CN109536513B - 白菜雌蕊发育相关基因BrCRF11a及其应用 - Google Patents
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Abstract
本发明提供了白菜雌蕊发育相关基因BrCRF11a及其应用,属于植物基因工程技术领域。所述白菜BrCRF11a的DNA序列如SEQ ID No.1所示,该基因人工miRNA序列如SEQ ID No.2所示。通过农杆菌介导法将该基因及针对该基因设计的人工miRNA分别转入‘油青四九’菜心中,得到BrCRF11a过表达和抑制表达的转基因菜心株系,结果发现白菜BrCRF11a的表达变化会导致菜心出现嵌套花的表型,该表型是由于花分生组织持续保持干细胞活性所致。这表明白菜BrCRF11a与雌蕊发育关系密切,将该基因应用于白菜类及其他园艺植物育种,具有良好的应用前景。
Description
技术领域
本发明属于植物基因工程技术领域,具体的说,涉及白菜BrCRF11a、其编码蛋白、其人工miRNA及其在花发育过程中的应用。
背景技术
白菜(Brassica rapa L.syn.B.campestris L.)属十字花科芸薹属芸薹种作物,主要包含有结球白菜、不结球白菜、芜菁、菜薹、紫菜薹、薹菜、乌塌菜、日本水菜等8个变种。它们不仅是重要的蔬菜和油料作物,在生产上有较高的经济价值,而且与十字花科模式植物拟南芥有较近的亲缘关系,在植物基础科学中也有重要研究意义。花的正常发育是被子植物的有性繁殖的基础,与植物的种子品质和数量息息相关。对于花发育相关机理的研究,在植物育种工作中具有重要的应用价值。
细胞***素响应因子(cytokinin response factor,CRF)是植物转录因子AP2/ERF大家族中的一个分支,因其对细胞***素的响应而得名。前人研究发现细胞***素在植物生长和发育的许多进程中均发挥重要作用,而CRF是植物体内细胞***素信号转导路径中的重要组分。目前,在许多细胞***素对植物的调控中均发现了CRF的参与,而CRF在花发育中的作用却鲜有报道。因此,研究CRF基因在植物花发育过程中的作用,对在基础科学研究中具有重要的理论意义,也在育种工作中有广泛的应用价值。
发明内容
本发明的目的是提供白菜BrCRF11a及其应用。
本发明提供了一种细胞***素响应基因BrCRF11a,该基因为:从来源于品种为‘Chiifu-401-42’的白菜的基因CRF11a克隆到的基因,其具有:
1)SEQ ID No.1所示的核苷酸序列;或
2)SEQ ID No.1所示的核苷酸序列经取代、缺失和/或增加一个或几个核苷酸;或
3)在严格条件下与1)限定的DNA序列杂交的核苷酸序列。
本发明提供了上述的白菜BrCRF11a的人工miRNA(amiR),其具有:
1)SEQ ID No.2所示的核糖核苷酸序列;或
2)与1)限定的RNA序列对应的脱氧核糖核苷酸序列。
本发明提供了含有上述白菜BrCRF11a或其amiR的生物材料,所述生物材料为表达载体,表达盒,宿主细胞或工程菌。
本发明提供了上述白菜BrCRF11a或其amiR或其相应的生物材料在调控白菜花发育中的应用。
本发明提供了上述白菜BrCRF11a或其amiR或其相应的生物材料在制备转基因白菜中的应用。
本发明提供了上述白菜BrCRF11a或其amiR或其相应的生物材料在白菜种质资源改良中的应用。
本发明提供的白菜BrCRF11a序列如SEQ ID No.1所示,针对其设计的amiR序列如SEQ ID No.2所示。通过农杆菌介导法将该基因或其amiR导入‘油青四九’菜心中,分别获得白菜BrCRF11a过表达和抑制表达的转基因菜心株系,结果发现白菜BrCRF11a的表达变化可以使花分生组织持续保持干细胞活性,从而使菜心出现了嵌套花的表型。这表明了白菜BrCRF11a与花发育关系密切,将该基因应用于白菜类蔬菜育种,具有良好的应用前景。
附图说明
图1为白菜BrCRF11a克隆PCR电泳图。其中左泳道为marker Fermentas SM0321,右泳道为目的片段。
图2BrCRF11a过表达及抑制表达载体示意图。
