CN116064567B - 一种玉米小籽粒突变体及其应用 - Google Patents
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Abstract
本发明属于生物科学技术领域，具体涉及一种玉米小籽粒突变体及其应用。具体的所述突变体为编码植物生长调控因子GRF5的突变体基因。所述GRF5的突变体基因从野生型B73和突变体叶片基因组及cDNA扩增获得。本发明研究发现野生型与突变体的表达型在种子大小上有明显的差异，与野生型比较，突变体表达型的种子明显变小，突变后提高了玉米的抗旱性，我们可以通过基因转化或基因编辑或分子标记辅助育种培育氮高效玉米新品种。
Description
发明领域
本发明属于生物科学技术领域，具体涉及一种玉米小籽粒突变体及其应用。
背景技术
玉米是世界三大主要农作物之一，20世纪90年代以来，世界玉米总产量首次超越水稻和小麦，成为第一位的粮食作物。玉米不仅是重要的粮食作物，也是重要的饲料来源和能源作物。随着人们对肉蛋奶需求的增加以及世界能源的日益匮乏，玉米作为主要的饲料来源和能源作物在提供饲料方面和提供液体燃料方面发挥着日益重要的作用。随着我国工业化、城市化的不断推进和加速，耕地面积急剧减少、人口数量持续增加，我国的粮食需求面临着重大的压力。提高玉米产量成为各国科学家和育种学家的重要目标。干旱是影响玉米生长发育及产量的最主要非生物因子之一，其造成的作物减产程度居于各种自然灾害的首位。据统计，山东省常年遭受干旱灾害影响的面积都在800到1000万亩之间，这极大地影响了农业生产及粮食安全。此外，全球其他国家与地区的农业也都不同程度的遭受着干旱影响，农业经济的发展被严重制约。
长期以来，玉米耐旱品种的选育一直是国内外学者的热点和难点。传统的作物育种不仅费时费力，而且育种材料的遗传背景对耐旱品种的选育有很大的限制性。同时抗逆育种需要特殊的选择环境和检测方法，因此传统的育种方法已经不能满足目前农业生产的需要。随着分子生物学技术的发展，遗传学手段为玉米遗传改良提供了一条具有竞争力的途径，也是目前分子育种研究的重点。在植物感知外界环境并做出相应反应过程中，转录因子发挥了重要的作用。探究转录因子基因在逆境下尤其是干旱胁迫中发挥抗性作用的分子机制，可深入了解其上下游基因信息和信号转导通路，为作物抗逆育种提供新的思路和候选。因此，研究调控玉米抗旱的分子机理和寻找抗旱的基因，对提高玉米产量具有非同寻常的意义。
植物生长调控因子(GRF)基因在植物的生长发育过程中发挥重要的调节作用，GRF的作用机制在植物中也已被广泛深入研究，特别是在模式植物拟南芥和水稻中。作为重要的粮食作物，玉米中也含有13个GRF蛋白，然而，关于玉米中GRF基因功能的研究还不是很深入，尤其在干旱胁迫中的研究比较少。因此本研究描述的是GRF缺失功能的突变体，研究发现GRF突变体玉米对干旱有更强的抗性，这表明GRF可能参与玉米的非生物逆境应答。为玉米的抗逆育种提供了重要的候选基因，为实践应用提供理论参考。
发明内容
针对玉米中GRF基因功能的研究现状，本发明提供了一种玉米小籽粒突变体及其应用，所述突变体为编码植物生长调控因子GRF5的突变体基因。本发明研究发现野生型与突变体的表型在种子大小上有明显的差异，与野生型比较，突变体表型的种子明显变小，突变后提高了玉米的抗旱性，我们可以通过基因转化或基因编辑或分子标记辅助育种培育氮高效玉米新品种。
为实现上述发明目的，本发明采用以下技术方案：
首先，本发明提供一种玉米小籽粒突变体，所述突变体为编码植物生长调控因子GRF5的突变体基因。
进一步地，所述突变体的GRF5的CDS序列如SEQ ID NO:1所示，所述突变体GRF5蛋白序列如SEQ ID NO:2所示；
突变体GRF5 CDS序列SEQ ID NO:1:
