CN116656635A - 甘薯9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1及其在调控株高中的应用 - Google Patents
甘薯9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1及其在调控株高中的应用 Download PDFInfo
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Abstract
本发明涉及分子生物学以及植物基因工程技术研究领域,具体涉及一种甘薯9‑顺式环氧类胡萝卜素双加氧酶基因IbNCED1及其在株高中的应用。该IbNCED1基因的核苷酸序列如SEQ ID NO.1所示。本发明通过将甘薯9‑顺式环氧类胡萝卜素双加氧酶基因IbNCED1转化到甘薯品种徐薯22中,获得超表达甘薯植株,结果发现,与野生型相比,超表达IbNCED1基因的甘薯ABA含量显著提高,赤霉素(GA)显著减低,植株显著降低。可见,甘薯IbNCED1参与ABA和GA的合成,进而调控甘薯株高,这将为为甘薯品质改良和新品种的选育提供参考。
Description
技术领域
本发明涉及分子生物学以及植物基因工程技术研究领域,具体涉及一种甘薯9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1及其在调控株高中的应用。
背景技术
甘薯(Ipomoeabatatas(L.) Lam)是我国重要的粮食、饲料、工业原料和新型能源作物,一年生或多年生双子叶,广泛种植于世界上100多个国家或地区,在保持粮食安全和能源安全方面起着至关重要的作用。
甘薯茎匍匐或半直立生长,在生产上,株高过长会造成茎相互缠绕,不利于机械收获,也会造成产量降低。控制株高的因素有很多,其中,赤霉素(GA)是一类双萜类化合物的生长调节剂, 可以促进细胞伸长,促进茎叶伸长,从而调控植物的植株。所有的GA成分中,GA3活性最高。
脱落酸(abscisic acid,ABA)是具有倍半萜结构的植物内源激素,广泛地参与到植物体内一系列重要的生理生化过程。在植物生长发育过程中,ABA和GA之间密切相关。9-顺式环氧类胡萝卜素双加氧酶是ABA合成途径中的关键限速酶,在植物生长发育过程中起着关键作用。
目前,甘薯株型改良及新品种的选育过程中,育种过程繁琐且时间长,耗费大量的人力物力等。
发明内容
针对现有技术中存在的研究空白,本发明提供了一种甘薯9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1。
本发明还提供了一种甘薯9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1在调控株高中的应用。
本发明为了实现上述目的所采用的技术方案为:
本发明提供了一种甘薯9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1,其核苷酸序列如SEQ ID NO.1所示。
本发明还提供了一种含有上述甘薯9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1的重组载体pCAMBIA1301-IbNCED1。
本发明进一步提供了一种含有上述甘薯9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1的宿主细胞。
本发明还提供了上述甘薯9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1在调控植物株高中的应用。
优选的,调控植物株高所采用的具体方法为:将包含所述甘薯IbNCED1基因连接到载体上,通过农杆菌介导转化到甘薯中,获得超表达甘薯IbNCED1转基因植株。
本发明还提供了一种上述植物超表达重组载体pCAMBIA1301-IbNCED1在调控植物株高中的应用。
