CN105671055B - 水稻生殖发育基因mmd2的应用及恢复水稻雄性不育的方法 - Google Patents
水稻生殖发育基因mmd2的应用及恢复水稻雄性不育的方法 Download PDFInfo
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Abstract
本发明涉及水稻育种技术领域的一种水稻生殖发育基因MMD2的应用及恢复水稻雄性不育的方法;所述的MMD2基因序列如SEQ ID NO.1所示,其编码的氨基酸序列如SEQ ID NO.2所示,所述的应用是:通过基因变异或抑制表达使得水稻雄性生殖发育异常,获得水稻雄性不育系并可以用来生产杂交种子;所涉恢复MMD2基因功能缺失所致的雄性不育的方法,通过引物扩增MMD2基因构建载体,使用遗传转化方法转化突变体,能够使突变体恢复到野生型表型。本发明获得的水稻突变体营养生长阶段没有任何异常,但生殖生长异常导致完全不育;应用于杂交育种中,可以免除母本去雄的工作,大大提高生产效率,降低人工成本,在农业生产上具有重要的应用。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种水稻生殖发育基因MMD2的应用及恢复水稻雄性不育的方法。
背景技术
水稻是世界上主要的粮食作物之一,更是我国最重要的粮食作物。中国是世界上最大的稻米生产国和消费国,约有60%以上的国人以稻米为主食。我国水稻年播种面积超过3000万公顷,占世界的20%,占全国粮食作物播种面积的30%。水稻产量的高低与稳定,直接关系着人类的生活和社会的安定。
水稻生殖发育,是水稻生活史上最重要的一段时期,也是水稻产量与品质形成的关键时期。在生殖发育中,雄蕊和雌蕊的发育占重要地位。植物适时释放出发育成熟的花粉,对于完成受精结实是至关重要的。花药是花粉发育的场所,首先造孢细胞增殖发育成花粉母细胞,经过减数分裂形成四分体,随后包裹在四分体周围的胼胝质降解,释放出小孢子,然后单核小孢子经过两次有丝分裂,形成一个包含两个精子、一个营养细胞的成熟花粉,最后花药开裂释放花粉完成受精作用。任何一个过程的变化,均有可能引起花药发育异常最终导致雄性不育。
雄性不育在自然界中普遍存在,是杂交育种的基础,研究雄性生殖器官花药的发育,对作物遗传育种和农业产量的提高等方面具有重要的作用。从育种战略上看,杂交水稻的发展可以分为三系法、两系法和一系法三个发展阶段。每进入一个新阶段,都是育种上的一次突破,从而会把水稻的产量提高到一个新台阶。现在生产上用的杂交水稻属于三系法品种间杂交优势利用的范畴,这种三系杂交稻一般要比常规水稻增产20%左右,当前仍处于方兴未艾时期。但是,三系法杂交水稻种子优势表现复杂,受恢复系和保持系关系限制,使优良组合的筛选比较困难。因此,科学家一直在筛选和培育新的不育系,以期扩展细胞质背景,为远缘杂交和杂种优势的利用奠定基础。
发明内容
本发明针对现有技术存在的上述不足,提供一种水稻生殖发育基因MMD2的应用及恢复水稻雄性不育的方法,具体是一种雄性生殖发育相关基因MMD2及恢复MMD2功能缺失导致水稻雄性不育的方法,利用MMD2基因及其蛋白参与调控水稻雄性生殖发育的特点,及其利用转基因技术控制水稻雄性生殖发育,通过突变该蛋白序列或抑制该蛋白的表达产生新的水稻雄性不育株系,在农业生产上具有十分重要的应用。
本发明是通过以下技术方案来实现的:
第一方面,本发明涉及一种水稻雄性生殖发育相关基因MMD2,其基因全长序列如SEQ ID NO.6所示、其CDS如SEQ ID NO.1所示。
第二方面,本发明涉及一种所述雄性生殖发育相关基因MMD2编码的蛋白,其氨基酸序列如SEQ ID NO.2所示。