atgggcatggcgatgccctttgcctccccgtctccggcagccgaccaccgcccctcctccctcctccccttctgccgcgccgcccctctctccgcggcgggggaggacgccgcgcagcagcacgcgatgagcggcaggtgggccgcgaggccggcgctcttcaccgcggcgcagtacgaggagctggagcaccaggcgctcatatacaagtacctcgtcgccggcgtgcccgtcccgccggacctcctcctccccctgcgccgaggcttcgtcttccaccagccacccgcccttgggtacgggccctacttcggcaagaaggtggacccggagcccgggcggtgccggcgtacggacggcaagaagtggcggtgctccaaggaggccgccccggactccaagtactgcgagcgccacatgcaccgcggccgcaaccgttcaagaaagcctgtggaagcgcagctcgcgcccccgccgcacgcccagccgcagcagcagcagcaggcccccgcgcccgctgctggcttccagaaccactcgctgtacccgtcgatcctcaccggcaacggcggcggcggggtaggtgctggtgctggtggtggcacgttcggcctggggcccacctctcagctgcacatggacagtgccgctgcctacgcgactgctgccggtggagggagcaaatatctcaggtactctgcatacggggtgaaatctctgtcggacgagcacagcacgctcttgtcgggcggcatggatccgtcgatgatggacaactcgtggcgcctgctgccatcccaaaccaacacattccaagccacaagctaccctgtgttcggcacgctgagtgggctagacgagagcaccatcgcgtcgctgccgaagacccagagggagcccctctctttcttcggcagcgacttcgtaaccgccgccaagcaggagaaccagacgctgcgccctttcttcgacgagtggcccaagtcgagggactcgtagccggagctgggcgaggacagcagcctcggcttctcggccacccagctctccatctccattcccatggcgacctccgacttctccaacaccagctccagatcgccgggtggaataccgtcgagatttctaaatttagcacggcctccaatcattgcagatgaacgagtaccgtgcatgtggatcccagcgtcttag
突变体GRF5蛋白序列SEQ ID NO:2：
MGMAMPFASPSPAADHRPSSLLPFCRAAPLSAAGEDAAQQHAMSGRWAARPALFTAAQYEELEHQALIYKYLVAGVPVPPDLLLPLRRGFVFHQPPALGYGPYFGKKVDPEPGRCRRTDGKKWRCSKEAAPDSKYCERHMHRGRNRSRKPVEAQLAPPPHAQPQQQQQAPAPAAGFQNHSLYPSILTGNGGGGVGAGAGGGTFGLGPTSQLHMDSAAAYATAAGGGSKYLRYSAYGVKSLSDEHSTLLSGGMDPSMMDNSWRLLPSQTNTFQATSYPVFGTLSGLDESTIASLPKTQREPLSFFGSDFVTAAKQENQTLRPFFDEWPKSRDS.PELGEDSSLGFSATQLSISIPMATSDFSNTSSRSPGGIPSRFLNLARPPIIADERVPCMWIPAS.
进一步地，所述GRF5的突变体基因(Zm00001d026240cDNA基因)使用如下引物：
ems0084-F：5′-CCGCAACGACAGATGTAAGA；
ems0084-R：5′-AGCTTTTGTGCTGGCACTTT。
进一步地，所述GRF5基因从野生型B73和突变体叶片基因组及cDNA扩增获得，直接连接克隆载体，测序，然后用序列分析软件DNAStar进行序列比对；所述野生型B73CDS序列如SEQ ID NO:3所示，所述野生型B73蛋白序列如SEQ ID NO:4所示；
野生型B73CDS序列SEQ ID NO:3：
atgggcatggcgatgccctttgcctccccgtctccggcagccgaccaccgcccctcctccctcctccccttctgccgcgccgcccctctctccgcggcgggggaggacgccgcgcagcagcacgcgatgagcggcaggtgggccgcgaggccggcgctcttcaccgcggcgcagtacgaggagctggagcaccaggcgctcatatacaagtacctcgtcgccggcgtgcccgtcccgccggacctcctcctccccctgcgccgaggcttcgtcttccaccagccacccgcccttgggtacgggccctacttcggcaagaaggtggacccggagcccgggcggtgccggcgtacggacggcaagaagtggcggtgctccaaggaggccgccccggactccaagtactgcgagcgccacatgcaccgcggccgcaaccgttcaagaaagcctgtggaagcgcagctcgcgcccccgccgcacgcccagccgcagcagcagcagcaggcccccgcgcccgctgctggcttccagaaccactcgctgtacccgtcgatcctcaccggcaacggcggcggcggggtaggtgctggtgctggtggtggcacgttcggcctggggcccacctctcagctgcacatggacagtgccgctgcctacgcgactgctgccggtggagggagcaaatatctcaggtactctgcatacggggtgaaatctctgtcggacgagcacagcacgctcttgtcgggcggcatggatccgtcgatgatggacaactcgtggcgcctgctgccatcccaaaccaacacattccaagccacaagctaccctgtgttcggcacgctgagtgggctagacgagagcaccatcgcgtcgctgccgaagacccagagggagcccctctctttcttcggcagcgacttcgtaaccgccgccaagcaggagaaccagacgctgcgccctttcttcgacgagtggcccaagtcgagggactcgtggccggagctgggcgaggacagcagcctcggcttctcggccacccagctctccatctccattcccatggcgacctccgacttctccaacaccagctccagatcgccgggtggaataccgtcgagatttctaaatttagcacggcctccaatcattgcagatgaacgagtaccgtgcatgtggatcccagcgtcttag
野生型B73蛋白序列SEQ ID NO:4：
MGMAMPFASPSPAADHRPSSLLPFCRAAPLSAAGEDAAQQHAMSGRWAARPALFTAAQYEELEHQALIYKYLVAGVPVPPDLLLPLRRGFVFHQPPALGYGPYFGKKVDPEPGRCRRTDGKKWRCSKEAAPDSKYCERHMHRGRNRSRKPVEAQLAPPPHAQPQQQQQAPAPAAGFQNHSLYPSILTGNGGGGVGAGAGGGTFGLGPTSQLHMDSAAAYATAAGGGSKYLRYSAYGVKSLSDEHSTLLSGGMDPSMMDNSWRLLPSQTNTFQATSYPVFGTLSGLDESTIASLPKTQREPLSFFGSDFVTAAKQENQTLRPFFDEWPKSRDSWPELGEDSSLGFSATQLSISIPMATSDFSNTSSRSPGGIPSRFLNLARPPIIADERVPCMWIPAS.
进一步地，所述PCR扩增程序具体为：95℃，5min；95℃，30s；58℃，45s；72℃，1min；共32个循环，最后72℃，10min；4℃，1h。
其次，本发明提供一种所述所述GRF5的突变体基因在培育玉米新品种中的应用。
进一步地，所述的应用具体为培育具有抗旱性的玉米小籽粒。
进一步地，所述应用具体可以通过基因转化或基因编辑或分子标记辅助育种培育氮高效玉米新品种。
与现有技术相比，本发明具有如下有益效果：
首先本发明提供了一种玉米小籽粒突变体，即编码植物生长调控因子GRF5的突变体基因，与野生型比较，突变体表达的种子明显变小，突变后提高了玉米的抗旱性。
其次，本发明提供的GRF5的突变体基因为玉米的抗逆育种提供了重要的候选基因，为实践应用提供理论参考，可以通过基因转化或基因编辑或分子标记辅助育种培育氮高效玉米新品种。
附图说明
图1.GRF5的突变体基因测序结果。
图2.野生型和突变体种子表型。
图3.野生型和突变体苗期表型。
图4.野生型和突变体苗期干旱处理表型。
具体实施方式
下面将以实施例的方式对本申请作进一步的详细描述，以使本领域技术人员能够实践本申请。应当理解，可以采用其他实施方式，并且可以做出适当的改变而不偏离本申请的精神或范围。为了避免对于使本领域技术人员能够实践本申请来说不必要的细节，说明书可能省略了对于本领域技术人员来说已知的某些信息。因此，以下详细描述不应以限制性的意义来理解，且本发明的范围仅由所附权利要求界定。以下的实施例便于更好地理解本申请，但并不用来限制本申请的范围。
