CN114621334B - 一种马铃薯StABI5基因在抗旱调节中的应用以及基于该基因调节马铃薯抗旱性的方法 - Google Patents
一种马铃薯StABI5基因在抗旱调节中的应用以及基于该基因调节马铃薯抗旱性的方法 Download PDFInfo
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Abstract
本发明提供了一种马铃薯StABI5基因在抗旱调节中的应用。本发明还提供了一种基于StABI5基因调节马铃薯抗旱性的方法,通过对马铃薯植株中StABI5基因的表达调控来降低或提升马铃薯植株对干旱的抗性;其中,所述StABI5基因的核苷酸序列如SEQ ID NO.1所示,编码的氨基酸序列如SEQ ID NO.2所示。本发明的优点在于:利用StABI5基因对马铃薯中脱落酸代谢途径中相关基因的调控作用来调节马铃薯对干旱胁迫的应答,对开发具有干旱抗性的马铃薯品种具有重要意义。
Description
技术领域
本发明涉及分子生物学、基因工程技术领域,尤其涉及一种马铃薯StABI5基因在抗旱调节中的应用以及基于该基因调节马铃薯抗旱性的方法。
背景技术
马铃薯(Solanum tuberosum L.)是重要的粮食作物,以其适应性强、产量高、营养成分全和产业链长等特点,受到了全世界的广泛重视。
干旱具有发生频繁、持续时间长以及影响范围大等特点。因此,通过育种手段培育作物抗旱新品种尤为迫切和重要。作物育种包括传统遗传育种和分子育种,传统遗传育种周期长,方向难以把握,分子育种则可以针对植物特定性状进行遗传改良,能够大大缩短育种周期。而分子育种的关键就是挖掘重要的功能基因,并明晰其具体的功能机制。
目前,分子遗传育种方法已在水稻、玉米等粮食作物中广泛应用,而在马铃薯育种中的研究还相对不足。在马铃薯中只克隆了少数与抗旱相关的基因,而它们抗旱机制仍然不清楚,有待进一步探究。bZIP类转录因子在植物非生物逆境应答反应中发挥重要调控作用,并且在水稻等模式植物中已有广泛研究应用,但马铃薯中该家族基因的功能研究几乎没有。
因此,筛选马铃薯中bZIP家族基因,并研究其在马铃薯非生物逆境应答反应中的作用,对于加快马铃薯抗旱育种具有重大意义。
发明内容
本发明所要解决的技术问题在于提供一种马铃薯StABI5基因在抗旱调节中的应用以及基于该基因调节马铃薯抗旱性的方法,其利用StABI5基因对马铃薯中脱落酸代谢途径中相关基因的调控作用来调节马铃薯对干旱胁迫的应答,对开发具有干旱抗性的马铃薯品种具有重要意义。
本发明采用以下技术方案解决上述技术问题:
一种马铃薯StABI5基因在抗旱调节中的应用,所述StABI5基因的核苷酸序列如SEQ ID NO.1所示,编码的氨基酸序列如SEQ ID NO.2所示。
一种基于StABI5基因调节马铃薯抗旱性的方法,通过对马铃薯植株中StABI5基因的表达调控来降低或提升马铃薯植株对干旱的抗性;其中,所述StABI5基因的核苷酸序列如SEQ ID NO.1所示,编码的氨基酸序列如SEQ ID NO.2所示。
作为本发明的优选方式之一,所述StABI5基因的获得方法为:提取马铃薯植株的总RNA并反转录成cDNA;以cDNA为模板,以ABI5-F、ABI5-R为引物进行PCR克隆;其中,ABI5-F、ABI5-R引物的序列分别如SEQ ID NO.3、SEQ ID NO.4所示。
作为本发明的优选方式之一,所述StABI5基因在马铃薯干旱胁迫过程中发挥负调控作用。
本发明相比现有技术的优点在于:
(1)本发明从马铃薯基因组中筛选了一个bZIP家族基因StABI5,经发现,该bZIP家族基因StABI5对马铃薯中脱落酸(ABA)代谢途径中相关基因具有调控作用,可调节马铃薯对干旱胁迫的应答;
(2)本发明通过基因工程方法获得过StABI5基因的过表达植株,经研究,StABI5基因的过表达植株对干旱的抗性低于对照野生型马铃薯植株;本发明为马铃薯分子育种创建抗旱优质材料提供新资源,对于加快马铃薯抗旱育种具有重大意义。
附图说明
图1是实施例1中StABI5基因在干旱和外源激素ABA诱导下表达模式分析(图中,左图为干旱诱导下StABI5基因的表达情况;右图为外源激素ABA诱导下StABI5基因的表达情况);
图2是实施例2中pHELLSGATE 8植物表达载体图谱;
图3是实施例3中StABI5转基因马铃薯植株的PCR检测结果(图中,M:DL2000Marker;1-6为不同转基因株系;+:以pHELLSGATE 8质粒为模板阳性克隆;-:以野生型马铃薯为模板阴性对照);
图4是实施例3中StABI5基因在不同转基因株系中表达水平检测结果(图中,CK为对照组;OE-1、OE-9、OE-13、OE-14、OE-15和OE-16为六种不同的转基因株系);