图3为转基因菜心植株的PCR阳性检测,从左到右依次,1泳道为marker FermentasSM0321,泳道2、3、4为空载对照组,泳道5、6、7为BrCRF11a过表达株系,泳道8、9、10为BrCRF11a抑制表达株系,泳道11为pBI121质粒,泳道12为未转基因菜心。
图4为转基因菜心株系中BrCRF11a的相对表达量示意图。
图5为BrCRF11a过表达植株与对照植株花期生长状况比较。A中左边为对照植株,右边为BrCRF11a过表达植株。B是A图中红框中部分的放大图,其中箭头指示的是部分嵌套花。
图6为BrCRF11a过表达植株与对照植株花的形态比较。A-E为对照植株的花及四轮花器官,F-J为BrCRF11a过表达植株的嵌套花及四轮花器官。A和F为整个花的形态,B和G为萼片的形态,C和H为花瓣的形态,D和I为雄蕊的形态,E和J为雌蕊的形态。比例尺长度为1mm。
图7为BrCRF11a抑制表达植株与对照植株花期生长状况比较。A中左边为对照植株,右边为BrCRF11a抑制表达植株。B是A图中红框中部分的放大图,其中箭头指示的是部分嵌套花。
图8为BrCRF11a抑制表达植株与对照植株花的形态比较。A-E为对照植株的花及四轮花器官,F-J为BrCRF11a抑制表达植株的嵌套花及四轮花器官。A和F为整个花的形态,B和G为萼片的形态,C和H为花瓣的形态,D和I为雄蕊的形态,E和J为雌蕊的形态。比例尺长度为1mm。
图9为转基因菜心植株的嵌套花比例示意图。
图10为转基因菜心植株的花器官大小比较示意图。
图11为雌蕊半薄切片示意图,其中A、D为正常花,B、C、E、F为嵌套花。图B、C中的箭头表示雌蕊中产生额外的花器官或组织,E、F中的箭头表示额外的花器官或组织从雌蕊基部开始生长。
具体实施方式
下面通过具体实施例对本发明进行说明,实施例中未作详细描述的技术手段属于本领域专业技术人员熟知的常规技术。实施例只用于说明本发明,但不限制本发明的范围,任何本领域的技术人员在不付出创造性劳动的情况下,以本发明的实施例为基础所获得的其他实施例均属于本发明的保护范围。
本发明实施例提供了一种细胞***素响应基因BrCRF11a,该基因为:从来源于品种为‘Chiifu-401-42’白菜中克隆到的基因,其基因序列如SEQ ID No.1所示,针对该基因设计的人工miRNA(amiR)如SEQ ID No.2所示。
本发明实施例还提供了上述白菜BrCRF11a在调控白菜花发育中的应用,下面对其进行具体描述。
实施例1白菜BrCRF11a过表达及抑制表达载体的构建
1、过表达载体构建
(1)取白菜‘Chiifu-401-42’的雌蕊组织样品用TRIzol试剂提取总RNA,cDNA的合成用TAKARA反转录试剂盒完成,具体方法如下:5×gDNA Eraser Buffer 2μL,gDNA Eraser1μL,RNA 1μg,RNase Free H2O补足至10μL,42℃,2min去除基因组DNA,在上一步的反应液中加入5×Primer Script Buffer 4μL,RT Primer Mix 1μL,Primer Script RT EnzymeMix 1μL,RNase Free H2O 4μL,吸打混匀后37℃20min,85℃5s即完成cDNA的合成,cDNA在-20℃冰箱保存。
(2)以白菜雌蕊cDNA为模板,用表1中的引物通过高保真酶扩增获得BrCRF11a基因目的片段(图1),将目的片段回收后连接P-clone并测序确认获得目的序列(SEQ ID No.1)后,以其为模板再次扩增回收后,用相应的限制性内切酶酶切。酶切体系如下:Buffer 4μL,回收产物约2μg,Xba I和BamH I酶各2μL,双蒸水补足至40μL。37℃水浴1h后回收酶切产物。pBI121载体也用同样方法酶切并回收后,与基因的酶切产物连接,反应体系如下:10×Buffer 1μL,T4连接酶1μL,加入基因片段与载体片段的摩尔比约为3:1,双蒸水补足至10μL,4℃连接过夜后转化大肠杆菌。取阳性菌落PCR检测并测序证明连接正确后,菌液扩繁并抽提质粒,-20℃保存备用。
表1过表达载体构建所用引物
2、人工miRNA抑制表达载体构建
用BrCRF11a的基因序列在WMD3网站(http://wmd3.weigelworld.org/cgi-bin/webapp.cgi)中比对,得到对应基因编号后,在Designer板块下,输入序列号设计人工miRNA(amiR),选择得分较高的候选amiR分别在psRNATarget网站(http://www.plantgrn.org/psRNATarget/)和数据库(http://brassicadb.org/brad/blastPage.php)中比对,获得只针对目的基因的特异性amiR(SEQ ID No.2),将amiR嵌入拟南芥miR164a的前体骨架中,并通过mfold网站(http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form)预测其可以正常折叠形成前体RNA,即完成amiR设计。
将设计的amiR前体序列两端加上Xba I和BamH I的酶切位点序列及保护碱基后,送invitrogen公司合成,酶切回收后,分别连接到相同酶切后的pBI121载体上,连接体系及载体检测和扩繁同上述(图2)。
实施例2菜心遗传转化
1、将实施例1中得到的载体pBI121-BrCRF11a、pBI121-amiRBrCRF11a及pBI121空载体转入农杆菌GV3101中,具体方法如下:取解冻的农杆菌感受态与5μL质粒混匀后,冰浴10min,液氮中反应5min,28℃水浴5min;在超净工作台上加入1mL不含任何抗生素的液体LB培养基,28℃摇床200rpm培养4~5h;离心10,000rpm,1min,弃大部分上清,剩余约100μL将菌体重悬后涂于含Rif和Kan的固体LB平板上;28℃正向放置30min后倒置培养1~2d。取阳性菌落PCR检测正确后,菌株用25%甘油LB重悬后,-75℃保存备用。
2、配制培养基
(1)播种培养基:4.43g MS粉,20g蔗糖,8g琼脂粉,定容至1L,用2mol·L-1NaOH溶液调pH至5.8,121℃高压蒸汽灭菌20min,在超净台上分装至灭菌的培养瓶中,每瓶约30mL。
(2)MS液体培养基:4.43g MS粉,20g蔗糖,定容至1L,用2mol·L-1NaOH溶液调pH至5.8,分装至干净的培养瓶中,121℃高压蒸汽灭菌20min。
(3)预培养和共培养培养基:4.43g MS粉,20g蔗糖,8g琼脂粉,2mg6-BA,0.45mgNAA,定容至1L,用2mol·L-1NaOH溶液调pH至5.8,121℃高压蒸汽灭菌20min,在超净台上加入7.5mg AgNO3,摇匀后分装至灭菌的培养皿中,每皿约30mL,冷凝后预培养培养基直接封口,共培养培养基在培养基表面放置一层无菌滤纸,然后封口保存。
(4)分化培养基和继代培养基:4.43g MS粉,20g蔗糖,8g琼脂粉,2mg 6-BA,0.45mgNAA,定容至1L,用2mol·L-1NaOH溶液调pH至5.8,121℃高压蒸汽灭菌20min,在超净台上加入7.5mg AgNO3,250mg羧苄,7.5mg Kan摇匀后,分化培养基分装至灭菌的培养皿中,每皿约40mL,继代培养基分装至灭菌的培养瓶中,每瓶约30mL。、
(5)生根培养基:4.43g MS粉,20g蔗糖,8g琼脂粉,0.3mg NAA,定容至1L,用2mol·L-1NaOH溶液调pH至5.8,121℃高压蒸汽灭菌20min,在超净台上分装至灭菌的培养瓶中,每瓶约30mL。
3、菜心播种
称取1.8g‘油青四九’菜心种子,用75%乙醇洗涤2次,每次2min,期间不断振荡;用灭菌的蒸馏水洗涤2次,每次2min;用0.1%的HgCl2溶液浸泡,在28℃摇床振荡10min;用灭菌的蒸馏水洗涤10次,每次1min;在无菌的滤纸上晾干种子,然后均匀撒播在6瓶播种培养基中。
4、预培养及共培养
播种5~6d后,幼苗刚好长出两片子叶,在保湿环境中,用刀片在茎尖生长点处快速准确地切下去,从而得到两片带有子叶柄的子叶,将子叶柄的切口处***预培养基中约0.5cm深度,重复此步骤,直到将所有子叶柄***预培养基中,将培养基封口,做好标记,在光照培养室中,子叶柄向上放置培养3~5d,待切口处微微膨大后用菌液侵染。
