CN116497042A - 一种燕子花anr基因克隆及其应用 - Google Patents
一种燕子花anr基因克隆及其应用 Download PDFInfo
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Abstract
本发明属于植物基因工程育种领域,具体涉及一种燕子花ANR基因的克隆及其应用。所述的燕子花ANR基因的核苷酸序列如SEQ ID No.1所示,该基因编码的氨基酸序列如SEQ ID No.2所示。首次发现燕子花ANR基因在烟草中过量表达会导致烟草花冠中花青素含量降低,花冠颜色变浅。本发明提供的燕子花ANR基因不仅可以为燕子花的花色调控研究提供重要的理论基础,更是为园林植物花色育种提供重要基因资源。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及一种燕子花ANR基因克隆及其应用。
背景技术
在植物育种过程中,花色是人们关注的重点性状之一,特别是在以观花为主的观赏植物育种领域,一个新的花色品种能产生巨大的经济效益。基因工程育种可定向修饰植物的某些目标性状而保留其它原有的性状,大大缩短育种周期,为花色育种提供了新思路。植物花色主要成分是花青素,花青素含量及成分的差异是不同品种花色多样性的决定因素。在花青素生物合成途径中的支路结构基因,可以与主路结构基因竞争上一步基因的反应产物,阻碍花青素的积累,使植物花色变浅,丰富植物花色多样性。
燕子花(Iris laevigata)是鸢尾科鸢尾属的多年生草本,花形优美,耐寒性强,在园林中常应用于水池边,疏林下,岩石旁,是一种优良的园林观赏花卉。自然界中燕子花主要为蓝紫色,花色较为单一,为满足园林应用中花色多样性的需求,燕子花的花色育种尤为必要。燕子花中正调控花青素合成的结构基因的作用机制及生物学功能在模式植物中得到验证,但支路基因ANR对植物花色的影响还尚未报道。
发明内容
有鉴于此,本发明提供了一种燕子花ANR基因、克隆方法、载体构建及其在观赏植物花色育种上的应用方法。
本发明的目的之一在于提供一种燕子花ANR基因。该基因全长1287bp,ORF区为1020bp,编码339个氨基酸。
本发明的目的之二在于提供一种燕子花ANR基因所编码的蛋白,该蛋白可以调控烟草花冠中花青素的合成。
本发明的目的之三在于提供上述燕子花ANR基因相关的生物材料。
本发明的目的之四在于提供上述燕子花ANR基因的应用。
本发明的目的通过以下技术方案实现:
一种燕子花ANR基因,其特征在于,为如下A1)或A2)所示的基因:
A1)核苷酸序列是序列表中SEQ ID No.1的cDNA分子或基因组DNA;
A2)与A1)限定的核苷酸序列具有90%或90%以上相似性,且编码权利要求2所述的燕子花ANR蛋白的cDNA分子或基因组DNA。
上述燕子花ANR基因所编码的蛋白,其特征在于,为具有如下B1)、B2)、B3)或B4)所示序列的蛋白质:
B1)由序列表中SEQ ID No.2所示的氨基酸序列组成的蛋白质;
B2)其氨基酸序列与SEQ ID No.2所示的氨基酸序列具有90%以上同源性且具有相同表达功能的蛋白质;
B3)在序列表中SEQ ID No.2所示的氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸残基得到的具有相同功能的由B1)衍生的蛋白质;
B4)在B1)、B2)或B3)的N端或/和C端连接标签得到的融合蛋白质。
与上述的燕子花ANR基因相关的生物材料,其特征在于,为下述C1)至C5)中的任一种:
C1)含有燕子花ANR基因的重组克隆载体;
C2)含有燕子花ANR基因的重组植物表达载体;
C3)含有燕子花ANR基因的N端或/和C端连接标签得到的重组植物表达载体;
C4)含有C2)或C3)所述的重组载体的生物工程菌;
C5)含有C2)或C3)所述的重组植物表达载体的转基因植物。
C2)和/或C3)中所述的含有上述燕子花ANR基因的植物表达载体是指能够在宿主细胞中表达上述燕子花ANR基因的DNA,该DNA不但可包含启动ANR基因转录的启动子,还可包含终止ANR基因转录的终止子。
本发明实施例中所述的克隆载体为PMD18-T vector。
本发明实施例中所述的植物表达载体为GV1300。
本发明实施例中所述的生物工程菌为农杆菌GV3101。
所述的与上述燕子花ANR基因在调控植物花色中的应用。
进一步地,利用燕子花ANR基因培育植物花色新品种的方法为:
D1)将燕子花ANR基因导入到受体植物中,得到转基因植物;