图5是实施例4中StABI5转基因植株抗旱性分析(图中,自上而下分别为植株正常状态图、植株干旱状态、干旱植株恢复浇水2天状态);
图6是实施例4中转基因植株在干旱处理后生理指标分析(图中,A图为干旱处理后马铃薯植株存活率;B图为植株内丙二醛含量);
图7是实施例4中StABI5转基因马铃薯植株内ABA含量表示图。
具体实施方式
下面对本发明的实施例作详细说明,本实施例在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
实施例1StABI5基因的逆境诱导表达模式分析
(1)马铃薯材料的准备及逆境处理
选择生长状态良好且大小相对一致的马铃薯试管苗,移栽至栽培槽内(含有蛭石和黑土混合物),然后放置于温室内生长,光周期期14/10h,室温25℃。待马铃薯试管苗长到4叶期时开始逆境胁迫处理,具体处理方式为:100μMABA喷洒马铃薯植株叶片;20%聚乙二醇(PEG6000)充分浇灌马铃薯植株模拟干旱胁迫。分别在处理后0、1、3、6和12h各时间点取样,液氮速冻后于-80℃超低温冰箱保存备用。取样过程中,每个样品取自3棵不同植株,且每个时间点重复取样3次。
(2)总RNA提取
将上述取得样品于研钵中加入液氮充分研磨,取0.1g迅速转移到1.5mL的离心管(液氮预冷)中,加入1mL Trizol试剂,充分混匀后室温放置10min,使其充分裂解;加入1/4体积4℃的氯仿试剂,并在旋涡仪上迅速震荡30秒,冰上放置3min,于4℃预冷的冷冻离心机中13000rpm离心10min;吸取上层水相,至另一个离心管中;加入等体积预冷的异丙醇混匀,上下颠倒混匀后置于冰上5-10min,然后4℃,13000rpm离心15min;弃去上清,RNA沉于管底;加入1mL预冷75%乙醇洗涤沉淀,用移液器吹打10s,4℃,13000rpm离心5min;重复上述步骤,然后用枪吸掉管壁上残留的乙醇溶液,并在通风橱里晾干;加入RNA free水溶解RNA,并用微量移液器吹打混匀。利用核酸检测仪测量RNA浓度及纯度,于-80℃冰箱保存备用。
(3)RNA反转录成cDNA
采用TaKaRa公司的反转录试剂盒,根据操作说明进行如下步骤:
DNA的去除:取5×gDNA Eraser缓冲液2μL、gDNA Eraser 1.0μL、RNA样品1μg,加入RNA free管中,再用RNase free H2O补充到10μL。在PCR仪中42℃反应2min,最后4℃保温;
cDNA链的合成:在上面的反应体系中分别加入PrimeScript RT Enzyme Mix I 1μL、5×PrimeScript 4μL、RT Primer Mix 1μL以及RNase free 4μL。然后转移到PCR仪中设置如下程序:37℃,15min;85℃5s;4℃保温。
(4)StABI5基因诱导表达分析
根据StABI5基因编码序列(如SEQ ID NO.1所示),设计一对荧光定量PCR引物qABI5-F(序列如SEQ ID NO.5所示)和qABI5-R(序列如SEQ ID NO.6所示),并在NCBI网站进行扩增特异性检测,扩增产物150bp。
以马铃薯elf基因(本领域的公开基因)作为内参。荧光定量PCR反应体系20μL:上游引物1.0μL,下游引物1.0μL,cDNA模板1.5μL,SYBR Green 10μL,ddH2O 6.5μL。PCR反应程序为:95℃,10min;95℃,15s;60℃,1min;反应设40个循环。所有循环结束后增加溶解曲线步骤。扩增信号及数据的处理采用比较Ct法(ΔΔCt法)。
结果如图1所示,StABI5基因受到干旱胁迫诱导表达,且被外源激素ABA诱导。
荧光定量PCR引物序列为:qABI5-F(SEQ ID NO.5)、qABI5-R(SEQ ID NO.6)、elf-F(SEQ ID NO.7)、elf-R(SEQ ID NO.8)。
实施例2StABI5基因的克隆及过表达载体构建
根据StABI5基因的全长CDS序列以及pHELLSGATE 8载体序列特征(载体图谱如图2所示),设计同源重组引物ABI5-F(序列如SEQ ID NO.3所示)和ABI5-R(序列如SEQ ID NO.4所示),进行PCR扩增。
PCR反应程序为:98℃,预变性3min;98℃,变性10s;60℃,退火5s;68℃,延伸20s,33个循环;68℃,复性7min;10℃保存。
将PCR扩增产物用质量比为2%的琼脂糖凝胶电泳检测,回收目的片段。同时,将pHELLSGATE 8载体用XhoI和XbaI双酶切,回收酶切后大片段。再利用同源重组酶将回收StABI5基因PCR扩增产物和线性化载体进行连接,连接产物转化大肠杆菌Trans 5α细胞中,在含有壮观霉素LB固体培养基上进行筛选,挑选生长状态好的单克隆送上海生工生物工程有限公司测序。测序结果用MEGA7.0软件进行比对。选择测序完全正确的克隆扩繁,并提质粒,构建完成StABI5-pHELLSGATE 8融合表达载体,用作马铃薯遗传转化。
