CN116837001A - ZmPIF2基因或其蛋白在调控玉米株高中的应用 - Google Patents
ZmPIF2基因或其蛋白在调控玉米株高中的应用 Download PDFInfo
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Abstract
本发明属于农业生物技术领域，公开了ZmPIF2基因或其蛋白在调玉米株高中的用途，本发明还公开了一种降低玉米株高的方法。本发明利用CRISPR/Cas9技术将ZmPIF2基因敲除后，玉米的株高显著降低，表明ZmPIF2基因能够显著调控玉米植株高度，对株高的遗传改良及培育矮杆玉米具有重要的理论和实际意义。
Description
技术领域
本发明属于农业生物技术领域，具体涉及ZmPIF2基因或其蛋白在调控玉米株高中的用途。
背景技术
近年来，随着人口的不断增长、环境的不断恶化以及耕地面积的不断减少，玉米生产力和种植面积下降，导致玉米产量正面临着严峻的危机。提高产量已成为玉米生产的重要任务，其中伴随高产的是倒伏问题。玉米倒伏后，植株冠层结构遭到破坏，光合能力减弱，结实率也随之降低。与此同时，还严重影响了玉米的外观、蒸煮食味和营养品质。玉米自身的抗倒伏能力受株高影响较大。因此，有关降低玉米株高的遗传机制一直是玉米遗传育种学家研究的热点。
玉米株高与玉米穗位是影响抗倒伏和玉米产量的一个重要影响因子，是典型的数量性状，由多个基因控制，国内外有许多学者对该性状进行了研究。经过多年研究，目前在玉米株高的分子遗传机理方面已取得一定成就，但基因资源相对匮乏，亟待更多优异等位基因的挖掘和利用，且通过基因工程技术降低玉米株高的报道仍相当有限，因此利用基因工程技术选择可供育种的基因是研究的重点。
ZmPIF2基因是玉米中光敏色素相互作用因子(PIF)家族的一员，由Gao等人(2015)通过同源性分析在玉米基因组中鉴定获得。目前已知该基因参与到玉米的光周期敏感调控网络，影响玉米开花时间，未见其与玉米株高相关的报道。
发明内容
本发明提供ZmPIF2基因或其蛋白在调控玉米株高中的应用，为玉米株高的遗传改良及培育矮杆玉米提供了良好的理论基础，也为进一步充分挖掘玉米生产潜力、提高玉米产量提供了科学依据。
本发明提供的技术方案如下：
如SEQ ID NO:1所示的氨基酸序列组成的ZmPIF2蛋白或SEQ ID NO:2所示的编码所述蛋白的基因或含有所述基因的表达盒、重组载体、重组微生物在调控玉米株高中的应用。
进一步的，所述调控玉米株高为降低玉米株高。
本发明还提供一种降低玉米株高的方法，抑制玉米ZmPIF2基因的表达，和/或减少ZmPIF2基因编码蛋白的活性。
本发明还提供一种培育矮杆玉米的方法，选出ZmPIF2基因表达量降低或无表达，ZmPIF2蛋白表达量降低或无表达，和/或ZmPIF2基因蛋白活性降低或无活性的玉米植株，获得矮杆玉米。
进一步的，玉米育种时，至少一个亲本为上述的矮杆玉米。
进一步的，将玉米中的ZmPIF2基因敲除。
本发明还提供一种培育低穗位玉米的方法，选出ZmPIF2基因表达量降低或无表达，ZmPIF2蛋白表达量降低或无表达，和/或ZmPIF2基因蛋白活性降低或无活性的玉米植株，获得低穗位玉米。
进一步的，将玉米中的ZmPIF2基因敲除。
进一步的，针对ZmPIF2基因设计2个靶点并构建CRISPR载体，所述sgRNA靶点序列如下：
靶点一：GCATGCCTCGGACACCACCAAGG
靶点二：CCCCGTCGAGTCCACGGTCGTCC。
进一步的，构建CRISPR载体后，进行玉米遗传转化并获得阳性苗。
本发明的目的在于提供玉米ZmPIF2基因的新用途。
本发明提供了ZmPIF2基因或其蛋白在调控玉米株高中的用途。
本发明还提供了一种降低玉米植株的方法，抑制玉米ZmPIF2基因的表达，和/或减少ZmPIF2基因编码蛋白的活性，即可。
本发明还提供了ZmPIF2基因或其蛋白在筛选矮杆玉米中的用途。
本发明还提供了一种筛选矮杆玉米的方法，包括以下步骤：
选出ZmPIF2基因表达量降低或无表达，ZmPIF2蛋白表达量降低或无表达，和/或ZmPIF2基因蛋白活性降低或无活性的玉米植株。
有益效果
玉米株高是一个影响玉米产量和抗倒伏性的关键因素，研究也发现，随着玉米植株高度降低，株型紧凑，穗位降低，更适合高密度种植，这些特性使得玉米具有较强的抗倒伏能力和光能利用效率，促进产量上升。我们发现，玉米中ZmPIF2基因被敲除后，株高显著降低，抗倒伏能力上升，产量也增加，为株高的遗传改良及培育玉米抗倒伏性和高产提供了良好的理论基础，也为进一步充分挖掘玉米生产潜力、提高玉米产量提供了科学依据，应用前景良好。
附图说明
图1为过表达株系OE10的电泳图：M：Marker DL2000；1-12:T2代纯系鉴定PCR产物条带,13:阴性对照，水；14:阳性对照，质粒；
图2为过表达株系OE2、OE7、OE10中ZmPIF2表达量分析；
图3为不同玉米株系的株高比较图；
图4为不同玉米株系的株高统计图；
图5为敲除位点以及测序峰图。
具体实施方式
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
实施例1 ZmPIF2过表达株系的构建及鉴定
一、ZmPIF2基因的过表达载体构建
前期已将ZmPIF2基因全长(SEQ ID NO:2)从野生型KN5585中克隆出来，ZmPIF2编码一个由505个氨基酸(SEQ ID NO:1)组成的蛋白。然后将含有BamHI+KpnI酶切位点的p3301载体双酶切，并在ZmPIF2两端加上带有载体酶切位点两端序列的同源臂，利用DNA回收试剂盒回收酶切后的载体和带有同源臂的ZmPIF2，将ZmPIF2与酶切后的载体同源重组，获得的载体命名为p3301-ZmPIF2，并送去百格基因公司进行玉米转化，获得T0代转基因苗，并鉴定出阳性苗。
野生型KN5585中的ZmPIF2全长序列(SEQ ID NO:2，1518bp)
ATGCCCGTTTCGATTTCCATCTGTAGGACTGGGCCGGGCGAGGAGCTGGCCGAGCTGCTGTGGGACCGTGGCCCGGCGCTGCGGAGGGCGCCGCCGCCGTTCCAGCCCTTCACCTGCAGCGCCGCCGGCAGCAGCAGGTCGCAGGAGCTGAAGCGGCATGCCTCGGACACCACCAAGGCGTCAGCGTTCGTGACCGCCGTCTCCGTCCCGCTGGGCACGCACGACGCCGGCTCCGGGCTCGGGCTCGCCGGCCTCCCCGTCCACGACGACGACGACGCCGTGCCGTGGTTGCATTGCCCCGTCGCCGACGACGGCGACGGCGACACGGCGCCGCTGCCGCCGGAGTTCTGCGCCGGCCTCCTGTCCGAGTACTCGGAGGTGGCCGCGCCGGCGCCGGCCTTCCACGCCGCGGCCACGCCGCCGGCCGAGGCCGCGGCCAACAAGCTGGCCCCGCCGAGCGCCGCCGGCGGAGGGGAGGGCGTCTTGAACTTCACCTTCTTCTCGCGGCCCCTCCAGCGACCGCAGGCGGCGGCGGCGCCCGCCGCCGCGGCCGCGAGCAACCCCGTCGAGTCCACGGTCGTCCAGGCGGCAGCGAACCGGCTGCGGAGCACGCCGCTGTTCTCCGAGCAGAGGATGGCGTGGCTGCAGCCACCCAAGGCGCCGCGCACCACAGCGGCAGCGGCGGCGCCACCTCCTCCTCCGCTGGCGCCTCTGCTCCCAGATAGCCGCCATGGGGAGACGGTCGGCACGGTAGCTCAACCTCAGCCCCGGTCGCAACCAGAAGCAAGACCTCCGGATGCGGCGGCGGTGACGACCTCTTCGGTCTGCTCCGGCAACGGTGGTCGGAGCCAGCTCAAGAGGAGCCGCCACCTGGCCGCGGACTGCTCGGTCAGTCCGGACGAGGACCTGGACGACGAGCCCGGCGCGACGAGGAGGTCGGCGGCGCGGAGCGCCAAGCGCTGCCGCACCGCCGAGGTGCACAACCTGTCGGAGAGGAGGAGACGGGACCGGATCAACGAGAAGATGCGCGCCCTGCAGGAGCTCATTCCCAACTGCAACAAGGTCGACAAGTCGTCGATGCTGGAGGAGGCGATCGAGTACCTGAAGACGCTGCAGCTGCAAGTGCAGATGATGTCGATGGGGACCGGGCTGTGCATGCCACCGGCGGCGATGCTGCTGCCAGCGATGCAGCAGCAGCTCCTGCACCACCACCCCATGGCGCACTTCCCCCATCTCGGCATGGGCCTGGGCTTCGGCATGGGCGCGGCGGCGGGGTTCGACATGCTCCCGTTCCCGTGCGTCGCGGCCGGCGCCCACTTCCCGTGCCCGCCGGGGGCCATGTTCGGCGTGCCGGGGCAGGCGATGCCCTCGCTGCCGGCGGCGTTCGCTCACATGTACGGCGCTGGCAGTGGCGCCGGGCCGGCTGGGCAGACGGAAGCTGCTGATGCGGCTGCTCCTGCACGGCCAGGAGAGGCAGAGCAGGGTGATCAGCAGGTGCAGCACGCGAAGCAGACGTGA
ZmPIF2氨基酸序列(SEQ ID NO:1)
MPVSISICRTGPGEELAELLWDRGPALRRAPPPFQPFTCSAAGSSRSQELKRHASDTTKASAFVTAVSVPLGTHDAGSGLGLAGLPVHDDDDAVPWLHCPVADDGDGDTAPLPPEFCAGLLSEYSEVAAPAPAFHAAATPPAEAAANKLAPPSAAGGGEGVLNFTFFSRPLQRPQAAAAPAAAAASNPVESTVVQAAANRLRSTPLFSEQRMAWLQPPKAPRTTAAAAAPPPPPLAPLLPDSRHGETVGTVAQPQPRSQPEARPPDAAAVTTSSVCSGNGGRSQLKRSRHLAADCSVSPDEDLDDEPGATRRSAARSAKRCRTAEVHNLSERRRRDRINEKMRALQELIPNCNKVDKSSMLEEAIEYLKTLQLQVQMMSMGTGLCMPPAAMLLPAMQQQLLHHHPMAHFPHLGMGLGFGMGAAAGFDMLPFPCVAAGAHFPCPPGAMFGVPGQAMPSLPAAFAHMYGAGSGAGPAGQTEAADAAAPARPGEAEQGDQQVQHAKQT
二、ZmPIF2基因的过表达纯系鉴定
采用CTAB法提取ZmPIF2转基因阳性玉米植株的DNA，具体步骤如下：
(1)取约200 mg新鲜叶片加入2 ml离心管中，用液氮冷冻研磨或研磨机研磨成粉末。