D2)将燕子花ANR基因的重组植物表达载体导入到受体植物中,得到转基因植物;
D3)敲除或沉默燕子花ANR基因,使其功能丧失或降低。
所述的含有燕子花ANR基因的重组植物表达载体通过使用农杆菌介导或基因枪转化植物细胞或组织,并将转化的植物组织培育成植株。
根据本发明的技术方案,所述受体植物为单子叶植物或双子叶植物,优选地,为燕子花或普通烟草。
本发明提供的技术方案带来的有益效果是:本发明从燕子花中克隆出ANR基因,成功构建了重组植物表达载体,采用农杆菌转化法将含有燕子花ANR基因的重组植物表达载体转化至模式植物烟草中发现,与野生型烟草相比,导入燕子花ANR基因使烟草花冠颜色变浅,花青素的相对含量比野生型烟草降低了2倍以上,这说明燕子花ANR基因具有阻碍花青素合成,降低花青素含量的功能。
本发明提供的燕子花ANR基因可以作为一个优良的基因资源,广泛应用到鸢尾属或其他花卉植物的遗传育种领域,对于花色育种具有重要意义。
附图说明
图1是燕子花ANR基因的克隆琼脂糖凝胶电泳图;其中泳道1为DL2000 marker,泳道2、3为ANR基因克隆PCR结果。
图2是重组植物表达载体鉴定电泳图,其中泳道1为DL15000 marker,泳道2为GV1300-IlANR-GFP重组质粒,泳道3为Sal I和BamHⅠ双酶切验证。
图3是转燕子花ANR基因烟草的筛选图,1、2、3为转基因烟草阳性苗。
图4野生型和转燕子花ANR基因烟草花冠的表型图以及花青素相对含量图,其中WT为野生型,OE1、OE2、OE3为转基因烟草的三个株系。
具体实施方式
通过以下详细说明可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明,而不以任何方式限制本发明揭示的其余内容。下述实施例中的实验方法,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,均可从商业途径得到。
实施例1:燕子花ANR基因克隆
采用OminiPlant RNA Kit(Dnase I)(康为世纪,中国)试剂盒提取燕子花总RNA,总RNA通过PrimeScriptTMRT reagent Kit with gDNA Eraser(Perfect Real Time)(Takara,日本)试剂盒反转录为cDNA。
利用Primer 5设计燕子花ANR基因克隆特异性引物,以上述得到的燕子花cDNA稀释10倍为模板,按照以下反应扩增出ANR基因的ORF区,特异性引物序列如下:
ANR-F1:5’-TTGGAAACTAAGCATGGCTCAGATATTGAC-3’
ANR-R1:5’-GCATAATACGTTTTCTCTCTTCAAGTTGCA-3’
50μL反应体系:包括2μL模板cDNA,25μL 2×PCR buffer for KOD FX,10μL 2mMdNTPs,上下游引物各1μL,1μL KOD FX和10μL ddH2O。
加样操作在冰上进行,加样完成后混匀并离心,按以下程序放置于PCR仪进行扩增反应:
94℃2min;98℃10s,58℃30s,68℃90s,循环35次;68℃10min。
经PCR扩增,得到扩增产物(图1),用1%的琼脂糖凝胶电泳(120V,20min)检测,然后利用凝胶成像***观察条带情况。
在紫外透射切胶台下用刀片切取含有目的条带的琼脂糖凝胶放置于预先准备好的离心管中,胶回收步骤参照TIANGEN试剂盒说明书。
取3μL胶回收产物和1μL 6×Loading Buffer混合,然后用1%的琼脂糖凝胶电泳检测回收情况。如果条带亮度大小合适,则在PCR管中按照下列反应体系配制连接反应液,加样完成后将反应液混匀,置于16℃过夜连接。
10μL反应体系:包括4.5μL胶回收产物,5μL SolutionⅠ,0.5μLPMD 18-T。
将上述连接产物转化大肠杆菌(DH5α),具体操作步骤如下:
将10μL反应液加入到50μL大肠杆菌感受态细胞中,轻柔混合,然后在冰中放置30min;
42℃水浴锅中放置30s,然后迅速转入冰中放2min;
加入600μL LB液体培养基(5g/L Yeast extract+10g/L Tryptone+10g/L NaCl),放置在恒温培养摇床上,37℃,200rpm,振荡培养1h;
将转化液离心,去掉上层少许LB培养液,用枪头将剩余转化液混匀,然后取100μL转化液涂在含有Amp的LB固体培养基(5g/L Yeast extract+10g/L Tryptone+10g/L NaCl+15g/L Agar)上,用封口膜封好,放于37℃恒温培养箱中过夜培养(倒置)。
