CN114134157A - 甘薯IbSAP15基因在调控甘薯叶型与花型中的应用 - Google Patents
甘薯IbSAP15基因在调控甘薯叶型与花型中的应用 Download PDFInfo
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Abstract
本发明公开了一种甘薯IbSAP15基因在调控甘薯叶型和花型中的应用。与徐紫薯8号相比，IbSAP15过表达株系叶片的缺刻更深、花冠开裂，具有较高的观赏价值。本发明将甘薯IbSAP15基因在甘薯中过表达，能够加深叶片缺刻，并使得漏斗状花冠开裂，改变甘薯的叶型和花型，适用于培育不同叶型、花型的甘薯种质，增加观花型/观叶型甘薯品种。
Description
技术领域
本发明属于植物基因工程技术领域，涉及一种甘薯IbSAP15基因在调控甘薯叶型和花型中的应用。
背景技术
锌指结构域中包含多个半胱氨酸和组氨酸残基，能通过这些残基与锌离子结合，自我折叠为“手指”状，故称锌指。包含锌指结构域的蛋白被称为锌指蛋白(Kluget al.,1987)。锌指蛋白可以与DNA、RNA或其他蛋白相互作用，调控基因的转录、翻译等过程。植物胁迫相关蛋白(Stress-associated protein,SAP)家族成员包含A20/AN1锌指结构域，具有泛素连接酶活性或转录因子活性，或可通过构象改变感受细胞内氧化还原状态的变化，调控植物的生物、非生物胁迫响应与生长发育。
甘薯是重要的粮食作物，同时也是重要的蔬菜、饲料和工业原料作物。21世纪以来，甘薯也逐渐被人们作为观赏植物。按照用于观赏的部位不同，观赏甘薯可分为观叶型、观藤型、观花型、观薯型4种(任韵等，2005)。甘薯的叶片形状多样，有心形、掌状、戟形等，观叶甘薯叶形以复缺刻、鸡爪形为主，或叶色为紫色、嫩绿色、混色。甘薯的花冠呈漏斗状，由5个花瓣联合组成，与牵牛花花型一致，花色主要为白色、淡紫、淡粉。
观花甘薯要求易开花、花量大、花期长，可通过短日照诱导开花(孟羽莎等，2019)，并通过施用肥料提高甘薯的花量(邱才飞等，2010)。观赏甘薯可在室内通过水培或基质栽培，装点室内环境。水培操作简单、成本低，且可以与观赏鱼、乌龟或其他水草等水生生物构成生机盎然的生态圈。盆栽甘薯则可用于客厅、阳台、办公室、会议厅等的装饰。室外种植观赏甘薯则可用于立体装饰、坡体绿化、行道绿化、岩石绿化。与室内种植不同，室外种植观赏甘薯的面积更大，可以通过不同叶色、株型的观赏甘薯搭配，或观赏甘薯与其他植被搭配，形成更具美感的景观(孟羽莎等，2019)。目前，对于观花型甘薯花型的研究较少，而且市场观花型甘薯品种较少。
发明内容
本发明提供一种甘薯IbSAP15基因在调控甘薯叶型与花型中的应用。
本发明所述的IbSAP15基因是从甘薯中克隆出的一种AN1锌指蛋白编码基因，其编码的蛋白包含两个保守的AN1锌指结构域，为具有特定序列的DNA分子，其CDS为579bp，核苷酸序列如SEQ ID NO.1所示。
本发明还提供上述IbSAP15基因编码的蛋白IbSAP15，其包含192个氨基酸残基，氨基酸序列如SEQ ID NO.2所示。
本发明首次发现将甘薯IbSAP15基因在甘薯中过表达，能够加深叶片缺刻，并使得漏斗状花冠开裂，改变甘薯的叶型和花型，适用于培育不同叶型、花型的甘薯种质，增加观花型/观叶型甘薯品种。
附图说明
图1是线性化的入门载体
结构示意图。
图2是重组入门载体
-IbSAP15结构示意图。
图3是植物过表达载体pGWB12结构示意图。
图4是重组植物表达载体pGWB12-IbSAP15的结构示意图。
图5是IbSAP15-OE株系的检测结果图，Zi8、-CK、+CK分别为栽培种、阴性对照和阳性对照的检测结果，IbSAP15-OE为不同IbSAP15-OE株系的检测结果。