本发明利用基因工程技术,从短蔓甘薯品种“济薯26”中克隆获得9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1全长,构建了克隆载体和植物表达载体,并成功转化长蔓甘薯品种“徐薯22”,获得超表达甘薯植株。研究发现,也对照植株相比,转基因甘薯植株的株高ABA含量显著提高、GA3含量显著降低,株高显著降低。这将为进一步研究IbNCED1基因在甘薯生长发育过程中的调控机理奠定了基础,也为利用分子手段进行甘薯株型改良及新品种的选育提供理论依据,同时极具应用前景。
本发明以短蔓甘薯“济薯26”的cDNA为模板克隆并分离得到9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1,其ORF序列全长为1764 bp,编码587个氨基酸。
本发明进一步构建了植物超表达重组载体PCAMBIA1301-IbNCED1,转化并获得了超表达IbNCED1甘薯植株,对转基因甘薯的植株进行观察,结果发现,相比于野生型,转基因甘薯株系的株高显著降低。进一步对激素进行了测定,结果表明,与野生型相比,转基因甘薯株系ABA含量显著提高,GA3含量显著降低。这为进一步研究IbNCED1在甘薯株高调控中的功能奠定基础,也为利用分子手段加快甘薯短蔓新品种的选育提供参考。
本发明的有益效果为:本发明通过将甘薯9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1转化到甘薯品种“徐薯22”中,经鉴定,共获得8个超表达甘薯IbNCED1基因阳性植株。转基因株系ABA含量显著提高,GA含量显著降低,株高显著降低。表明,甘薯9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1参与调控ABA和GA3激素合成途径,进而调控甘薯的株高,这将为为甘薯短蔓改良和新品种的选育提供技术参考。
附图说明
图1IbNCED1基因在甘薯不同组织中的表达情况;
图2转基因甘薯株系DNA检测(A)和荧光定量PCR检测IbNCED1基因的表达情况(B);其中,M为DNA分子Marker,W为阴性对照水,P为阳性对照(pCAMBIA1301-IbNCED1),WT为野生型甘薯植株的基因组DNA,L1-L7为转IbNCED1基因甘薯阳性植株;
图3 转基因甘薯植株的表型及株高;其中;(A1-A2)为生长4 w的组培苗的表型及株高;(B1-B2)为生长4 w的盆栽苗的表型及株高。
具体实施方式
下面结合具体实施例对本发明做进一步的说明。
实施例1 IbNCED1基因在甘薯不同组织中的表达水平检测
本实施例所采用的材料是“济薯26”收获期各组织的植物材料,采后速冻于液氮中,超低温冰箱(-80℃)保存。
1)甘薯各组织Total RNA的提取
按照TaKaRa植物总RNA提取试剂盒的说明书进行,具体操作为:将超低温冻存的“济薯26”甘薯各组织
迅速转移至用液氮预冷的研钵中,用研杵研磨组织,其间不断加入液氮,直至分别研磨成粉末状;将研磨成粉状的样品分别加入到含有450 μl Buffer PE 的1.5 mL灭菌tube中,用移液器反复吹打直至裂解液中无明显沉淀;将裂解液12,000 rpm,4℃离心5min;将上清液小心吸取到新的1.5 mL灭菌tube 中。加入上清液1/10体积的Buffer NB,Vortex 振荡混匀,12,000 rpm,4℃离心5 min;将上清液小心吸取到新的1.5 mL灭菌tube中,加入450μL的Buffer RL,使用移液枪将溶液混合均匀;加入混合液1.5倍体积的无水乙醇,使用移液枪将溶液混合均匀后,立即将混合液全部转入到RNA Spin Column中;12,000rpm,离心1 min,弃滤液,将RNA Spin Column 放回到2 ml Collection Tube 中;将600μL的80%乙醇加入至RNA Spin Column 