次日,在LB固体平板上挑选阳性克隆10-20个单克隆菌株于30μL LB液体培养基的离心管中,37℃,200rpm,振荡培养1h;然后以振荡培养的菌液为模板,T载体的通用引物M13F和M13R为检测引物,按照下列反应体系配制PCR反应液,通过PCR反应来验证***目的基因大小。
20μL反应体系:包括2μL菌液模板,6μL ddH2O,上下游M13引物各1μL,10μL Es-TaqMaster Mix。
将PCR管放置在冰上,在冰上加样,加样完成后混匀并离心,然后放置于PCR仪中进行PCR扩增,反应程序如下:
95℃10min;94℃30s,55℃45s,72℃90s,循环30次;72℃10min。
PCR反应结束后,将反应液点在1%的琼脂糖凝胶上,电泳(120V,15min)检测,然后在凝胶成像仪上观察***的目的条带是否正确,如果正确的话,在15mL摇菌管中加入10mLLB液体培养基,10μL Amp以及剩余的所有菌液,将摇菌管置于37℃摇床上,200rpm振荡过夜。次日,于超净工作台中取1mL菌液送至生物公司测序,将剩余菌液各取200μL保存于同等体积(200μL)的60%的甘油中,放在-80℃冰箱保存。
根据生物公司的测序结果,将序列正确的菌液用于提取质粒,具体操作步骤参照质粒提取试剂盒(TIANGEN,中国)说明书。
获得燕子花ANR基因ORF区序列如下:
ATGGCTCAGATATTGACCAAAACTGCGTGTGTCACCGGAGGAAACGGGTTCCTCGCGACCATCTTGATCAAGCAACTGTTGGAGAAGGGCTACGCCGTCAATGCCACCGTTCGCGACCCCGAGAACAAGGCGAGGGTCGGTCACCTCTTGGACCTGCAAAGCCTGGGCGATCTCAAGCTCTTCCGAGCGGAACTGACCGAAGAAGGAAGCTTCGACGAGGCGATAAGCGGCTGCGAATACGTCTTCCATCTCGCCACCCCGGTGAACATCTTCTCCCAAGACCCAGAGAATGAGTTGATCAAACCTGCAATCGAAGGAACCCTGAATGTCATGAAGTCGTGCCTCAAGGCGAAGGTCAAGCGGGTCGTCCTGACGTCGTCGGCAGCCGCCGTGTCGGTGAACAAGCTCAGAGGGACGGACCTCATGATGGACGAGGAGTGCTGGTCGGACGTCGAGTTCCTGACCGCCGAAAAGCCCCCGACATGGGGCTACCTGGTCTCGAAGACGCTCGCGGAGAAGGAAGCGTGGAAATTCGCACGAGAGAACGGCATCGACCTCGTGACCATAGTACCGGTCTTGATGGTCGGCCCTCCGTTGAGCGGCGACGTCCCATCGAGTGTAGGCATGGCTTTGTGCTTGCTCCTTGGAGATGATCCCCGCATTGGCGGTCTGAAGATCATGCAATCGCTCTCGGGCTCGGTCTCGCTCGTGCACGTGGAGGACGCGAGCAGGGCCCAGATATTCGTCGCGGAGACCGAGTCGGCCTCCGGTCGATACATCTGCTGCGCCGTCAACACGAGCCTACCGGAGCTCGCGGAGTTCCTCTCGAAGAGATACCCAGAGTACAAAGTCCCTACTAATATCACCGACGTACCGAAGAAAGCTAAACTGAAACTCTCCTCCGAGAAGCTCATCAAGGAAGGGTTCAACTTCGAGAAGAAAGAACTTGGAGAGATCTACGACGGAGCCATCAGCTATGCTAAGAAAGCAGGATTTTTTCCCAAAAATGGTGCAACTTGA
燕子花ANR基因ORF区包含1020个碱基,编码339个氨基酸:
MAQILTKTACVTGGNGFLATILIKQLLEKGYAVNATVRDPENKARVGHLLDLQSLGDLKLFRAELTEEGSFDEAISGCEYVFHLATPVNIFSQDPENELIKPAIEGTLNVMKSCLKAKVKRVVLTSSAAAVSVNKLRGTDLMMDEECWSDVEFLTAEKPPTWGYLVSKTLAEKEAWKFARENGIDLVTIVPVLMVGPPLSGDVPSSVGMALCLLLGDDPRIGGLKIMQSLSGSVSLVHVEDASRAQIFVAETESASGRYICCAVNTSLPELAEFLSKRYPEYKVPTNITDVPKKAKLKLSSEKLIKEGFNFEKKELGEIYDGAISYAKKAGFFPKNGAT