图6是IbSAP15-OE株系及对照株系中IbSAP15的相对表达量结果图。
图7是IbSAP15-OE株系与栽培种株系叶型的比较图，箭头所指为缺刻变深的部位，比例尺为2cm。
图8是IbSAP15-OE株系与栽培种株系花型的比较图，图8A为IbSAP15-OE株系与栽培种株系花的主视图，图8B为IbSAP15-OE株系与栽培种株系花的俯视图，比例尺为2cm。
具体实施方式
下面结合附图和具体实施例对本发明做进一步详细描述。
下述实施例中所用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径获得。
实施例1：IbSAP15的克隆及过表达载体的构建
实施例中使用的甘薯品种为徐紫薯8号(Zi8)，通过杂交育种选育，亲本为徐紫薯3号和万紫56，是产量高、干率高、花青素含量高的鲜食品种。徐紫薯8号叶型为戟形，易开花，也是优质的观赏甘薯。
通过在甘薯的转录组测序数据中得到IbSAP15的CDS序列(见GenBank登录号MW661075，https://www.ncbi.nlm.nih.gov/nuccore/2026807425)。根据IbSAP15的CDS序列设计特异引物tIbSAP15-F(SEQ ID No.3)和tIbSAP15-R(SEQ ID No.4)，通过PCR从甘薯各组织混合样cDNA中扩增到IbSAP15的CDS全长，并连接到入门载体
上，构建得到重组入门载体
-IbSAP15。通过LR反应，将测序正确的
-IbSAP15上的IbSAP15片段置换到植物表达载体pGWB12上，得到重组植物表达载体pGWB12-IbSAP15。具体步骤为：
1、甘薯混合样RNA提取及完整性检测：收取水培甘薯叶片、不定根、花的样品，分别在液氮中研磨成粉末，等比例混合。取约100mg样品粉末，使用华越洋通用快速植物RNA提取试剂盒，参照说明书提取混合样总RNA。使用Nano drop 1000紫外分光光度计检测RNA的浓度，并取1μg总RNA通过1％琼脂糖凝胶电泳检测完整性。
2、第一链cDNA合成：使用ReverTra
qPCR RT Master Mix with gDNARemover(Toyobo)cDNA合成试剂盒进行第一链cDNA的合成。以500ng RNA为模板，总体系10μL，具体合成步骤参照试剂盒说明书。
3、IbSAP15的扩增和入门载体构建：使用引物对tIbSAP15-F(SEQ ID NO.3)和tIbSAP15-R(SEQ ID NO.4)，以甘薯混合样cDNA为模板进行PCR扩增，所使用的DNA聚合酶为TaqTMVersion 2.0plus dye(TaKaRa)。反应总体系50μL，包括Taq Version 2.0plus dye(2×)25μL，cDNA模板1μL，10mmol L-1正反向引物各2μL，ddH2O 20μL。PCR扩增程序为：98℃预变性30s；98℃变性10s，60℃退火15s，72℃延伸15s，35个循环；72℃充分延伸2min，4℃保温。PCR产物进行电泳检测和胶回收，通过TA克隆将胶回收产物连接在
(Invitrogen)上，构建入门载体
-IbSAP15，转化大肠杆菌DH5α。挑取阳性克隆送至生工生物工程(上海)股份有限公司测序，选取序列及方向均正确的单克隆提取
-IbSAP15质粒用于后续实验。
tIbSAP15-F(SEQ ID NO.3)：5’-ATGGGAGGAGGAACAGAAGCT-3’，
tIbSAP15-R(SEQ ID NO.4)：5’-TCAAAAAGCTTTAACAGAAGGTATGGTAGTTGG-3’。
4、IbSAP15表达载体构建：取100ng
-IbSAP15与100ng pGWB12质粒，混合均匀，加入2μL Gateway LR Clonase II Enzyme Mix(Invitrogen)，补水至10μL，25℃孵育1h。加入1μL蛋白酶K，37℃10min终止反应。取1μL反应液转化至大肠杆菌DH5α中，挑取阳性克隆提取质粒，获得植物表达载体pGWB12-IbSAP15。