第三方面,本发明涉及一种所述水稻雄性生殖发育相关基因MMD2的应用,所述的应用包括:通过基因变异或抑制表达获得水稻雄性不育系或/和用所述水稻雄性不育系生产种子。
优选地,所述抑制表达包括采用RNAi手段。
第四方面,本发明还涉及一种恢复所述基因MMD2功能缺失导致的水稻雄性不育的方法,包括如下步骤:将所述基因MMD2转入水稻雄性不育株系,即可恢复所述不育株系的野生型表型;
其中,所述水稻雄性不育株系是通过MMD2基因变异或抑制其表达获得的;
所述转入均采用常规遗传学手段。
优选地,所述方法具体为:将含MMD2互补构建的根癌农杆菌(Agrobacteriumtumefaciens)EHA105转入所述水稻雄性不育株系,培育,即得;
其中,MMD2互补构建含有如SEQ ID NO.3所示的核苷酸序列。
优选地,所述方法更具体为:
(a)从水稻BAC克隆中扩增出MMD2基因的如SEQ ID NO.3所示的序列片段;
(b)将所述序列片段导入根癌农杆菌,得MMD2互补根癌农杆菌;
(c)将所述根癌农杆菌与水稻雄性不育株系的细胞或组织或器官接触,从而使编码如SEQ ID NO.2所示氨基酸的核苷酸转入细胞,并且整合到水稻雄性不育株系的染色体上;
(d)选择转入所述核苷酸的水稻细胞或组织或器官,再生,即可。
优选地,步骤(a)中,所述扩增采用的引物如SEQ ID NO.4、SEQ ID NO.5所示。
优选地,步骤(b)中,所述导入的载体采用双元载体pCAMBIA1301。
优选地,步骤(c)中,所述编码如SEQ ID NO.2所示氨基酸的核苷酸序列如SEQ IDNO.1所示。
优选地,所述根癌农杆菌具体指根癌农杆菌(Agrobacterium tumefaciens)EHA105 CGMCC No.4819。
第五方面,本发明提供一种抑制所述水稻雄性生殖发育相关基因MMD2表达的RNAi载体,是通过如下方法构建:扩增基因MMD2编码区序列第677位至第965位特异性片段,插入pBluescript SK载体,验证正确后将其切下,再把特异性片段连入pHB载体,即得。
与现有技术相比,本发明具有如下的有益效果:本发明通过控制水稻雄性生殖发育相关基因MMD2及其编码蛋白获得水稻雄性生殖发育的变异株,实现控制水稻生殖过程;本发明获得的水稻突变体在营养期与来源亲本无明显差异,进入生殖生长阶段后雄性生殖发育异常,花粉败育,得到完全不育的植株,在杂交水稻构建和农业生产上具有十分重要的应用。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述,本发明的其它特征、目的和优点将会变得更明显:
图1 pHB载体及MMD2干扰构建示意图;
图2mmd2突变体植株的形态学观察示意图;
图3MMD2表达模式图;
图4互补突变体得到野生型表型示意图。
具体实施方式
下面结合具体实施例,进一步阐述本发明。这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等分子克隆:实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。
所述MMD2基因为编码如SEQ ID NO.2所示氨基酸序列的核苷酸序列。
实施例1、水稻雄性不育株系的创制
1.1通过物理诱变手段创制mmd2水稻雄性不育株系
