CN114807072B - 番茄SlDAO2基因及其应用 - Google Patents
番茄SlDAO2基因及其应用 Download PDFInfo
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- CN114807072B CN114807072B CN202210631071.6A CN202210631071A CN114807072B CN 114807072 B CN114807072 B CN 114807072B CN 202210631071 A CN202210631071 A CN 202210631071A CN 114807072 B CN114807072 B CN 114807072B
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Abstract
本发明属于生物技术领域，公开了番茄SlDAO2基因及应用。本发明首次从番茄中获得番茄生长素氧化酶SlDAO2基因，并且通过利用农杆菌介导的方法将SlDAO2在番茄AC(Ailsa Craig)中过量表达，得出SlDAO2过量表达可同时促进番茄种子萌发、引起植株矮化。
Description
技术领域
本发明属于生物技术领域，具体涉及番茄SlDAO2基因在促进种子萌发和引起植株矮化中的应用。
背景技术
番茄是全世界栽培最广泛的蔬菜作物之一，也是植物科学研究的重要模式植物。
番茄作为设施种植的主要作物之一，已实现周年生产供应。集约化育苗是番茄育苗的主要方式。集约化育苗对种子质量要求较高，种子发芽率低、萌发慢、出芽不整齐，既不利于机械化播种和后期育苗的自动化、智能化管理，还会大幅增加苗期人工管理成本，增加因人工选苗、移苗、补苗而产生的人工成本，同时增加种子、基质和设施等成本，降低育苗生产效益。
株高是评价番茄壮苗的重要指标之一。集约化育苗条件下，幼苗株间距小，幼苗易徒长。徒长苗俗称“高脚苗”，主要表现为植株高，茎细弱，组织柔嫩，徒长苗根系不发达，定植后缓苗慢以至于生育推迟、坐果率及前期和总产量降低等，徒长苗抗逆性差，不适于机械化移栽，易倒伏，不利于搬运和运输。合理降低番茄苗株高对于培育番茄壮苗具有重要意义。
生长素是调控植物种子萌发和植株高度的重要激素信号。吲哚-3-乙酸(Indole-3-acetic acid，IAA)是植物内源生长素最主要的形式。IAA可被氧化形成oxIAA，进而被降解，使内源IAA含量降低。IAA双加氧酶(DIOXYGENASEASE for AUXIN OXIDATION，DAO)酶催化IAA氧化过程。DAO催化的IAA氧化降解是不可逆的过程，对于维持局部IAA稳态起着非常重要的作用。
发明内容
本发明人通过大量研究，获得番茄吲哚乙酸双加氧酶SlDAO2基因，并意外地研究发现，该基因与番茄种子萌发和植株矮化有关，从而提出本发明。
具体而言，本发明提供一种蛋白质，为如下1)或2)所示：
1)SEQ ID No.2所示的蛋白质；
2)将SEQ ID No.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且功能相同的蛋白质。
本发明还提供上述编码蛋白质的基因。
一种基因，为如下至少一种：
1)SEQ ID No.1所示的DNA分子；
2)在严格条件下与1)限定的DNA分子杂交且编码所述蛋白质的DNA分子；
3)与1)或2)限定的DNA分子具有90％以上的同一性且编码所述蛋白质的DNA分子。
本发明还提供上述含有上述任一所述基因的重组载体、表达盒、转基因细胞系或重组菌。
本发明中，SEQ ID No.2所示的蛋白质也称番茄吲哚乙酸双加氧酶SlDAO2基因所编码的蛋白质。
本发明中，SEQ ID No.1所示的DNA分子也称番茄吲哚乙酸双加氧酶SlDAO2基因。
本发明还提供一种方法，包括：使植物中上述蛋白质的表达或活性被提高，或植物中上述任一所述编码基因的表达被提高，进而在促进植物种子萌发和/或引起植物植株矮化。进一步地，所述方法包括：将含有上述基因的过量表达载体或含有上述基因的基因编辑载体导入出发植物中，得到转基因植物。
进一步地，所述方法中，所述植物为番茄。
本发明还提供上述蛋白质或上述基因在在促进植物种子萌发和/或引起植物植株矮化的应用。进一步地，所述植物为番茄。
本发明首次构建了番茄SlDAO2基因过表达和基因编辑转基因植株，并进行功能研究。通过测定种子萌发速率和株高，发现SlDAO2基因在番茄种子萌发和植株矮化中起正向调控作用。基因SlDAO2用于构建转基因番茄，过量表达的转基因番茄能够促进番茄种子萌发并引起植株矮化。
附图说明
图1是SlDAO2基因过表达载体pCAMBIA35S-4×Myc-SlDAO2载体图谱；
图2是SlDAO2基因编辑载体pTX041-SlDAO2载体图谱；
图3是SlDAO2基因过表达的株系中SlDAO2基因的表达量，图3中，WT代表未转基因的野生型番茄Ailsa Craig，SlDAO2oe-#13代表SlDAO2的一个过表达株系，SlDAO2oe-#23代表SlDAO2基因的另一个过表达株系；
图4是SlDAO2基因编辑的株系中SlDAO2的突变情况；
图5是SlDAO2基因编辑株系中SlDAO2蛋白序列与野生型番茄AC中的比较；