将燕子花ANR基因序列登录至GenBank,登录号为OQ433948。
实施例2:燕子花ANR基因的植物过表达载体的构建
利用同源重组的方法,将去掉终止密码子的燕子花ANR基因ORF区构建到植物表达载体GV1300的Sal I和BamH I酶切位点之间,得到重组植物表达载体GV1300-IlANR-GFP(图2,泳道2)。载体构建采用ClonExpress II One Step Cloning Kit(诺唯赞,中国)同源重组试剂盒,按照说明书进行,载体同源臂引物(下划线部分为Sal I和BamH I酶切位点)如下:
ANR-F2:5’-TTGATACATATGCCCGTCGACTGGAAACTAAGCATGGCTCAGATATTGAC-3’
ANR-R2:5’-CCCTTGCTCACCATGGATCCGCATAATACGTTTTCTCTCTAGTTGCA-3’
为进一步验证重组质粒的连接情况,用限制性内切酶Sal I和BamHⅠ对重组植物表达载体GV1300-IlANR-GFP进行双酶切验证,验证结果显示两条带且条带位置正确(图2,泳道3),表明燕子花ANR基因与GV1300-GFP载体连接,植物表达载体构建成功。
双酶切鉴定20μL反应体系:包括6μL重组质粒GV1300-IlANR-GFP,2μL buffer,10μL ddH2O,限制性内切酶Sal I和BamHⅠ各1μL。
实施例3:燕子花ANR基因遗传转化烟草
通过冻转法将重组质粒GV1300-IlANR-GFP转入到农杆菌GV3101感受态细胞中,转化步骤参照说明书。
采用叶盘法将转化重组质粒GV1300-IlANR-GFP植物过表达载体的农杆菌遗传转化烟草,具体方法如下:
(1)从-80℃冰箱中取出GV1300-IlANR-GFP农杆菌液在YEP固体培养基(10g/LYeast extract+10g/L Tryptone+5g/L NaCl+15g/L Agar)上划线培养,放置28℃培养箱中倒置培养36h,挑取单克隆菌株进行PCR鉴定,再将鉴定正确的农杆菌在50mLYEP液体培养基(10g/L Yeast extract+10g/L Tryptone+5g/L NaCl)中扩大培养。
(2)将农杆菌摇至OD600达到0.7后用于侵染烟草叶盘。
(3)挑选长势良好的烟草无菌苗,将幼嫩的叶片切成1cm2的方形,然后放在预培养基(MS+20g/L蔗糖+7.8g/L Agar+1mg/L 6-BA+0.05mg/L NAA)上暗培养2d。
(4)4000rpm离心5min收集菌体,弃上清,在超净工作台中用重悬液(1/2MS+20g/L蔗糖)重悬菌体,然后把预培养的烟草叶片浸泡在重悬液中轻轻地晃动侵染5min,将叶片放至滤纸上晾干后再重新放回预培养培养基上(叶片上表面朝上),于28℃黑暗倒置共培养2d,同时设置未经农杆菌侵染的叶盘作为阴性对照。
(5)把共培养过的叶片放到筛选培养基(1/2MS+20g/L蔗糖+1mg/L 6BA+0.05mg/LNAA+20mg/L Hyg+500mg/L Cef)上光照培养,每15d换一次培养基,此时培养基可适当降低Cef的浓度,目的是促进转基因烟草叶片长出抗性芽又有抑制农杆菌的作用。非转基因烟草同时也做抗性培养,正常情况下,非转基因烟草叶片会逐渐死亡。
(6)叶片分化出不定芽时,将不定芽切下转移生根培养基(1/2MS+20g/L蔗糖+0.1mg/L NAA+25mg/L Hyg+200mg/L Cef)中进行生根培养,大约10d左右可以长出不定根。
(7)将生根的烟草幼苗移到营养土中继续培养,并取部分叶片提取DNA,采用ANR同源臂引物进行PCR验证,将阳性烟草幼苗继续培养直到收获种子,继续筛选下一代阳性植株(图3)。
实施例4:对获得的T3代转基因烟草进行花青素含量鉴定,具体过程如下:
将收获的T1烟草种子烘干后,同时消毒并播种在含25mg/L的Hyg的1/2MS筛选培养基上,消毒方法为:用75%酒精消毒1min,然后用无菌水清洗3遍,再用2%的次氯酸钠溶液振荡消毒10min,最后用无菌水清洗5遍;放在4℃春化2d后,然后在组培室正常培养,当烟草长出四片叶片稍大时,移到营养土(草炭土:蛭石=3:1)中培养,光照环境为光照/黑暗14h/10h,每周浇一次水,收获T2代种子,用相同的种子消毒和筛选方法获得T3代转基因烟草。
