
gaaggcgcct cagagaagaa attgactcca attgaagaaa tgacgtctct tgacttgaaa 1020
actggtatcg agatcactga agctctgcga ctgcagatgg aagttcagaa gcgactgcat 1080
gaacagcttg agattcaaag aaatctgcag ttacgaatag aagaacaagg gaagtatctt 1140
caaatgatgt ttgagaagca atgcaagtca ggcatcgaca cgctgaaccc atcatcatcc 1200
aatttggacg acccctccgc tcagccttca gatgcaacgc aagtttgtct cgacaaaagt 1260
gaaccggagt cttccaagtt gggccaaggc gagactcaaa ctgatccagt taaagccaac 1320
tccacatcat caggtggttc acaggaaccg gaagggaagc agaaggcacc tgaaacggaa 1380
actgttcccc agaatcccga gccagatgtc ggtgaggcca gttcccaacc tccaaggcgt 1440
gccaaaatca aagaatag 1458
<210> 2
<211> 485
<212> PRT
<213> Malus domestica
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Gln Val Ser Met Glu Arg Glu Leu Met Thr Arg Pro Val Val His Ala
50 55 60
Gly His Leu Thr Ser Asn Ser Gly Val Val Gly His Ile Phe Ser Ser
65 70 75 80
Ser Ser Gly Phe Ser Thr Asp Leu His Tyr Ser Thr His Ser Pro His
85 90 95
Glu Lys Gln Gln Lys Asn Ser Pro Phe Ile Ser Gln Ser Pro His Ser
100 105 110
Gly Phe Leu Gln Ser Thr Glu Ser Cys Pro Tyr Pro Lys Glu Asn Ser
115 120 125
Gly Ser Trp Cys Thr Asp Pro Leu Pro Gly Phe Leu Asp Phe Pro Val
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Asn Asn His Ile Glu Asn Ser Gln Ile Glu Ser Ser Ser Cys Ser Gly
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Ile Met Ala Ala Asp Glu Phe Ala Lys Arg His Asp Trp Gln Glu Trp
165 170 175
Ala Asp Gln Leu Ile Thr Asp Asp Asp Ala Leu Thr Ser Asn Trp Asn
180 185 190
Glu Leu Leu Val Asp Asn Val Thr Asp Leu Glu Gln Lys Met Lys Tyr
195 200 205
Gln Ala Pro Lys Pro Ser Pro Asn Phe Ser Val Gln Gln Ser Gln Val
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His Gln Gln Gln Pro Ala Ser Ser Gly Glu Ile Ile Pro Ala Pro Ser
225 230 235 240
Arg Glu Ile Ile Ser Val Thr Ala Pro Ser Ser Ala Asn Ser Ala Thr
245 250 255
Ala Lys Ala Arg Met Arg Trp Thr Pro Glu Leu His Glu Ser Phe Val
260 265 270
Glu Ala Val Asn Gln Leu Gly Gly Ser Glu Arg Ala Thr Pro Lys Gly
275 280 285
Val Leu Lys Leu Met Lys Val Glu His Leu Thr Ile Tyr His Val Lys
290 295 300
Ser His Leu Gln Lys Tyr Arg Thr Ala Arg Tyr Arg Pro Glu Ser Ser
305 310 315 320
Glu Gly Ala Ser Glu Lys Lys Leu Thr Pro Ile Glu Glu Met Thr Ser
325 330 335
Leu Asp Leu Lys Thr Gly Ile Glu Ile Thr Glu Ala Leu Arg Leu Gln
340 345 350
Met Glu Val Gln Lys Arg Leu His Glu Gln Leu Glu Ile Gln Arg Asn
355 360 365
Leu Gln Leu Arg Ile Glu Glu Gln Gly Lys Tyr Leu Gln Met Met Phe
370 375 380
Glu Lys Gln Cys Lys Ser Gly Ile Asp Thr Leu Asn Pro Ser Ser Ser
385 390 395 400
Asn Leu Asp Asp Pro Ser Ala Gln Pro Ser Asp Ala Thr Gln Val Cys
405 410 415
Leu Asp Lys Ser Glu Pro Glu Ser Ser Lys Leu Gly Gln Gly Glu Thr
420 425 430
Gln Thr Asp Pro Val Lys Ala Asn Ser Thr Ser Ser Gly Gly Ser Gln
435 440 445
Glu Pro Glu Gly Lys Gln Lys Ala Pro Glu Thr Glu Thr Val Pro Gln
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Asn Pro Glu Pro Asp Val Gly Glu Ala Ser Ser Gln Pro Pro Arg Arg
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Ala Lys Ile Lys Glu
485
<210> 3
<211> 26
<212> DNA
<213> Artificial
<400> 3
gtcgacatgg aggcacgccc tgctat 26
<210> 4
<211> 23
<212> DNA
<213> Artificial
<400> 4
gaattcttct ttgattttgg cac 23
Claims (6)
1.一种苹果MdPHR1基因,其特征在于,所述苹果MdPHR1基因的核苷酸序列如SEQ.ID.NO.1所示。
2.一种利用权利要求1所述苹果MdPHR1基因编码出的多肽,其特征在于,所述苹果MdPHR1基因编码的多肽的氨基酸序列如SEQ.ID.NO.2所示。
3.一种如权利要求1所述苹果MdPHR1基因的制备方法,其特征在于,包括以下步骤:
(1)提取皇家嘎啦苹果组培叶片中的RNA并对其进行反转录得到嘎啦基因组cDNA;
(2)cDNA全长序列的获得:根据NCBI查到的拟南芥中At PHR1基因保守氨基酸序列并进行同源序列比对获得苹果中MdPHR1基因的核苷酸序列,以MdPHR1-F和MdPHR1-R为引物,以反转录合成的嘎啦基因组cDNA为模板进行PCR扩增,得到cDNA全长序列;其中,
MdPHR1-F序列如SEQ.ID.NO.3所示,
MdPHR1-R序列如SEQ.ID.NO.4所示;
(3)PCR产物进行凝胶回收、载体连接、大肠杆菌转化,测序得到苹果MdPHR1基因。
5.根据权利要求3所述的苹果MdPHR1基因的制备方法,其特征在于,步骤(2)中的反应条件:在95℃预变性5min;循环参数为:95℃变性30sec、58℃退火30sec、72℃延伸1min,运行35个循环;再在72℃延伸10min。
6.一种如权利要求1所述的苹果MdPHR1基因在转基因制备苹果王林愈伤组织和拟南芥中的应用,所述应用为,促进磷酸盐吸收。
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