侵染前一晚,取30μL经过两次活化的表达载体农杆菌菌液,加入30mL不含抗生素的液体LB培养基中,28℃200rpm振荡培养过夜;当菌液OD600达到0.4~0.5时,在超净工作台中,取出预培养的外植体,将其浸没于菌液中,8~12min后取出,在无菌滤纸上晾干多余菌液,最后将侵染过的外植体放入共培养基中暗培养48h,注意子叶柄切口处需接触培养基。
5、分化及继代培养
暗培养结束后,在超净工作台上将外植体取出,将子叶柄切口一端***分化培养基中约0.5cm深度,全部转移后,将培养基封口,做好标记,在光照培养室中,子叶柄向上放置培养约20d;待愈伤组织处生长出幼芽约1cm时,仔细将幼芽切下,将基部***继代培养基中,注意将愈伤组织全部去除,在光照培养室中培养,此后每隔约20d继代培养一次,期间不断除去假阳性的白化苗,留下绿色抗性苗。
6、生根及移栽
当外植体生长为4~5cm高的幼苗时,在超净工作台上将其基部愈伤组织全部切除,***生根培养基中培养8~12d,待其生长出3条约3cm幼根后,用清水轻轻去除幼根表面培养基,定植于灭菌处理的基质中,在光照培养箱中培养,用保鲜膜保湿2~3d后,去除保鲜膜,此后每周浇水和营养液各1次。
7、转基因菜心阳性检测
(1)PCR检测
取样:继代培养过程中,每个芽系的多个芽的叶片混合取样检测;生根及移栽过程中,每个单株的叶片取样检测。
采用DNA简易提取法提取DNA。首先,配制DNA提取缓冲液,取20%SDS溶液0.5mL,0.5mol·L-1EDTA溶液0.5mL,1mol·L-1Tris-HCl缓冲液(pH 9.0)2mL,2mol·L-1LiCl溶液2mL,双蒸水补足至10mL,混合均匀即可使用。然后,取约0.1g样品放入2mL离心管中,加入200μL提取缓冲液及1个磁珠,放入磨样机中研磨(65Hz,120s),取出离心管中的磁珠后,13,000rpm离心5min;转移100μL上清液至新的1.5mL离心管中,加入100μL异丙醇后,迅速温和颠倒混匀,室温静置5min后,13,000rpm离心10min;去掉上清液,用1mL 70%乙醇洗涤沉淀后,13,000rpm离心3min,弃上清液;重复洗涤一次后,将离心管倒置于吸水纸上,吹干乙醇后,加入50μL双蒸水溶解DNA。
转基因阳性植株基因组中应有NPTII基因的***,因此,所有转基因植株均用NPTII基因的引物(表2)检测,PCR体系如下:T5Mix 12.5μL,NPTII正反向引物各0.5μL,DNA模板2μL,双蒸水补足至25μL,98℃3min,35个循环(98℃10s,55℃10s,72℃15s),72℃3min,4℃保温。PCR产物通过1.2%琼脂糖凝胶电泳鉴定扩增片段的长度,只有阳性植株用于进行后续实验(参见图3)。
表2转基因菜心PCR检测所用引物
引物名称 | 引物序列(5’-3’) |
NPTII-F | GTCACTGAAGCGGGAAGGG(SEQ ID No.5) |
NPTII-R | CGGCGATACCGTAAAGCAC(SEQ ID No.6) |
(2)实时荧光定量PCR分析转基因菜心植株中BrCRF11a基因的相对表达量
取移栽后通过PCR检测的阳性株系中每个株系至少3株进行混合取材,为排除干扰,所有植株均取第3片叶子,做好标记后,迅速置入液氮中固定,全部取材完成后,提取总RNA并合成cDNA后,进行qRT-PCR分析。qRT-PCR分析所用引物通过Primer Premier 5设计,如表3所示。反应体系为15μL:7.5μL of SYBR Green Master Mix,正反向引物各0.3μL,模板1μL,5.9μL双蒸水。qRT-PCR反应流程:95℃:30s,40个循环(95℃:5s,55℃:45s)。通过熔融曲线确定反应的特异性,内参基因为BrActin1,基因的相对表达量通过2-ΔΔCt方法计算。
表3转基因菜心植株qRT-PCR分析所用引物
引物名称 | 引物序列(5’-3’) |