实施例2：在甘薯中过表达IbSAP15基因
为研究IbSAP15基因的功能，将基因IbSAP15在甘薯中过表达，通过对比过表达株系和栽培种的表型来分析IbSAP15的功能。农杆菌介导的甘薯遗传转化方法包括侵染、共培养、筛选鉴定、诱导成苗等，后续通过组培快繁和移栽后扦插来进行扩繁。所用抗生素和激素有2,4-二氯苯氧乙酸(2,4-Dichlorophenoxyacetic acid，2,4-D)、脱落酸(AbscisicAcid，ABA)、卡那霉素(Kanamycin，Kan)、潮霉素(homomycin B，Hyg)、利福平(Rifampin，Rif)、头孢(Cefotaxime sodium，Cef)。所用培养基有：YEB(酵母提取物1g L-1，牛肉浸膏5gL-1，蔗糖5g L-1，蛋白胨5g L-1，MgSO47H2O 0.5g L-1)、MS(购买自北京酷来搏科技有限公司)、MSD(MS+2mg·L-1 2,4-D)。其他试剂有：乙酰丁香酮(Acetosyringone，AS)。具体步骤如下：
1、农杆菌介导的甘薯遗传转化：
通过液氮冻融法将植物表达载体pGWB12-IbSAP15转入农杆菌菌株GV3101感受态中，挑取阳性克隆接种到1mL含有50mg L-1Kan和20mg mL-1Rif的YEB液体培养基中，28℃过夜培养。吸取500μL过夜培养的菌液，转接到新的50mL含有50mg L-1Kan和20mg mL-1Rif的YEB液体培养基中，培养至OD600＝0.6左右。4000rpm离心5min，收集菌体，以MSD液体培养基重悬，调整OD600在0.1～0.2之间，并加入AS至终浓度30mg L-1。将甘薯愈伤研磨成1～2mm的颗粒，使用MSD清洗3次，放入10mL重悬的农杆菌菌液中，40rpm、25℃避光培养30min，超声波处理15s。将愈伤取出，放置于垫有滤纸的MSD+30mg L-1AS培养基中，避光共培养。3d后，使用MSD清洗至上清清澈，转移至MSD+300mg L-1Cef液体培养基中，室温避光慢摇1h，接种到MSD+200mg L-1Cef固体培养基上共培养2周。
2、抗性愈伤的筛选、诱导与移栽
将共培养2周后的愈伤转移至MSD+10mg L-1Hyg+200mg L-1Cef固体培养基上，2周后挑选状态良好的愈伤继代到新的培养基上再培养2周。将愈伤转移到MS+10mg L-1Hyg+200mg L-1Cef+1mg L-1ABA固体培养基上诱导体细胞胚，每2周继代一次。体胚形成芽之后转移到固体培养基MS+10mg L-1Hyg+200mg L-1Cef上成苗。试管苗长至6cm以上后切段转移至MS固体培养基上扩繁，在组培苗数量较多后，选取一部分长势较好的进行移栽。用水清洗干净组培苗上的培养基，将其移栽至营养土中，用保鲜膜保湿，置于28℃培养室中光照培养。1d后去除保鲜膜，正常培养。
实施例3：IbSAP15-OE株系的鉴定与IbSAP15表达量的检测
1、IbSAP15-OE转基因株系的鉴定
使用改良的CTAB法提取阳性株系及栽培种甘薯的基因组DNA，使用IbSAP15-OE检测引物FLAG-F(pGWB12)(SEQ ID NO.5)和ATTB2-R(SEQ ID NO.6)进行PCR扩增，检测是否为IbSAP15转基因，并以pGWB12-IbSAP15质粒为阳性对照，以栽培种徐紫薯8号基因组DNA为阴性对照，以ddH2O为空白对照。所使用的DNA聚合酶为2×PCR Master Mix(CWBIO)。反应总体系20μL，包括2×PCR Master Mix 10μL，模板1μL，10mmol L-1正反向引物各0.5μL，ddH2O 8μL。PCR扩增程序为：94℃预变性2min；94℃变性30s，60℃退火30s，72℃延伸30s，35个循环；72℃充分延伸2min；4℃保温。反应结束后使用琼脂糖凝胶电泳分析，栽培种(Zi8)和阴性对照(-CK)未扩增出条带，阳性对照(+CK)扩增出950bp大小的条带，8个拟转基因株系中有7个扩增出了与阳性对照一致的条带，确定这7个株系为IbSAP15-OE转基因株系(图5)。