本实施例的mmd2突变材料是用60Coγ射线诱变常规粳稻品种武育粳7号(又名9522),处理剂量为280Gy(陈亮,储黄伟,袁政,et al.60Coγ-Ray射线诱变水稻突变体的分离和遗传学初步分析[J].厦门大学学报:自然科学版,2006,(S1):82-85)。对诱变的突变体三代回交,获得隐性核单基因控制的稳定遗传的mmd2突变体。进一步将mmd2突变体与9522回交,所有F1代的表型都与9522一致,表现为可育。突变体与野生型杂交的F1代自交后产生的F2代群体中,可育与不育植株的分离比约为3:1(可育:不育=87:24,χ2=0.68,P>0.05),表明这是一个由隐性单基因突变导致的突变体不育的表型。
1.2水稻雄性生殖发育相关基因MMD2的克隆
利用发明人构建的包含育性控制蛋白基因MMD2及其突变基因mmd2(其序列如SEQID NO.7所示)组成的、本领域内技术人员清楚的水稻基因定位克隆(map-based cloning或position cloning)群体,按分子标记定位于1个小的基因组片段内。在此基础上,用常规方法分离包含该片段的基因组DNA克隆。经测序和进一步的杂交鉴定确定其中一个含完整水稻雄性生殖发育控制蛋白MMD2。
经全核苷酸序列分析结果表明:水稻雄性生殖发育相关基因MMD2基因全长2093bp(SEQ ID NO.6,包含调控区和内含子);经软件分析和cDNA克隆,其CDS为1428bp,如SEQ IDNO.1所示;编码一个全长为475个氨基酸的F-box蛋白,其序列如SEQ ID NO.2所示。
1.3水稻雄性生殖发育蛋白基因的点突变
本实施例的mmd2突变材料是由常规粳稻品种武育粳7号(又名9522)经过对MMD2基因的序列变异获得,经过对MMD2突变基因mmd2的序列比较,水稻雄性生殖发育控制蛋白的移码和提前终止会使得水稻雄性生殖器官不能正常发育,造成植株不育;本实施例MMD2突变基因是在编码区的2个碱基的缺失(其序列如SEQ ID NO.7所示)引起水稻雄性生殖发育控制蛋白翻译得提前终止和功能丧失。
1.4通过RNAi手段降低水稻品种中的MMD2的表达水平
为了对MMD2蛋白进行应用,构建了MMD2基因的RNAi载体,并转化野生型9522植株,以期降低MMD2的表达,从而达到改变水稻育性的目的。
从水稻cDNA中用两对引物
Q69-RNAi-R-F(SEQ ID NO.8):5’GCTCTAGAACATCGAGAGGTGCTCGTA3’(XbaI)和
Q69-RNAi-R-R(SEQ ID NO.9):5’CGAGCTCGCAACCAAGAAGTCAACCTT3’(SacI)
Q69-RNAi-L-F(SEQ ID NO.10):5’AACTGCAGACATCGAGAGGTGCTCGTA3’(PstI)和
Q69-RNAi-L-R(SEQ ID NO.11):5’ACTGGATATCGCAACCAAGAAGTCAACCTT3’(EcoRV)
分别扩增出MMD2基因编码区序列的第677位至第965位共289bp的特异性片段;将这两个片段分别通过XbaI/SacI和EcoRV/PstI正反向插入连入加入含有水稻Intron序列的pBluescript SK载体;测序验证正确,再用SacI和HindIII酶切切下含有MMD2正反向特异性片段和Intron和片段,连入经同样酶切的pHB载体中。再次测序检验核苷酸序列是否正确,成功构建pHB-MMD2-RNAi质粒(图1)。
将含有pHB-MMD2-RNAi构建的农杆菌在含有Kan(50μg/μl)的YEB平板上划线,获的单菌落。挑单菌落接种到3ml含抗生素的YEB液体培养基中于28℃振荡培养过夜,第2天按1%接种量转接入50ml含抗生素的YEB液体培养基中,200rpm继续振荡培养至OD600为0.6至0.8左右时,将新鲜的农杆菌菌液于5000rpm、离心5分钟,收集并重悬于1/3体积的AAM液体培养基中,此时即可用于转化水稻各种受体材料。