(2)加入600μl 65℃预热的2×CTAB，混匀并在65℃水浴中保温20 min，期间上下震荡2-3次，使细胞裂解充分。
(3)加入600μl氯仿/异戊醇(24:1)，上下颠倒混匀1 min，之后静置3-5min，使其分层。
(4)10000 r/min离心10 min，小心取上清至新的1.5ml离心管中。
(5)加入等体积-20℃预冷的异丙醇，上下颠倒混匀，置于-20℃环境中30min，使DNA沉淀。
(6)12000 r/min离心10 min，倒掉上清液，加入0.5 ml 70％乙醇溶液，上下颠倒洗涤DNA沉淀(若DNA沉淀产生滑动、漂浮等，洗涤后可以用12000 r/min离心5 min，使得DNA贴附管底)，之后倒掉乙醇溶液(注意不要把离心管底的白色DNA沉淀倒掉)。
(7)倒掉乙醇后，室温晾干DNA沉淀。
(8)加入200μl纯水溶解DNA，保存于4℃备用。
2XCTAB溶液配置如下：
CTAB粉末:4克，NaCl：16.364克，1M Tris-HCl：20ml(PH＝8.0)，0.5MEDTA:8ml，用超纯水溶解，再定容至200ml，高压灭菌后加巯基乙醇。
对获得的基因组DNA用检测引物进行PCR扩增，取2μl转基因玉米DNA作为模板，以检测引物：
F:5’-TGACGACCTCTTCGGTCTG-3’(SEQ ID NO.3)，
R:5’-TGATAATCATCGCAAGACCGG-3’(SEQ ID NO.4)进行PCR扩增，
扩增条件为：95℃预热5 min；95℃,45 s,61℃,30s,72℃,60s,共38个循环；72℃,10 min。
扩增体系为：2xEs Tag MasterMix(康为世纪)10μl，上下游引物各1μl，ddH2O7μl，DNA 1μl。
扩增出长度为913bp的目的片段(见图2)，经测序证明该片段为重组载体p1011-ZmPIF2的片段，证明重组载体已转化入玉米中，且根据纯系计算方法，10株以上单株为阳性即该株系为纯系，因此将其命名为OE2，OE7，OE10。
二、ZmPIF2基因的敲除纯系鉴定
在百格公司进行CRISPR靶点设计，针对ZmPIF2基因设计2个靶点并构建CRISPR载体，本材料sgRNA靶点设计如下：
靶点一：GCATGCCTCGGACACCACCAAGG(SEQ ID NO.5)
靶点二：CCCCGTCGAGTCCACGGTCGTCC(SEQ ID NO.6)
然后进行玉米遗传转化并获得阳性苗，经测序获得两个ZmPIF2敲除株系纯系，将其命名为KO-2,KO-7，敲除位点以及测序峰图如图5所示：
WT：靶点一GCATGCCTCGGACACCACCAAGG(SEQ ID NO.7)
靶点二CCCCGTCGAGTCCACGGTCGTCC(SEQ ID NO.8)
KO-2：靶点一GCATGCCTCGGACACC...AAGG(SEQ ID NO.9)
(172bp处缺失三个碱基A,C,C)
靶点二CCCCGTACGAGTCCACGGTCGTCC(SEQ ID NO.10)
(567bp处增加一个碱基A)
KO-7：靶点一GCATGCCTCGGACACCAACCAAGG(SEQ ID NO.11)
(172bp处增加一个碱基A )
靶点二CCCCGTCCGAGTCCACGGTCGTCC(SEQ ID NO.12)
(567bp处增加C碱基)
实施例2 ZmPIF2过表达株系表达量测定
一、玉米总RNA的提取以及cDNA的合成
