CN111333706B - 大豆GmSPLE基因及其编码蛋白和应用 - Google Patents
大豆GmSPLE基因及其编码蛋白和应用 Download PDFInfo
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Abstract
大豆GmSPLE基因及其编码蛋白和应用，涉及一种大豆基因及其编码蛋白和应用。本发明提供一种大豆GmSPLE基因及其编码蛋白和应用。大豆GmSPLE基因的核苷酸序列如序列表中SEQ ID NO：1所示。其编码蛋白的氨基酸序列如序列表SEQ ID NO：2所示。本发明通过将大豆GmSPLE基因在大豆中过量表达，确定了大豆GmSPLE基因对大豆的株高具有调控作用，具有减少大豆主茎节数，促进大豆节间距变小，从而使大豆矮化的功能。该基因在抗倒伏，或通过调控株高增加大豆产量中具有重要的应用。
Description
技术领域
本发明涉及一种大豆基因及其编码蛋白和应用。
背景技术
解决全球人口逐年增加与可耕地面积日益减少的矛盾，只能通过提高单位面积的产 量。大豆是重要的粮食与经济作物，大豆为人类提供重要的植物蛋白质和油份。株高是影响大豆产量的主要性状之一，与大豆的抗倒伏性及产量等密切相关。上世纪60年代初 的第一次绿色革命爆发，其主要特征是把水稻与小麦的高秆变矮秆，由于矮秆基因的导 入解决了由于倒伏导致的粮食减产问题，可见株高在产量决定中的关键作用。大豆的荚 着生于节处，节的多少决定着单株荚数，从而决定着大豆的产量。通过分子育种控制大 豆的株高，培育抗倒伏，节数多，荚密的大豆品种是提高大豆产量的有效手段。
发明内容
本发明的目的是提供一种大豆GmSPLE基因及其编码蛋白和应用。
本发明大豆GmSPLE基因的核苷酸序列如序列表中SEQ ID NO：1所示。
本发明大豆GmSPLE基因的编码蛋白的氨基酸序列如序列表SEQ ID NO：2所示。
本发明提供大豆GmSPLE基因在调控大豆株高中的应用。
本发明的有益效果：
本发明公开了大豆GmSPLE基因及其编码蛋白和应用。本发明通过将大豆GmSPLE基因在大豆中过量表达，确定了大豆GmSPLE基因对大豆的株高具有调控作用，具有减 少大豆主茎节数，促进大豆节间距变小，从而使大豆矮化的功能。因此大豆GmSPLE基 因在抗倒伏，或通过调控株高增加大豆产量中具有重要的应用前景。
附图说明
图1为GmSPLE基因在过表达转基因大豆及非转基因大豆中的半定量RT-PCR分析；
图2为GmSPLE基因在过表达转基因大豆及非转基因大豆中的Western blot分析；
图3为GmSPLE过表达转基因大豆及非转基因大豆株高表型；
图4为GmSPLE过表达转基因大豆及非转基因大豆花期表型；
图5为GmSPLE过表达转基因大豆及非转基因大豆主茎节数统计；
图6为GmSPLE过表达转基因大豆及非转基因大豆节间距表型；
图7为GmSPLE过表达转基因大豆及非转基因大豆节间距数据统计。
具体实施方式
下面对本发明的实施例做详细说明，以下实施例在以本发明技术方案为前提下进行 实施，给出了详细的实施方案和具体的操作过程，但本发明的保护范围不限于下述的实施 例。
实施例1：GmSPLE基因过表达载体的构建
一、以大豆品种Kariyutaka的cDNA为模板，以GmSPLEmyc-F1和GmSPLEmyc-R1 为引物，采用购买自北京全式金生物科技有限公司的高保真酶FastPfu Fly DNA Polymeras，进行PCR扩增。PCR反应条件：94℃预变性5min，(94℃变性30s，58℃退 火30s，72℃延伸60s)×6循环，94℃变性30s，68℃退火30s，72℃延伸60s，26循环， 再72℃延伸5min，采用购买自Promega公司的
SV Gel and PCR Clean Up System 进行凝胶回收。通过PCR扩增获得GmSPLE基因的扩增条带，并通过此次PCR扩增在 GmSPLE基因的上游引入BamHⅠ的酶切位点，在下游引物SacⅠ的酶切位点。扩增所得 的PCR产物及载体pBA-myc同时经过BamHⅠ与SacⅠ双酶切后，T4 DNA连接酶过夜 连接，转化大肠杆菌感受态细胞。经过菌落PCR鉴定阳性克隆，将阳性克隆送交测序公 司测序，测序结果如序列表SEQ ID NO：1所示，该基因片段即为大豆GmSPLE基因， 由417bp个碱基组成。其编码具有序列表中SEQ ID NO：2的氨基酸序列的蛋白质。测序 结果表明已成功构建好GmSPLE基因在大豆中遗传转化的载体，pBA-myc:GmSPLE载体 含有Bar筛选标记基因(草铵膦抗性)，35S启动子驱动GmSPLE基因，该载体在GmSPLE 基因的N端加上了6×cmyc标签，便于在蛋白水平上检测。
引物GmSPLEmyc-F1：5’-CGGGATCCTGCATATGGACGAAAG-3’