实施例3StABI5转基因植株的获得
(1)马铃薯遗传转化
将构融合表达载体StABI5-pHELLSGATE 8通过农杆菌介导遗传转化方法转化到马铃薯品种鄂马铃薯3号中。经过预培养、浸染、共培养、筛选具有卡那霉素抗性的愈伤、分化、生根、炼苗、移栽,得到转基因植株。具体转化方法为本领域常规方法,在此不再赘述。
(2)阳性转基因植株鉴定
通过卡那霉素抗性筛选,总共获得抗性转化植株6株,提取转基因植株的基因组,通过PCR方法进行阳性检测。根据pHELLSGATE 8载体上的序列特征,以35S启动子作为目标基因,设计特异性引物35S-F(序列如SEQ ID NO.9所示)和35S-R(序列如SEQ ID NO.10所示)进行PCR扩增,以pHELLSGATE 8载体作为阳性对照,野生型马铃薯基因组作为阴性对照。PCR反应程序如实施例2,结果如图3所示,转基因植株和阳性对照均能扩增出500bp左右条带,阴性对照无条带。回收PCR产物,并送生工测序,结果与35S基因序列一致,表明转基因植株均为阳性植株。
(3)阳性转基因植株中StABI5表达水平检测
为验证StABI5基因在转基因马铃薯中是否表达成功,提取转基因植株RNA,通过荧光定量PCR方法,检测StABI5基因在不同转基因植株中表达水平。
RNA提取、反转录、荧光定量体系、反应程序以及扩增引物见实施例1。实验结果如图4所示,StABI5基因在不同转基因株系中表达成功,但表达水平存在一定差异,其中OE-1、OE-15和OE-16表达水平较高,可用于抗旱性分析。
实施例4StABI5转基因植株抗旱性鉴定
将三个基因表达水平比较高的转基因株系OE-1、OE-15和OE-16及对照株系的试管苗在实验室扩繁,待植株长到10cm左右时,选择生长势强,大小相对一致的试管苗移栽到培养盆中,每个盆中装有相同重量的营养土,每个盆中栽一株试管苗。每个株系播种15株,设置3个重复。在生长期内,所有植株采用相同管理模式,每次每个盆种中浇相同体积的水。在日光温室中培养1个月后进行干旱处理。具体处理方式将培养一个月的转基因马铃薯植株和野生型植株,浇入饱和水,以此时为处理0d,干早胁迫15d,直到植株出现明显的干旱胁迫表型,恢复浇水,培养2d,观察表型(如图5)。统计处理前后转基因植株存活率并检测植株体内丙二醛含量。
结果如图6所示,干旱处理后三个转基因株系的存活率低于野生型对照,植株体内的丙二醛含量要高于野生型,表明StABI5基因在马铃薯干旱胁迫过程中发挥负调控作用。
此外,对转基因植株内ABA含量进行检测,结果如图7所示,三个转基因株系中ABA含量均低于野生型植株,表明StABI5基因通过抑制马铃薯体内ABA的合成,从而调控马铃薯对干旱的响应。
SEQUENCE LISTING
<110> 安徽省农业科学院园艺研究所
<120> 一种马铃薯StABI5基因在抗旱调节中的应用以及基于该基因调节马铃薯抗旱
性的方法
<130> 2022
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 1284
<212> DNA
<213> 马铃薯(Solanum tuberosum)
<400> 1
atgggagtac cagaatcaga gatggtgtca caaagtgagg ttcaatcacc attgcaacca 60
gaccaaaacc agaacaagaa caacccgttt ccgtccctcg gtcgacaagc gtcgatttac 120
tccctcacac ttgatgaatt ccaacacact gtttgcgaga gtgggaagaa ttttgggtcg 180
atgaacatgg atgaatttct taacagcatt tggactgctg aagaaaacca agcccacgcc 240
catgcccacg tgcacgctca gcctcactgc caggctgcaa gtactgggga agcaactagc 300
gcgccacgtt ttgcattagg acagggcaat gtttcgttac agaaagctat tgtcgagcag 360
ccaagcttgc caagacaagg ctcacttacg cttcccgcac cgttgtgtag taaaactgta 420
gatgaagttt ggtcagaaat tcataagacc caacaagagc agcaacagaa caacgggtgc 480
agcatacaga acaccggtaa cggaagttcc actcaacgac agactacgtt cggtgagatg 540
acgctcgagg attttttggt taaagcaggg gtcgtgcgcg aacagggcaa ttcagcccct 600