取0.1g盛开的T3代转基因烟草花冠,加液氮研磨,然后加入10mL 0.1mol/L盐酸乙醇(8.3mL浓盐酸用95%乙醇稀释成1L)于60℃水浴条件下浸提30min。12000rpm离心后取上清检测提取液在530nm、620nm和650nm波长下的光密度值。其中,野生型烟草植株作为对照,设置3次生物学重复。花青素含量计算方法如下:
计算花青素的光密度值:ODλ=(OD530-OD620)-0.1(OD650-OD620)
花青素含量(nmol/g)=ODλ/ε×v/m×1000000
ODλ:花青素在530nm波长下的光密度
ε:花青素摩尔消光系数4.62×106
v:提取液体积(mL)
m:取样质量(g)
1000000:计算结果换算成nmol的倍数
实验结果表明,野生型烟草花冠中的花青素含量为114.52nmol/g,而3个转ANR基因烟草株系花冠中的花青素含量分别为63.39nmol/g、49.60nmol/g、53.38nmol/g(图4)。以上数据充分说明转ANR基因烟草花冠中的花青素含量有了显著降低,进而导致烟草花色变浅。
Claims (10)
1.一种燕子花ANR基因,其特征在于,为如下A1)或A2)所示的基因:
A1)核苷酸序列是序列表中SEQ ID No.1的cDNA分子或基因组DNA;
A2)与A1)限定的核苷酸序列具有90%或90%以上相似性,且编码权利要求2所述的燕子花ANR蛋白的cDNA分子或基因组DNA。
2.权利要求1所述的燕子花ANR基因所编码的蛋白,其特征在于,为如下B1)、B2)、B3)或B4)所示的蛋白质:
B1)由序列表中SEQ ID No.2所示的氨基酸序列组成的蛋白质;
B2)其氨基酸序列与SEQ ID No.2所示的氨基酸序列具有90%以上同源性且具有相同表达功能的蛋白质;
B3)在序列表中SEQ ID No.2所示的氨基酸序列中经过取代和/或缺失和/或添加一个或几个氨基酸残基得到的具有相同功能的由B1)衍生的蛋白质;
B4)在B1)、B2)或B3)的N端或/和C端连接标签得到的融合蛋白质。
3.一种与权利要求1所述的燕子花ANR基因相关的生物材料,其特征在于,为下述C1)至C5)中的任一种:
C1)含有燕子花ANR基因的重组克隆载体;
C2)含有燕子花ANR基因的重组植物表达载体;
C3)含有燕子花ANR基因的N端或/和C端连接标签得到的重组植物表达载体;
C4)含有C2)或C3)所述的重组载体的生物工程菌;
C5)含有C2)或C3)所述的重组植物表达载体的转基因植物。
4.根据权利要求3所述的与燕子花ANR基因相关的生物材料,其特征在于:所述的克隆载体为PMD18-Tvector。
5.根据权利要求3所述的与燕子花ANR基因相关的生物材料,其特征在于:所述植物表达载体为GV1300。
6.根据权利要求3所述的与燕子花ANR基因相关的生物材料,其特征在于:所述生物工程菌为农杆菌GV3101。
7.权利要求3~6任一项所述的与燕子花ANR基因相关的生物材料在改变植物花色中的应用。
8.根据权利要求7所述的调控植物花色的方法,其特征在于:
D1)将权利要求1所述的燕子花ANR基因导入到受体植物中,得到转基因植物;
D2)将权利要求3所述的燕子花ANR基因的重组植物表达载体导入到受体植物中,得到转基因植物;
D3)敲除或沉默权利要求1所述的燕子花ANR基因,使其功能丧失或降低。
9.根据权利要求8所述的将重组植物表达载体导入受体植物的方法,其特征在于:所述的含有ANR基因的重组植物表达载体通过使用农杆菌介导或基因枪转化植物细胞或组织,并将转化的植物组织培育成植株。
10.根据权利要求3所述的生物材料或权利要求7所述的用途或权利要求8所述的方法,其特征在于,所述植物为双子叶植物或单子叶植物,优选地,为燕子花或普通烟草。
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Cited By (2)
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CN117051014A (zh) * | 2023-09-22 | 2023-11-14 | 东北林业大学 | 一种燕子花抗寒基因myb97的克隆及应用 |
CN117051014B (zh) * | 2023-09-22 | 2024-05-31 | 东北林业大学 | 一种燕子花抗寒基因myb97的克隆及应用 |
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