FLAG-F(pGWB12)(SEQ ID NO.5)：5’-ATGAGCGACTACAAGGATGACGAT-3’，ATTB2-R(SEQ ID NO.6)：5’-ACCACTTTGTACAAGAAAGCTGGG-3’。
2、IbSAP15-OE转基因株系IbSAP15表达量的检测
参照实施例1提取IbSAP15-OE转基因株系叶片的RNA，并反转录为cDNA。使用RT-qPCR检测IbSAP15的表达量。使用SYBR Green Realtime PCR Master Mix(Toyobo)试剂盒进行RT-qPCR检测，反应体系为10μL，其中2×SYBR Green Realtime PCR Master Mix(Toyobo)50μL，20倍稀释的cDNA模板2μL，10mmol L-1正反向引物qIbSAP15-F(SEQ ID NO.7)和qIbSAP15-R(SEQ ID NO.8)各0.5μL，ddH2O 2μL。所采用的RT-qPCR程序为：第一阶段：95℃预变性10min；第二阶段：95℃变性15s，60℃退火15s，72℃延伸20s，40个循环；第三阶段：65℃-95℃检测溶解曲线。以甘薯ADP核糖基化因子(ADP-ribosylation factor，IbARF)基因(Park et al.,2012)为内参，引物序列为qIbARF-F(SEQ ID NO.9)和qIbARF-R(SEQ IDNO.10)。通过2-ΔΔCt法计算IbSAP15在各个株系中相对栽培种徐紫薯8号的表达量。其中3个IbSAP15-OE株系的表达量如图6所示，分别为栽培种徐紫薯8号的123.56、30.78、16.16倍，分别命名为OE1、OE2和OE3。
qIbSAP15-F(SEQ ID NO.7)：5’-GATCACGCTTGCAAAGGCAG-3’，
qIbSAP15-R(SEQ ID NO.8)：5’-CGTAGAATCCCTGCTCTTGTTTCC-3’，
qIbARF-F(SEQ ID NO.9)：5’-CTTTGCCAAGAAGGAGATGC-3’，
qIbARF-R(SEQ ID NO.10)：5’-TCTTGTCCTGACCACCAACA-3’。
实施例4：IbSAP15-OE株系与栽培种徐紫薯8号叶型、花型的比较
过表达株系的叶片相比栽培种徐紫薯8号的叶片，其叶型发生了变化。OE1、OE2两个株系的叶片相比于栽培种更小，且缺刻变深，特别是OE1株系，叶片近似鸡爪状，更具观赏价值(图7)。
将IbSAP15-OE株系和栽培种徐紫薯8号栽种于花盆中，正常日照培养，待开花后比较花型。期间可通过短日照处理加速开花，即8h光照/16h黑暗培养。栽培种的花冠为漏斗状，每个花冠的5个花瓣相互联合在一起，IbSAP15-OE株系的花冠则出现了不同程度的开裂。OE1株系的花筒完全开裂，OE2株系花筒部分开裂，这在甘薯及其近缘种中都是极其少见的，具有极高的观赏价值(图8)。
序列表
<110> 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心)
<120> 甘薯IbSAP15基因在调控甘薯叶型与花型中的应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 579
<212> DNA
<213> 甘薯(Ipomoea batatas L.)