实施例1
玉米基因组DNA提取采用CTAB法，具体操作步骤如下：
(1)取适量三叶一心野生型B73和突变体叶片材料于研钵中，加入液氮磨成粉末后，装入2ml离心管中；
(2)加入800μL 2％CTAB Buffer，充分混匀后，65℃温育30min，混匀几次；
(3)加入等体积的氯仿，颠倒混匀后室温放置5min，12,000rpm离心10min；
(4)取550μL上清液于新的1.5ml离心管中，加入等体积预冷异丙醇，颠倒混匀，沉淀10min；
(5)12,000rpm离心10min，沉淀用70％乙醇洗2次；
(6)12,000rpm离心5min，弃上清，室温干燥10min，溶于100μL去离子水中，-20℃保存。
玉米总RNA提取采用诺唯赞公司的Trizol试剂，具体操作方法如下：
(1)取约2-3g三叶一心野生型B73和突变体叶片材料于液氮预冷的研钵中，用液氮迅速磨成粉末，快速转移预冷过的2ml离心管中；
(2)加入1ml Trizol，快速颠倒混匀，涡旋震荡震荡1min，冰上静置5min；
(3)加入250μL氯仿，涡旋震荡震荡1min，室温静置10min；
(4)4℃，12,000rpm离心10min；
(5)取500μL上清液于1.5ml RNase-free离心管中，加入等体积异丙醇，颠倒混匀冰上放置10min；
(6)12,000rpm离心10min，去上清液，用75％DEPC-乙醇洗2次；
(7)室温晾干，将沉淀溶解于30-50μL DEPC处理过的ddH2O中；
(8)用紫外分光光度计Nano-drop测定RNA的浓度和质量(OD260/OD280)。
反转录：
体外合成cDNA第一链。具体操作步骤如下：
(1)RNA的变性：取总量2μg总RNA并加入Nuclease-free Water至总体积15μL，置于65℃温育5min，然后迅速放在冰上急速冷却；
(2)去除基因组DNA反应：在上面体系中加入5μL 4×DN Master Mix(预先加入gDNA Remover)，置于37℃温育5min后，迅速在冰上冷却2min；
(3)逆转录反应：在(2)的体系中加入5μL 5×RT Master MixⅡ至终体系为25μL；
按照如下参数进行反应，37℃，30min；50℃，5min；98℃，5min；16℃，5min。
Zm00001d026240基因的克隆
以玉米自交系B73和突变体为实验材料，提取三叶期玉米叶片组织，提取DNA和RNA并反转录成cDNA备用。从玉米数据库检索获取Zm00001d026240的生物学信息。以基因序列为模板设计特异性扩增引物，特异性扩增引物序列如下所示：ems0084-F：5′-CCGCAACGACAGATGTAAGA；ems0084-R：5′-AGCTTTTGTGCTGGCACTTT。以gDNA和cDNA为模板，ems0084-F和ems0084-R为引物，利用诺唯赞公司的高保真酶对其进行扩增，扩增后，利用将其连入PeasyB-T载体上，用M13F/R进行测序。
PCR反应体系:
PCR扩增程序:
95℃，5min预变性；95℃，30s变性；58℃，35s退火；72℃，30s延伸；共30个循环，最后，72℃下继续延伸10min完成反应，获得的PCR产物用质量比为1％的琼脂糖凝胶电泳检测拍照，将目标基因用诺唯赞公司的胶回收试剂盒进行切胶回收，与pEASY-BT载体连接后转化到DH5α大肠杆菌感受态细胞中，筛选阳性克隆子，并送去测序。
玉米材料干旱表型考察：
玉米材料在装有蛭石的穴盘中发苗到一叶一心叶期，之后挑选长势一致的幼苗移栽到浅盆中，将实验组和对照组种植；待植株生长到三叶一心叶期开始干旱处理，在表型差异明显时拍照；由于生长条件和材料的差异性处理时长会有所不同，在本研究中通常处理14天左右。
Claims (2)
1.一种玉米小籽粒突变体，其特征在于，所述突变体为编码植物生长调控因子GRF5的突变体基因；
所述突变体GRF5的CDS序列如SEQ ID NO:1所示，所述突变体GRF5蛋白序列如SEQ IDNO:2所示。
2.一种权利要求1所述的GRF5突变体基因在培育玉米新品种中的应用，其特征在于，所述的应用具体为培育具有抗旱性的玉米小籽粒。
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