中,12,000 rpm 离心30 s,弃滤液;向RNA SpinColumn膜中央加入50μLDNase I 反应液,室温静置15 min;向RNA Spin Column膜中央加入350μL的Buffer RWB,12,000 rpm离心30 s,弃滤液;将600μL的80%乙醇加入至RNA SpinColumn 中,12,000 rpm 离心30 s,弃滤液;将RNA Spin Column 重新安置于2mLCollection Tube 上,12,000 rpm 离心2 min;将RNA Spin Column 安置于1.5 mL的RNaseFree Collection Tube上,在RNA Spin Column 膜中央处加入30μL的RNase Free dH2O室温静置5 min,12,000 rpm 离心2 min洗脱RNA。所得RNA经浓度和纯度检测后存于-80℃冰箱保存备用。
吸取2μL RNA利用1%琼脂糖凝胶电泳检测,结果显示28S和18S条带较为清晰,28S条带亮度约为18S的两倍,RNA质量较好。通过微量核算蛋白测定仪检测RNA纯度,OD260/OD280和OD260/OD230均在1.8~2.1之间,完整性较好,可用于反转录。
2)反转录cDNA第一链的合成
RNA进行反录前进行电泳检测是否降解,并用RNA/DNA calculator 测算RNA 浓度,根据RNA反转录试剂盒对RNA的要求进行。反转录cDNA第一链采用TaKaRa反转率试剂盒PrimeScriptTMRT reagent Kit(Perfect Real Time),具体操作参照试剂盒说明书进行。
3)荧光定量分析
根据甘薯IbNCED1测序结果,运用NCBI中BLAST设计甘薯IbNCED1基因的荧光定量引物,以ACTIN作为内参基因,将反转录cDNA稀释10倍,取1μL作模板,所用荧光定量引物如下:
IbNCED1-F:5’- ATTCCCACTTCAATATCCACTGCC -3’
IbNCED1-R:5’- TTGCCGCCGCTCTTTGC -3’
ACTIN-F:5’- AGCAGCATGAAGATTAAGGTTGTAGCAC -3’
ACTIN -R:5’- TGGAAAATTAGAAGCACTTCCTGTGAAC -3’
利用SYBR Green Pro TaqHSqPCR Kit试剂盒(艾克瑞生物公司)的说明书进行反应溶液的配制,在Roche Lightcycler®480荧光定量仪上运行PCR程序:95℃ 2 min;95℃15 s,55℃ 15 s,72℃ 15 s,循环40次;37℃ 1 s。待反应结束,获得扩增曲线,导出数据,利用Excel进行数据分析,根据CT值用2-ΔΔCq相对定量法计算相对表达量,数据分析结果如图1所示。
本实施例1根据对荧光定量结果的分析,测定了甘薯IbNCED1基因在收获期的各组织的表达水平。从图1可以看出甘薯IbNCED1基因在老组织中的表达量比在幼嫩组织中的表达量较高,在块根中的表达量也较高。
实施例2 基因的克隆与重组质粒的构建
本实施例所用的植物材料为“济薯26”和“徐薯22”,由本实验保存。试验中所用植物表达载体为pCAMBIA1301,由本实验保存; 所用的大肠杆菌菌株为Trans5α,农杆菌菌株为EHA105,购自北京擎科生物科技有限公司,用于载体构建和转化甘薯。
1)目的基因引物的设计及克隆
根据sweetpotato garden中公布的甘薯IbNCED1基因的CDS序列(见SEQ NO.1),利用Prime5.0设计扩增引物,并在两端加入酶切位点(Kpn I、Sal I),其引物序列为:
IbNCED1-Kpn I -F:5'- GGGGTACCATGGCCAACACCATT-3'(下划线部分为KpnI酶切位点),
IbNCED1- Sal I -R:5'- CGGTCGACAGCTTGGGTGGATAG-3'(下划线部分为EcoRI酶切位点)。