引物GmSPLEmyc-R1：5’-CGCGAGCTCTCAAAGTTCGTGGTAT-3’
所述载体pBA-myc已于文章(Wenfeng Ning,et al.2017)中公开。
将构建好的载体pBA-myc:GmSPLE通过根癌农杆菌介导大豆子叶节的方法转入大豆 东农50品种基因组中，获得GmSPLE基因过表达转基因大豆株系。
实施例2：GmSPLE基因过表达转基因大豆的分子鉴定
1、通过对大豆的遗传转化，在T0代共获得28个独立的转化事件，通过使用160mg/L的 草铵膦涂抹实验，发现在T1代仍然表现出草铵膦抗性的转化事件有四个(35S:GmSPLE#L3、 35S:GmSPLE#L9、35S:GmSPLE#L17、35S:GmSPLE#L22)，提取这四个转化事件的大豆的成熟叶片的RNA，RNA提取方法参见Invitrogen公司的TRIzol试剂盒的操作手册。
2、采用DNase处理步骤一提取的总RNA，处理后的总RNA采用购买自Toyobo公司的NanoDrop检测其DNA含量及质量；
3、取经步骤2NanoDrop检测到达标准后的2μg总RNA，采用购买自北京全式金生物科 技有限公司的
One-Step gDNARemoval and cDNASynthesis SuperMix试剂盒，以 及试验盒配带的dT18引物，按照试剂盒的操作手册合成cDNA；
4、取2μl步骤3稀释5倍后的cDNA作为RT-PCR模板，以qGmSPLE-F1和qGmSPLE-R1 为引物，采用购买自北京全式金生物科技有限公司的EasyTaq，进行PCR扩增。PCR反应条 件：94℃预变性5min，94℃变性30s，60℃退火30s，72℃延伸30s，内参基因TUA5 27个循 环，GmSPLE基因29个循环，终延伸72，℃5min，大豆内参基因TUA5的引物序列为qTUA5-F1 和qTUA5-R1，内参基因TUA5的引物序列为已发表论文总的公开序列(Ruibo Hu,et al.2019)。取5μl PCR产物进行琼脂糖凝胶电泳，结果如图1所示，与非转基因大豆东农50相比，GmSPLE基因的表达量在4个独立的转化事件中均显著高于非转基因大豆。说明GmSPLE基 因在大豆中成功实现了过量表达。
引物qGmSPLE-F1：5’-CTTCATGTCAAGTTGATGGTTGTAG-3’
引物qGmSPLE-R1：5’-GTGCTGGTCTCCAATGAGTACG-3’
引物qTUA5-F1：5’-TGCCACCATCAAGACTAAGAGG-3’
引物qTUA5-R1：5’-ACCACCAGGAACAACAGAAGG-3’
5、提取其中三个转化事件的大豆的成熟叶片的蛋白，取完全展开的大豆叶片，在液氮 中用研钵磨碎后，迅速把粉末转移到1.5ml离心管中，加入1mL蛋白质裂解提取液(50mM/L Tris-HCl,pH8.0,120mM/L NaCl，10％甘油，0.2％Triton X-100，1.5g/L的DTT，0.1％SDS， 1mM/L的PMSF，1×Roche proteinase inhibitor)，4℃摇床摇晃20min，16,100×g离心10min， 移取上清液至干净的离心管中。取适量蛋白加入5×loading buffer，95℃水浴5min，凉至室 温后短暂离心，用SDS-PAGE分离，采用anti-cmyc(Thermo Fisher,货号MA1980)抗体做 Western Blot杂交。Western blot结果如图2所示，非转基因大豆东农50没有目的条带，而3 个独立的转化事件由于转入了带有cmyc标签的GmSPLE基因，能杂出目的条带，说明 GmSPLE基因成功在大豆中实现过量表达。
实施例3：GmSPLE基因过表达转基因大豆的表型分析
将T2代GmSPLE基因过表达转基因大豆与非转基因对照品种东农50播种于盆钵中，每个株系40-50株，置于自然光下种植(长日照)，对植株的表型进行详细调查。结果发 现，转基因大豆的结荚习性发生了改变(图3与图4)，转基因大豆顶端提前开花(图4)， 结束顶端生长，变为有限结荚习性，而非转基因大豆的结荚习性为亚有限结荚习性。结荚 习性的改变导致GmSPLE基因过表达转基因大豆主茎节数减少(图5)，使植株明显变矮。 除了结荚习性，节间距也也是影响大豆株高的重要因素，我们进一步调查了转基因大豆及 非转基因大豆的节间距。与非转基因植株相比，GmSPLE基因转基因大豆的节间距明显变 短(图6、图7)。
序列表
<110> 中国科学院东北地理与农业生态研究所
<120> 大豆GmSPLE基因及其编码蛋白和应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 417
<212> DNA
<213> 大豆属(Glycine max L. Merr.)