本实施例采用常规的农杆菌转化方法转化水稻9522的幼胚愈伤。取授粉后12-15天的9522未成熟种子经70%乙醇浸泡1分钟后,于NaClO溶液中(与水1:3混合,加2-3滴吐温20)消毒90分钟以上,用无菌水冲洗4-5次,然后用解剖刀和镊子挑出幼胚并接种月N6D2培养基上诱导愈伤组织,在26±1℃、避光条件下培育,4天后可用于转化。将幼胚愈伤浸泡入新鲜的AAM农杆菌菌液中并不时摇动,20分钟后将水稻材料移出,在无菌滤纸上吸去过多的菌液,随即转移到N6D2C培养基上,于26℃共培养3天。共培养时,在共培养培养基中加入乙酰丁香酮,使用浓度为100μM。3天后,从共培养培养基上取出愈伤组织,切去胚芽并转入含有25mg/L Hyg的选择培养基上进行选择培养。7-12天后将抗性愈伤组织转到含有50mg/LHyg的选择培养基上继续筛选。10-12天后生长旺盛的抗性愈伤组织转移到预分化培养基上培养一周左右,再移至分化培养基上分化(12小时光照/天)。再生的小苗在1/2MS0H培养基上生根壮苗,随后移入人工气候室营养液栽培。
获得的再生植株移栽成活后以除草剂再次筛选转化植株;阳性植株提取叶片总DNA,经PCR进一步鉴定转化植株。RT-PCR分析阳性植株中MMD2基因的表达水平,表达水平降低到野生型20%以下为有效RNA干扰植株。
1.5 MMD2蛋白活性丧失或表达水平导致水稻雄性发育异常
对mmd2突变体植株的形态学观察。与野生型相比,mmd2突变体在营养生长阶段没有明显的表型缺陷(图2A)。进入生殖生长后,mmd2突变体的小穗和花器官发育正常(图2B,图2C,图2D)。但mmd2突变体的花药呈现出很浅的黄色,且比野生型花药小(图2D,图2E),成熟的花药内无任何花粉粒产生(图2F,图2G)。
实施例2、MMD2基因表达特征分析
利用mmd2突变株的来源亲本9522各个器官组织,提取RNA,进行反转录得到cDNA第一链,利用荧光定量PCR的方法确定MMD2基因的表达模式,结果表明,在根、茎、叶、内外稃和不同发育时期的花药中均可以检测到MMD2基因的表达,而在叶中和花药发育的前期(Stage1-Stage10)表达量较高(图3)。
实施例3、恢复mmd2突变体雄性不育性状的方法
将编码MMD2基因的基因组核苷酸序列转入突变体mmd2植株,能够使突变体恢复到野生型表型。
从水稻BAC克隆(OSJNBa0060P14)中用引物:
Q69hb-F(SEQ ID NO.4):5’CGGTACCCGGGGATCCGTACCGCAATCAACAAACAG 3’,
Q69hb-R(SEQ ID NO.5):5’ACCTGTAATTCACACGTGTATTCGTCACCGTATTCGTT 3’,
扩增出MMD2基因的5434bp(SEQ ID NO.3)的基因组序列片段。
将该片段与用于经过BamH I和PmaCI双酶切过的用于转化水稻的双元载体pCAMBIA1301载体通过in-fusion连接;测序验证正确,该载体通过电击导入根癌农杆菌(Agrobacterium tumefaciens)EHA105,得到MMD2互补根癌农杆菌(Agrobacteriumtumefaciens)EHA105,使用遗传转化手段转化突变体mmd2成熟胚愈伤,从而使编码如SEQID NO.2所示氨基酸的核苷酸转入水稻细胞,并且整合到水稻细胞的染色体上;再生,获得水稻植株;以观察是否会使突变体恢复到野生型表型。T0代获得互补植株,互补植株可以产生花粉,并被I2/KI染色,即表现出的野生型表型(图4E,图4F)。