以cDNA为模板,利用LA Taq高保真酶进行甘薯IbNCED1基因的克隆。PCR扩增体系(50μL)为:0.5 μlLA Taq,10 μlMg2+Plus mix,8 μLdNTP Mixture,1 μL Forward Primer,1μL Reverse Primer,2 μL Template DNA,25.5 μL ddH2O。PCR程序为:反应条件为94℃预变性3min,95℃变性30s,55℃退火30 s,72℃延伸2 min,34个循环,72℃总延伸10 min,4℃保温。
PCR反应完成后,进行琼脂糖凝胶电泳检测并切割目的片段,凝胶回收纯化PCR目的扩增产物。采用艾克瑞生物公司的DNA凝胶回收试剂盒,进行目的片段纯化回收,具体操作为:将单一目的条带从琼脂糖凝胶中切下,放入干净的离心管中,称取重量;向胶块中加入3倍体积溶液GSB(如果凝胶为0.1g,其体积可视为100μL,则加入300μLGSB溶液),55℃水浴放置,其间不断温和上下翻转离心管,直至胶块完全溶解;将融化的凝胶溶液降至室温,加入1倍体积异丙醇(如果凝胶为0.1g,则加入100μL异丙醇),轻柔混匀;将混合液加入离心柱中,室温放置1 min,12000rpm离心1min,弃流出液,后将离心柱放回收集管中;向离心柱内加入650μL溶液WB,12000rpm离心1min,弃流出液;12000rpm离心2min,尽量除尽残留的WB,将吸附柱置于室温开盖放置5min,彻底晾干;将离心柱放到一个干净离心管中,向吸附膜中间位置悬空滴加30μL ddH2O(ddH2O需提前置于60~70℃水浴预热),室温静置2min,12000rpm离心2min收集DNA溶液。取2μL回收纯化后的产物,使用1.5%琼脂糖进行凝胶电泳检测,其余放置在-20℃冰箱,后续将用于与pCAMBIA1301载体连接,构建过表达载体。
3)质粒的提取:
按照天根质粒小提中量试剂盒说明书提取质粒,具体步骤如下:
取过夜培养的10mL菌液,12000 rpm离心1min,去掉上清液;取500μL P1溶液(含RNase A)加至留有菌体沉淀的离心管中,使用涡旋仪彻底悬浮菌体沉淀;取500μL P2溶液加至离心管中,温和上下翻转时菌体裂解充分,取700μL P3溶液加至离心管中,立即温和上下翻转,充分混匀,当出现白色絮状沉淀后,12000rpm离心10min;取500μL平衡液BL加至吸附柱CP4中,12000rpm离心1min,弃掉收集管中的废液,将吸附柱放回收集管,将收集的上清液分批加入过滤柱CS中,12000rpm离心2min,小心将收集管中收集的溶液分批加入吸附柱CP4中,12000rpm离心1min,弃掉收集管中的废液,将吸附柱CP4放回收集管;取500μL去蛋白液PD加至吸附柱CP4中,12000rpm离心1min,弃掉收集管中废液,将吸附柱CP4重新放回收集管;取600μl漂洗液PW(含无水乙醇)加至吸附柱CP4中,12000rpm离心1min,弃掉收集管中的废液,将吸附柱CP4放回收集管,12000rpm离心2min,去除吸附柱中残余的漂洗液;将吸附柱CP4移至新的1.5ml离心管中,向吸附膜中间加入60μL ddH2O;室温静置2min,12000rpm离心1min,离心管中收集的溶液即为质粒。最后测定质粒浓度,为下一步实验做准备。
4)双酶切反应
将提取的pCAMBIA1301质粒用Kpn I和Sal I在37℃条件下酶切30 min,电泳回收线性载体,-20℃保存备用。双酶切反应体系为50μL:pCAMBIA1301质粒20μL,5×buffer 5μL,Kpn I 1μL,Sal I 1μL,ddH2O 23μL。
5)重组反应
琼脂糖凝胶电泳检测酶切后所回收到的目的基因和载体pCAMBIA1301,根据所检测出的纯度和浓度,按连接体系加入各试剂。连接反应体系为:线性化pCAMBIA1301载体7μL,插入片段3μL,T4 buffer 4μl,T4 2μl,ddH2O Up to 20μL。在37℃下反应30min,常温放置(勿立即置于冷却),10 min后转化至大肠杆菌感受态Trans5α。