<400> 1
atgggaggag gaacagaagc tttcccagat ttaggacgcc attgcgaatt ctccgattgc 60
cgccaactcg attttctccc cttccaatgc gacgcttgcc gtcacgtttt ctgtgtagac 120
caccgatcat ataaatccca cgcctgccca aaatccgacc gccatagccg caaggttgtg 180
gtgtgcgacg cctgttccac gtcgatcgag accaccggct gcggcggcga agacgaggag 240
aaggccatat tgcagaggca ccagaaatta gggcactgtg atcccgcgaa gaagaagaaa 300
cctacgtgcc ccgtgaggcg gtgcaaggaa cctttaacct tctcaaatac tagcgtctgt 360
aagggctgcc agattccggt gtgcttaaaa caccgttttc cggcggatca cgcttgcaaa 420
ggcagagcca cttcttctcc ggcgccgccg gcgctcaggg gcggcgtcaa taacaagttt 480
ctggttgcgt ttgctgcaag gaacccgaaa gattgtggaa acaagagccg ggattctacg 540
tcttctccaa ctaccatacc ttctgttaaa gctttttga 579
<210> 2
<211> 192
<212> PRT
<213> 甘薯(Ipomoea batatas L.)
<400> 2
Met Gly Gly Gly Thr Glu Ala Phe Pro Asp Leu Gly Arg His Cys Glu
1 5 10 15
Phe Ser Asp Cys Arg Gln Leu Asp Phe Leu Pro Phe Gln Cys Asp Ala
20 25 30
Cys Arg His Val Phe Cys Val Asp His Arg Ser Tyr Lys Ser His Ala
35 40 45
Cys Pro Lys Ser Asp Arg His Ser Arg Lys Val Val Val Cys Asp Ala
50 55 60
Cys Ser Thr Ser Ile Glu Thr Thr Gly Cys Gly Gly Glu Asp Glu Glu
65 70 75 80
Lys Ala Ile Leu Gln Arg His Gln Lys Leu Gly His Cys Asp Pro Ala
85 90 95
Lys Lys Lys Lys Pro Thr Cys Pro Val Arg Arg Cys Lys Glu Pro Leu
100 105 110
Thr Phe Ser Asn Thr Ser Val Cys Lys Gly Cys Gln Ile Pro Val Cys
115 120 125
Leu Lys His Arg Phe Pro Ala Asp His Ala Cys Lys Gly Arg Ala Thr
130 135 140
Ser Ser Pro Ala Pro Pro Ala Leu Arg Gly Gly Val Asn Asn Lys Phe
145 150 155 160
Leu Val Ala Phe Ala Ala Arg Asn Pro Lys Asp Cys Gly Asn Lys Ser
165 170 175
Arg Asp Ser Thr Ser Ser Pro Thr Thr Ile Pro Ser Val Lys Ala Phe
180 185 190
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgggaggag gaacagaagc t 21
<210> 4
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tcaaaaagct ttaacagaag gtatggtagt tgg 33
<210> 5
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgagcgact acaaggatga cgat 24
<210> 6
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
accactttgt acaagaaagc tggg 24
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gatcacgctt gcaaaggcag 20
<210> 8
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cgtagaatcc ctgctcttgt ttcc 24
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ctttgccaag aaggagatgc 20
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tcttgtcctg accaccaaca 20
Claims (2)
1.甘薯IbSAP15基因在调控甘薯叶型与花型中的应用，所述的甘薯IbSAP15基因核苷酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的应用，其特征在于，所述的IbSAP15基因编码的蛋白IbSAP15的氨基酸序列如SEQ ID NO.2所示。
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| NL2032335A NL2032335B1 (en) | 2021-12-03 | 2022-06-30 | Use of sweet potato ibsap15 gene in regulating leaf shape and flower shape of sweet potato |
| PCT/CN2022/132557 WO2023098481A1 (zh) | 2021-12-03 | 2022-11-17 | 甘薯IbSAP15基因在调控甘薯叶型与花型中的应用 |
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| Title |
|---|
| LIU,Y.ET AL.,: ""GenBank: MW661075.1"", 《GENBANK》 * |
| 杨强强等: ""甘薯IbSAP15 基因提高拟南芥耐盐性"", 《2018中国遗传学会第十次全国会员代表大会暨学术讨论会论文摘要汇编》 * |
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