BrCRF11aF | GCTCCCTGTGATTCAACTGC(SEQ ID No.7) |
BrCRF11aR | CTCGTCAATGAAGCAACCC(SEQ ID No.8) |
BrActin1F | CGCTTAACCCGAAAGCTAAC(SEQ ID No.9) |
BrActin1R | TACGCCCACTAGCGTAAAG(SEQ ID No.10) |
结果表明,在BrCRF11a过表达植株(OE11a-1、OE11a-2、OE11a-8)中BrCRF11a的表达量高于对照组植株(CK1、CK3、CK4),而在BrCRF11a干涉表达植株(amiR11a-4、amiR11a-6、amiR11a-7)中BrCRF11a的表达量低于对照组植株(图4)。
实施例3转基因菜心植株表型观察
1、在转基因菜心植株花期观察发现,BrCRF11a过表达及抑制表达的转基因菜心植株均出现了嵌套花的表型,而对照组转基因菜心植株中没有出现嵌套花(图5、7)。进一步的观察发现,嵌套花是由雌蕊中再生花序所产生(图6、8)。
2、转基因植株嵌套花的比例及花器官大小统计
待转基因植株抽薹开花后,每一株系取10株植株,统计其嵌套花与正常花的数量,并计算嵌套花所占比例(图9)。结果表明,BrCRF11a的表达被扰乱后,可以使转基因植株产生一定比例的嵌套花。
取转基因植株嵌套花及对照植株的正常花各10朵,取其萼片、花瓣和雄蕊在体式显微镜下拍照并用标尺标记,用软件Image J分别统计萼片、雄蕊的长度及花瓣的面积大小(图10)。结果表明,嵌套花的萼片、花瓣和雄蕊的大小与正常花的没有明显差异。
3、转基因菜心植株雌蕊的半薄切片
取转基因菜心中嵌套花及正常花的雌蕊,放入2.5%的戊二醛溶液中,用真空器处理直至雌蕊全部浸没在液面以下,4℃固定过夜;去掉固定液,用0.1mol·L-1磷酸缓冲液(pH7.0)洗涤样品3次,每次15min;用1%的锇酸溶液固定样品2h,小心将样品取出放入新的离心管中,用磷酸缓冲液洗涤3次,每次15min;依次用浓度为30%、50%、70%、80%、90%和95%的乙醇溶液处理样品,每一浓度处理15min;用无水乙醇处理样品20min,用纯丙酮处理样品20min;用1:1体积比的包埋剂与丙酮混合液处理样品1h;用3:1体积比的包埋剂与丙酮混合液处理样品3h;用纯包埋剂处理样品过夜,用新的包埋剂包埋样品,70℃处理过夜。
所得样品在切片机(PYRAMITOME-11800)上切成2μm的半薄切片,放在滴有二甲基蓝(Dimethyl Blue)的载玻片上,加热蒸干染液后,在荧光显微镜白光下观察并拍照(图11)。结果表明,与正常花的雌蕊相比,嵌套花的雌蕊中出现了额外的花器官或组织,且这些器官或组织是从雌蕊基部开始生长的。因此,嵌套花的产生是BrCRF11a的表达发生变化后,扰乱了花分生组织干细胞活性的适时终止所造成的。
以上所述为本发明较佳的具体实施方式,但在其基础上可以进行一些改进或修改,这对任何熟悉本领域的技术人员而言是显而易见的。因此,在本发明基础上所作的这些修改和改进,均属于本发明要求保护的范围。
序列表
<110> 浙江大学
无锡迪茉得生物种业科技有限公司
<120> 白菜雌蕊发育相关基因BrCRF11a及其应用
<141> 2018-12-29
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 957
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aagatccgtt tcgttgtgaa tgatccttac gctactgact actcatcagg cgaggaagat 180
gttgaaatga ctcggaggag gaaacgttat gtctgtgaga tcgaccgtcc tttctctcaa 240
gccactgctc aagcagagtc tgaaagctct tatgttcagg agagtcgtaa caatggtgga 300
agcaaaaagg ctttaagcgg gaaagcctct caggttgttg ggcgctctag tgtcacgaag 360