6)连接产物转入大肠杆菌
从超低温冰箱中取出感受态细胞Trans5α菌株,置于冰上融化。吸取10 μL重组产物加入到100μL感受态细胞中;将离心管置于冰上冰浴10 min;42℃水浴锅中水浴,热激50s,期间不要摇动;后立即置于冰上冰浴2 min;在超净台中加入500μL无抗生素的液体培养基,37℃、200 rpm培养60 min复苏;6000 rpm 离心1 min,吸去350μL上清;将沉淀的菌体重悬,涂布于LB平板(Kana的浓度为50 mg/L),37℃培养过夜。
7)重组子的鉴定
挑取平板上的单菌落接种到含有抗生素(Kana)的LB液体培养基中,37℃、200 rpm震荡培养过夜。使用目的基因全长引物进行菌液PCR,以筛选阳性克隆。筛选后的阳性克隆送至青岛擎科生物有限公司测序。测序结果正确的阳性克隆,扩大培养后,使用天根质粒提取试剂盒提取质粒以备转化农杆菌感受态。
本实施例2以“济薯26”的cDNA为模板克隆并分离得到9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1,其ORF序列全长为1764 bp,编码587个氨基酸,并成功构建重组载体pCAMBIA1301-IbNCED1,用于甘薯的遗传转化。
实施例3 甘薯的遗传转化与阳性株系的鉴定
1)根癌农杆菌EHA105感受态的制备与转化
感受态制备操作步骤:挑取单菌落接种于10 mL含有抗生素利福平的YEP液体培养基中,28℃,180~250 rpm的摇床上震荡培养过夜。取2 mL菌液转入50 mL含有抗生素利福平的YEP液体培养基中,继续培养至OD值为0.3~0.4。将菌液转入无菌离心管中,冰浴30 min。5000 rpm,离心10 min,去上清。加入2 mL预冷的含有15%甘油的0.1 mol·L-1的CaCl2溶液,轻轻悬浮。将农杆菌悬浮液分装到1.5 mL无菌离心管管中,每管200 μL,用液氮迅速冷冻并保存于-80℃冰箱备用。
感受态转化具体步骤:取10 μL质粒DNA,加入到200 μL冰上解冻的农杆菌感受态中,冰浴5 min,液氮中迅速冷冻5 min,37℃水浴5 min。加入800 μL YEP液体培养基,28℃,100 rpm,摇2~4 h。5000 rpm离心机离心,倒去大部分上清,留50 μL左右,重悬菌体。将菌液涂布于含有含有抗生素利福平及卡那霉素的YEP固体培养基上,28℃,倒置培养48~72 h,至平板上长出单菌落。挑取单菌落,提取质粒,进行目的基因的PCR鉴定。鉴定后的阳性克隆送至青岛擎科生物有限公司测序,选取测序结果正确的阳性菌落进行摇菌,加入适量无菌50%甘油,于-80℃保存备用。
2)甘薯品种徐薯22胚性愈伤组织的诱导及胚性细胞悬浮系的建立:
收获的徐薯22薯块用于提供甘薯茎尖,剥取茎尖分生组织接种于2.0 mg/L 2,4-D的MS固体培养基上,室温27±1℃,暗培养诱导愈伤组织,然后进行扩繁和继代,建立胚性细胞悬浮系,用于转化。
3)农杆菌的培养:在抗性平板上活化农杆菌菌液,挑取单菌落接种于5 mL的已加入对应抗生素的YEP液体培养基中,28℃下200 rpm振荡培养,直到OD600值在0.8~1.0范围内。
4)悬浮细胞系的准备和根癌农杆菌的侵染:选择生长8~12w状态良好的悬浮细胞系进行研磨,继代培养3d后,取其中直径约0.7~1.4 mm的胚性悬浮细胞团用于农杆菌的侵染转化。
5)共培养和延迟培养:农杆菌侵染后的悬浮细胞系转移至含有30 mg/L乙酰丁香酮(AS)和2 mg/L 2,4-D 的MS固体培养基上进行共培养,黑暗环境,温度为27±1℃。共培养3d后,细胞团用含200 mg/L 头孢霉素(CS)和2 mg/L 2,4-D的MS液体培养基洗1次,用含100mg/L CS和2 mg/L 2,4-D的MS液体培养基静置浸泡30 min,最后用含2 mg/L 2,4-D的MS液体培养基延迟培养1w。培养条件为27±1℃,500 Lux光照(每天13h光照),100 rpm 摇晃培养。