综上所述,本发明通过控制水稻雄性生殖发育相关基因MMD2及其编码蛋白获得水稻雄性生殖发育异常的变异株,实现控制水稻雄性生殖发育和育性;本发明获得的水稻突变体在营养生长时期与来源亲本无明显差异,进入生殖生长阶段后,雄性生殖器官发育异常、花粉败育引起植株不育,在农业生产上具有十分重要的应用。
综上所述,本发明通过控制水稻雄性生殖发育相关基因MMD2及其编码蛋白获得水稻雄性生殖发育的变异株,实现控制水稻生殖过程;本发明获得的水稻突变体在营养期与来源亲本无明显差异,进入生殖生长阶段后雄性生殖发育异常,花粉败育,得到完全不育的植株,在杂交水稻构建和农业生产上具有十分重要的应用。
以上对本发明的具体实施例进行了描述。需要理解的是,本发明并不局限于上述特定实施方式,本领域技术人员可以在权利要求的范围内做出各种变形或修改,这并不影响本发明的实质内容。
Claims (8)
1.一种水稻雄性生殖发育相关基因MMD2的应用,其特征在于,所述的应用包括:通过基因变异或抑制表达获得水稻雄性不育系或/和用水稻雄性不育系生产种子;所述变异或抑制的目的基因为MMD2;
所述水稻雄性生殖发育相关基因MMD2的基因全长序列如SEQ ID NO.1所示的核苷酸序列,其编码的氨基酸序列如SEQ ID NO.2所示。
2.一种恢复基因MMD2功能缺失导致的水稻雄性不育的方法,其特征在于,包括如下步骤:将所述基因MMD2转入水稻雄性不育株系,即可恢复所述不育株系的野生型表型;
其中,所述水稻雄性不育株系是通过MMD2基因变异或抑制其表达获得的;
所述水稻雄性生殖发育相关基因MMD2的基因全长序列如SEQ ID NO.1所示的核苷酸序列,其编码的氨基酸序列如SEQ ID NO.2所示。
3.根据权利要求2所述的恢复所述基因MMD2功能缺失导致的水稻雄性不育的方法,其特征在于,所述方法具体为:将含MMD2互补构建的根癌农杆菌转入所述水稻雄性不育株系,培育,即得;
其中,MMD2互补构建含有如SEQ ID NO. 3所示的核苷酸序列。
4.根据权利要求3所述的恢复所述基因MMD2功能缺失导致的水稻雄性不育的方法,其特征在于,所述方法具体为:
(a)从水稻BAC克隆中扩增出MMD2基因的如SEQ ID NO.3所示的序列片段;
(b)将所述序列片段导入根癌农杆菌,得MMD2互补根癌农杆菌;
(c)将所述根癌农杆菌与水稻雄性不育株系的细胞或组织或器官接触,从而使编码如SEQ ID NO.2所示氨基酸的核苷酸转入细胞,并且整合到水稻雄性不育株系的染色体上;
(d)选择转入所述核苷酸的水稻细胞或组织或器官,再生,即可。
5. 根据权利要求4所述的恢复所述基因MMD2功能缺失导致的水稻雄性不育的方法,其特征在于,步骤(a)中,所述扩增采用的引物如SEQ ID NO.4、SEQ ID NO.5所示。
6.根据权利要求4所述的恢复所述基因MMD2功能缺失导致的水稻雄性不育的方法,其特征在于,步骤(b)中,所述导入的载体采用双元载体pCAMBIA1301。
7. 根据权利要求3或4所述的恢复所述基因MMD2功能缺失导致的水稻雄性不育的方法,其特征在于,所述根癌农杆菌具体指根癌农杆菌(Agrobacterium tumefaciens)EHA105CGMCC No.4819。
8.一种抑制水稻雄性生殖发育相关基因MMD2表达的RNAi载体,其特征在于,通过如下方法构建:扩增基因MMD2编码区序列第677位至第965位特异性片段,插入pBluescript SK载体,验证正确后将其切下,再把特异性片段连入pHB载体,即得。
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