种植KN5585、OE2、OE7、OE10至三叶一心期，并按照Takara公司RNAiso Plus的产品使用说明书进行。将玉米样本在液氮中快速研磨成粉状，加入RNAiso Plus，剧烈震荡之后于冰上放置一段时间，离心并吸取上清液，再加入氯仿抽提，将上清液与等量异丙醇混匀，以沉淀RNA。离心去掉异丙醇之后用75％的酒精洗涤RNA沉淀，待酒精挥发之后用DEPC水溶解RNA。然后用抽提的总RNA反转录cDNA。cDNA用Takara公司的PrimeScriptTM RT MasterMix(Perfect Real Time)试剂合成。
二、荧光定量PCR分析基因表达
使用ABI公司的7300荧光定量仪进行荧光定量分析基因表达。野生型KN5585、OE2、OE7，OE10中ZmPIF2表达量见图2。
实施例3 ZmPIF2调控玉米株高的效果验证
将已鉴定的ZmPIF2敲除株系KO-2,KO-7和过表达株系OE2、OE7、OE10分别种植于扬州大学生物科学与技术学院试验田，待其成熟之后进行收获并统计株高。
统计数据采用软件为prism5.0进行分析，分析方法为单因素方差分析(One-wayANOVA)。
统计结果见表1。
表中标注：与野生型KN5585相比，*为差异显著；**为差异极显著。
表1不同玉米株系的株高统计
| 株系 | 株高(cm) |
| KN5585 | 165.50±3.26 |
| OE2 | 181.75±5.43** |
| OE7 | 181.17±5.43** |
| OE10 | 191.80±5.27** |
| KO-2 | 141.38±6.24** |
| KO-7 | 150.60±8.36** |
由表1可知，与野生型品种相比，ZmPIF2过表达株系的株高显著变高，范围为9～16％；而ZmPIF2敲除株系的株高显著变矮，范围为9～15％；表明ZmPIF2对调控玉米株高发挥重要作用。
综上，ZmPIF2基因能够显著调控玉米株高，可用于株高的遗传改良及提高抗倒伏性和产量。
Claims (10)
1.如SEQ ID NO:1所示的氨基酸序列组成的ZmPIF2蛋白或SEQ ID NO:2所示的编码所述蛋白的基因或含有所述基因的表达盒、重组载体、重组微生物在调控玉米株高中的应用。
2.根据权利要求1所述的应用，其特征在于，所述调控玉米株高为降低玉米植株高度。
3.一种降低玉米株高的方法，其特征在于，抑制玉米ZmPIF2基因的表达，和/或减少ZmPIF2基因编码蛋白的活性。
4.一种培育矮杆玉米的方法，其特征在于，选出ZmPIF2基因表达量降低或无表达，ZmPIF2蛋白表达量降低或无表达，和/或ZmPIF2基因蛋白活性降低或无活性的玉米植株，获得矮杆玉米。
5.根据权利要求4所述的培育矮杆玉米的方法，其特征在于，玉米育种时，至少一个亲本为权利要求4所述的矮杆玉米。
6.根据权利要求4所述的培育矮杆玉米的方法，其特征在于，将玉米中的ZmPIF2基因敲除。
7.一种培育低穗位玉米的方法，其特征在于，选出ZmPIF2基因表达量降低或无表达，ZmPIF2蛋白表达量降低或无表达，和/或ZmPIF2基因蛋白活性降低或无活性的玉米植株，获得低穗位的玉米。
8.根据权利要求7所述的培育低穗位玉米的方法，其特征在于，将玉米中的ZmPIF2基因敲除。
9.根据权利要求6或8所述的方法，其特征在于，针对ZmPIF2基因设计2个靶点并构建CRISPR载体，所述sgRNA靶点序列如下：
靶点一：GCATGCCTCGGACACCACCAAGG
靶点二：CCCCGTCGAGTCCACGGTCGTCC。
10.根据权利要求9所述的方法，其特征在于，构建CRISPR载体后，进行玉米遗传转化并获得阳性苗。
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