6)抗性细胞团的筛选:延迟培养后,将细胞团转移到含5.0 mg/L 潮霉素(Hyg)、100 mg/L CS和2 mg/L 2,4-D的MS固体培养基上进行暗培养,温度为27±1℃,每2w更换1次新培养基。4w后将抗性细胞团转移到10.0 mg/L Hyg、100 mg/L CS和2 mg/L 2,4-D的MS固体培养基上,共培养4~8w。
7)体细胞胚的诱导:将生长状态良好的抗性细胞团转移至含有1.0 mg/L ABA和100 mg/L CS的MS培养基上,诱导体细胞胚生长。培养条件为27±1℃,3000 Lux光照(每天13h光照)。
8)拟转基因植株的再生和鉴定:将诱导2~4w后在ABA 培养基上变绿的成熟体细胞胚连同愈伤组织一起转移到MS固体培养基上,培养至完整植株形成,温度为27±1℃,每天13h光照,光照强度为3000 Lux,获得拟转基因植株。
转IbNCED1基因植株的鉴定使用PCR检测和qRT-PCR检测相结合的方法。
PCR检测方法如下:提取甘薯品种徐薯22和拟转基因株系的DNA,进行PCR鉴定。使用pCAMBIA1301-IbNCED1载体质粒为阳性对照,水和野生型甘薯品种徐薯22为阴性对照,引物如下:
pCAMBIA1301-F:5'- GACGCACAATCCCACTATCC -3'
IbNCED1-R:5'- AGCTTGGGTGGATAG-3'
将扩增得到的PCR产物在1%(w/v)的琼脂糖凝胶中进行电泳分离,PCR阳性植株具有一条特异的电泳条带,记录下PCR阳性植株的株系号。
qRT-PCR检测方法如下:提取甘薯转基因阳性植株的RNA,反转录得到cDNA,进行qRT-PCR,以徐薯22为对照WT。
本实施例3通过农杆菌介导的遗传转化方法,将已连有甘薯IbNCED1基因的pCAMBIA1301-IbNCED1过表达重组载体转入甘薯中,结果如图2所示,结果显示只有阳性对照和拟转基因株系L1-L7在1800 bp附近出现了电泳条带,野生型甘薯和阴性对照没有出现条带,初步确定了本发明获得了甘薯转基因阳性植株L1-L7(图2A)。qRT-PCR检测结果表明,阳性转基因甘薯株系中IbNCED1基因的表达量显著提高(图2B)。
实施例4 甘薯IbNCED1调控株高的功能鉴定
对转基因植株和对照进行表型观察,结果如图3所示,相比于野生型,转基因甘薯株系的株高显著降低(图3)。进一步对植株的激素含量进行测定,结果表明,与野生型甘薯WT相比,转基因甘薯株系的ABA含量显著提高,GA3含量显著降低(表1)。
表1转IbNCED1基因甘薯植株和野生型甘薯植株激素含量测定
Claims (6)
1.一种甘薯9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1,其特征在于,其核苷酸序列如SEQ ID NO.1所示。
2.一种含有如权利要求1所述的甘薯9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1的植物超表达重组载体pCAMBIA1301-IbNCED1。
3.一种含有如权利要求1所述的甘薯9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1的宿主细胞。
4.一种如权利要求1所述的甘薯9-顺式环氧类胡萝卜素双加氧酶编码基因IbNCED1在调控植物株高中的应用。
5.根据权利要求4所述的应用,其特征在于,具体方法为:将包含所述甘薯IbNCED1基因连接到载体上,通过农杆菌介导转化到甘薯中,获得超表达甘薯IbNCED1转基因植株。
6.一种如权利要求2所述的植物超表达重组载体pCAMBIA1301-IbNCED1在调控植物株高中的应用。
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