gcacctcctc agcagcaatc atatatgatg tatccaaaca gtgcaaatcc cactatggct 660
cggcctgtta tcggccttgg cggagtcacg ggcggtgttg gcgtcggtgt cgcgattcct 720
ggttatccgc cacttccaca aaccggggtg gtcgaggcgc ctgtgtaccc tatgagcatg 780
aaaagaggca gtggattccc acaacagcca accccagtct acggtggtag aatgggaaac 840
ggtagtgggg ttggctacgg gcaagtagtg caaagcgtga ccggaatggg gtcaccacta 900
agtccggtgt cgtcagatgg actatgcgtg aatcaaatcg ataatgtggg ccaatacggg 960
ttggaaatag gaatgagagg tgggcgaaaa cgcgtactag acggtccagt agagaaagta 1020
gttgaaagga ggcaaaggag gatgatcaag aacagagaat cagcagcaag atcaagagca 1080
aggaaacagg cttacactgt tgaacttgag gcagaattga accagctaaa agaagaaaat 1140
gcacatctaa aacaggccct ggcggagctt gagaggaaaa ggaaacaaca gtactttgat 1200
gaagcgaaaa cgaaagctca aacaaaggcg caaaaggcga atggcaaatt aagagggatg 1260
agaaggagct ttagttgccc ttga 1284
<210> 2
<211> 427
<212> PRT
<213> 马铃薯(Solanum tuberosum)
<400> 2
Met Gly Val Pro Glu Ser Glu Met Val Ser Gln Ser Glu Val Gln Ser
1 5 10 15
Pro Leu Gln Pro Asp Gln Asn Gln Asn Lys Asn Asn Pro Phe Pro Ser
20 25 30
Leu Gly Arg Gln Ala Ser Ile Tyr Ser Leu Thr Leu Asp Glu Phe Gln
35 40 45
His Thr Val Cys Glu Ser Gly Lys Asn Phe Gly Ser Met Asn Met Asp
50 55 60
Glu Phe Leu Asn Ser Ile Trp Thr Ala Glu Glu Asn Gln Ala His Ala
65 70 75 80
His Ala His Val His Ala Gln Pro His Cys Gln Ala Ala Ser Thr Gly
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Glu Ala Thr Ser Ala Pro Arg Phe Ala Leu Gly Gln Gly Asn Val Ser
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Leu Gln Lys Ala Ile Val Glu Gln Pro Ser Leu Pro Arg Gln Gly Ser
115 120 125
Leu Thr Leu Pro Ala Pro Leu Cys Ser Lys Thr Val Asp Glu Val Trp
130 135 140
Ser Glu Ile His Lys Thr Gln Gln Glu Gln Gln Gln Asn Asn Gly Cys
145 150 155 160
Ser Ile Gln Asn Thr Gly Asn Gly Ser Ser Thr Gln Arg Gln Thr Thr
165 170 175
Phe Gly Glu Met Thr Leu Glu Asp Phe Leu Val Lys Ala Gly Val Val
180 185 190
Arg Glu Gln Gly Asn Ser Ala Pro Ala Pro Pro Gln Gln Gln Ser Tyr
195 200 205
Met Met Tyr Pro Asn Ser Ala Asn Pro Thr Met Ala Arg Pro Val Ile
210 215 220