<400> 1
atggacgaaa gttggagtga gggaaaaagg agcatgagtt acaaggagga ggatgagtac 60
gaagaagagg aagaggagga ggtgagtgag tatagagatg atggtaggaa aaagaaggtg 120
gtgagtagta agagagggtc caaagctgga ggctcagtgc caccttcatg tcaagttgat 180
ggttgtagcg ctgatctaag tgaagctaag ccctaccata ggcgtcacaa ggtttgtgag 240
taccatgcca aggctcctgc cgtactcatt ggagaccagc accaacggtt ttgccaacaa 300
tgtagtaggt ttcatgagct atcagaattc gatgactcaa aaaggagttg cagaagacgt 360
ttggctggac ataatgagag gcgtcgcaaa aatgcatctg aataccacga actttga 417
<210> 2
<211> 138
<212> PRT
<213> 大豆属(Glycine max L. Merr.)
<400> 2
Met Asp Glu Ser Trp Ser Glu Gly Lys Arg Ser Met Ser Tyr Lys Glu
1 5 10 15
Glu Asp Glu Tyr Glu Glu Glu Glu Glu Glu Glu Val Ser Glu Tyr Arg
20 25 30
Asp Asp Gly Arg Lys Lys Lys Val Val Ser Ser Lys Arg Gly Ser Lys
35 40 45
Ala Gly Gly Ser Val Pro Pro Ser Cys Gln Val Asp Gly Cys Ser Ala
50 55 60
Asp Leu Ser Glu Ala Lys Pro Tyr His Arg Arg His Lys Val Cys Glu
65 70 75 80
Tyr His Ala Lys Ala Pro Ala Val Leu Ile Gly Asp Gln His Gln Arg
85 90 95
Phe Cys Gln Gln Cys Ser Arg Phe His Glu Leu Ser Glu Phe Asp Asp
100 105 110
Ser Lys Arg Ser Cys Arg Arg Arg Leu Ala Gly His Asn Glu Arg Arg
115 120 125
Arg Lys Asn Ala Ser Glu Tyr His Glu Leu
130 135
<210> 3
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cgggatcctg catatggacg aaag 24
<210> 4
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cgcgagctct caaagttcgt ggtat 25
<210> 5
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cttcatgtca agttgatggt tgtag 25
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gtgctggtct ccaatgagta cg 22
<210> 7
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tgccaccatc aagactaaga gg 22
<210> 8
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
accaccagga acaacagaag g 21
Claims (3)
1.大豆GmSPLE基因在调控大豆株高中的应用；所述大豆GmSPLE基因的核苷酸序列如序列表中SEQ ID NO：1所示。
2.根据权利要求1所述的应用，其特征在于所述调控大豆株高具体为减少大豆主茎节数，减小大豆节间距，从而促使大豆矮化。
3.根据权利要求1所述的应用，其特征在于所述GmSPLE基因导入植物细胞、组织或器官，再将被转化的植物细胞、组织或器官培育成植株，使所述GmSPLE基因在植物中表达，得到调控大豆株高的转基因植物；所述植物为大豆。
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| CN110527687A (zh) * | 2019-07-25 | 2019-12-03 | 南京大学 | 一种水稻转录因子基因Osspl10及其应用 |
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| EP0979873A1 (en) * | 1998-08-07 | 2000-02-16 | Director General of National Institute of Agrobiological Resources, Ministry of Agriculture, Forestry and Fisheries | Gene for petunia transcription factor PetSPL2 and its use |
| CN105695478A (zh) * | 2014-12-09 | 2016-06-22 | 中国科学院上海生命科学研究院 | 调节植物株型和产量的基因及其应用 |
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