cctgttggtg taaggcagag gaaatggggc aaatgggctg ctgagatcag acatccaatc 420
accaagacaa gaacttggtt gggtacttac gagacacttg aacaagctgc tgatgcttat 480
gctaccaaga agctcgagtt tgatgctctg gctgctgcct cttccgctgc tgctccctgt 540
gattcaactg cttcaaatga gactgtttct atggtctcca atgttgaagc tgtgtcaagc 600
attgatcgtg atatgaaggt aactgattca aagaagccga gttttgactt caacttcgca 660
gatctacaga ttcctgatat gggttgcttc attgacgagt cactgatccc aaatgcttgt 720
gagcttgatt tcttcttaac tgaagagaag gacgaccagc tcttggatga ttactgcggt 780
atagatgata tgaacatcat tggtcttgaa tgtgacggtc caagcgaact tccagactat 840
gatttctcag acgtggagat cgatcttggt ctcattggaa ccaccattga caagtttgct 900
ttcgtcgatc atcttgcaac gagtacaacc actcctctta atatcgcgtg cccataa 957
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<210> 4
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 4
cgggatcctt atgggcacgc gatattaag 29
<210> 5
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 5
gtcactgaag cgggaaggg 19
<210> 6
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 6
cggcgatacc gtaaagcac 19
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 7
gctccctgtg attcaactgc 20
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 8
ctcgtcaatg aagcaaccc 19
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 9
cgcttaaccc gaaagctaac 20
<210> 10
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 10
tacgcccact agcgtaaag 19
Claims (1)
1.一种白菜雌蕊发育相关基因BrCRF11a、白菜BrCRF11a的人工miRNA或包含白菜雌蕊发育相关基因BrCRF11a、白菜BrCRF11a的人工miRNA任意一种基因的生物材料在调控菜心花发育中的应用,其特征在于,所述白菜雌蕊发育相关基因BrCRF11a具有:
1)SEQ ID No.1所示的核苷酸序列;
所述白菜BrCRF11a的人工miRNA具有:
2)SEQ ID No.2所示的核糖核苷酸序列;
3)与2)限定的RNA序列对应的脱氧核糖核苷酸序列;
所述生物材料为表达载体,表达盒或工程菌;
所述应用具体为:通过提高或抑制白菜雌蕊发育相关基因BrCRF11a的表达,扰乱花分生组织干细胞活性的适时终止使转基因菜心产生一定比例的嵌套花。
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