Gly Leu Gly Gly Val Thr Gly Gly Val Gly Val Gly Val Ala Ile Pro
225 230 235 240
Gly Tyr Pro Pro Leu Pro Gln Thr Gly Val Val Glu Ala Pro Val Tyr
245 250 255
Pro Met Ser Met Lys Arg Gly Ser Gly Phe Pro Gln Gln Pro Thr Pro
260 265 270
Val Tyr Gly Gly Arg Met Gly Asn Gly Ser Gly Val Gly Tyr Gly Gln
275 280 285
Val Val Gln Ser Val Thr Gly Met Gly Ser Pro Leu Ser Pro Val Ser
290 295 300
Ser Asp Gly Leu Cys Val Asn Gln Ile Asp Asn Val Gly Gln Tyr Gly
305 310 315 320
Leu Glu Ile Gly Met Arg Gly Gly Arg Lys Arg Val Leu Asp Gly Pro
325 330 335
Val Glu Lys Val Val Glu Arg Arg Gln Arg Arg Met Ile Lys Asn Arg
340 345 350
Glu Ser Ala Ala Arg Ser Arg Ala Arg Lys Gln Ala Tyr Thr Val Glu
355 360 365
Leu Glu Ala Glu Leu Asn Gln Leu Lys Glu Glu Asn Ala His Leu Lys
370 375 380
Gln Ala Leu Ala Glu Leu Glu Arg Lys Arg Lys Gln Gln Tyr Phe Asp
385 390 395 400
Glu Ala Lys Thr Lys Ala Gln Thr Lys Ala Gln Lys Ala Asn Gly Lys
405 410 415
Leu Arg Gly Met Arg Arg Ser Phe Ser Cys Pro
420 425
<210> 3
<211> 41
<212> DNA
<213> 人工序列
<400> 3
agaggacacg ctcgagatgg gagtaccaga atcagagatg g 41
<210> 4
<211> 42
<212> DNA
<213> 人工序列
<400> 4
cattaaagca ggactctaga tcaagggcaa ctaaagctcc tt 42
<210> 5
<211> 20
<212> DNA
<213> 人工序列
<400> 5
aatgagaggt gggcgaaaac 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列
<400> 6
gccttttgcg cctttgtttg 20
<210> 7
<211> 19
<212> DNA
<213> 人工序列
<400> 7
caaggatgac ccagccaag 19
<210> 8
<211> 20
<212> DNA
<213> 人工序列
<400> 8
ttccttacct gaacgcctgt 20
<210> 9
<211> 25
<212> DNA
<213> 人工序列
<400> 9
caggactaat tgcatcaaga acaca 25
<210> 10
<211> 25
<212> DNA
<213> 人工序列
<400> 10
tctccaaatg aaatgaactt cctta 25
Claims (3)
1. 一种马铃薯StABI5基因在抗旱调节中的应用,其特征在于,所述StABI5基因的核苷酸序列如SEQ ID NO.1所示,编码的氨基酸序列如SEQ ID NO.2所示;通过过表达马铃薯植株中StABI5基因来降低马铃薯植株对干旱的抗性。
2. 一种基于StABI5基因调节马铃薯抗旱性的方法,其特征在于,通过过表达马铃薯植株中StABI5基因来降低马铃薯植株对干旱的抗性;其中,所述StABI5基因的核苷酸序列如SEQ ID NO.1所示,编码的氨基酸序列如SEQ ID NO.2所示。
3. 根据权利要求2所述的基于StABI5基因调节马铃薯抗旱性的方法,其特征在于,所述StABI5基因的获得方法为:提取马铃薯植株的总RNA并反转录成cDNA;以cDNA为模板,以ABI5-F、ABI5-R为引物进行PCR克隆;其中,ABI5-F、ABI5-R引物的序列分别如SEQ ID NO.3、SEQ ID NO.4所示。
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