图6是SlDAO2基因过表达株系和基因编辑株系的种子萌发率；
图7是SlDAO2基因过表达株系和基因编辑株系的株高；
图3和图7中的星号表示与野生型对照AC(WT)比较，t-检验存在显著性差异(*P<0.05，**P<0.01)。
具体实施方式
下面结合附图对本发明的具体实施方式作进一步详细说明。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例
一、获得番茄SlDAO2基因全长序列
用Primer Premier 6.0设计全基因扩增引物，取种植在中国农业科学院蔬菜花卉研究所玻璃连栋温室中的野生型番茄Ailsa Craig(AC)的叶片cDNA为模板，设计特异性引物SlDAO2-F和SlDAO2-R，利用高保真酶2×Phanta Max Master Mix(诺唯赞)进行PCR获取SlDAO2基因cDNA片段。
引物序列为：
SlDAO2-F：5'-AAGAAGACCAACAAATAAACTCC-3'；
SlDAO2-R：5'-ATTGCTCCACCACACATA-3'
PCR扩增反应体系：2×Phanta Max Master Mix 10μl、cDNA 1μl、上下游引物各1μl、ddH2O 7μl。PCR反应程序为：94℃预变性5min；94℃变性30s，52℃退火30s，72℃延伸2min，30个循环，72℃延伸7min，冷却至4℃保存。PCR产物经琼脂糖凝胶电泳分离，切胶回收，利用琼脂糖凝胶DNA回收试剂盒(天根，DP209)进行产物回收，回收产物连接到 Zero(CB501-01，全式金)T载体上，转化大肠杆菌DH5α感受态细胞，进行菌液PCR鉴定阳性克隆，阳性克隆菌液送生工生物工程股份有限公司进行测序。
所得的基因SlDAO2的核苷酸序列如SEQ ID No.1所示；该基因编码的蛋白质的氨基酸序列如SEQ ID No.2所示。
二、SlDAO2基因过量表达载体构建
SlDAO2过表达载体的构建，以N端带有Myc标签的pCAMBIA35s-4×Myc-MCS-3×Flag质粒作为背景质粒，构建具有CaMV35S重组型过表达启动子的pCAMBIA35s-4×Myc-SlDAO2载体。
通过CE Design软件设计SlDAO2基因CDS片段插入的同源重组引物35S-SlDAO2-F和35S-SlDAO2-R，以携带SlDAO2基因CDS全长的T载体为模板，利用PCR制备含同源臂和酶切位点的CDS全长序列。重组载体构建参照Clon Ultra One Step克隆试剂盒(C115-01，诺维赞)说明书进行。将线性化载体和插入片段按比例混合，在Exnase催化作用下，50℃反应15min，立即置于冰上冷却，完成重组反应，实现两线性化DNA的体外环化。将重组质粒转化Fast-T1感受态细胞，涂板抗性(50mg/l Kan)培养基上，挑取单克隆，阳性克隆鉴定，菌液测序。测序正确的菌液可以进行重组质粒提取，即构建好过表达的载体pCAMBIA35s-4×Myc-SlDAO2(图1)。
引物序列为：
35S-SlDAO2-F：5'-cggtacccggggatccATGGAGAGTGAAAATTCAGTGCC-3'
35S-SlDAO2-R：5'-cgactctagaggatccTTACAGGTTAGTTCGCATAAGATC-3'三、SlDAO2基因编辑载体构建
根据SlDAO2基因的编码序列(SEQ ID NO.1)，利用在线软件CRISPR-P v 2.0(http://crispr.hzau.edu.cn/CRISPR2/)进行设计gRNA，设计包含2个靶点的引物Cas9-SlDAO2-P-F和Cas9-SlDAO2-P-R，利用cp043质粒为扩增模板进行PCR扩增，得到产物片段Target1_U6-26t_SlU6p_Target2，进行片段纯化回收。再利用BasI对pTX041质粒进行酶切制备线性质粒，进行纯化回收。将回收片段与pTX041线性质粒进行连接反应。连接产物转化大肠杆菌感受态DH5α，将菌液涂板至抗性LB培养基上(50mg/L Kan)培养，挑取单克隆，菌液PCR进行阳性克隆鉴定，阳性克隆菌液测序。测序正确的单克隆即为SlDAO2基因编辑载体pTX041-SlDAO2(图2)。
引物序列为：
Cas9-SlDAO2-P-F：
5'-atatatggtctcgATGGCAGAGATGAAGGAAGGTTTTAGAGCTAGAAATAGC-3'
Cas9-SlDAO2-P-R：
5'-attattggtctcgAAACACGCATTCATCATCCTGTACAAACTACACTGTTAGATTC-3'
四、番茄遗传转化
将所得的过量表达载体和基因编辑载体转化农杆菌感受态LBA4404，并通过农杆菌介导的叶盘法进行番茄遗传转化，番茄背景材料为AC，操作步骤参考Van Eck等(2019)的方法[Van Eck J,Keen P,Tjahjadi M,2019.Agrobacterium tumefaciens-MediatedTransformation of Tomato.Methods in molecular biology,1864:225-234]。
五、SlDAO2过量表达转基因株系的筛选与鉴定
将获得的转基因番茄苗通过潮霉素抗性(5mg/L)筛选和DNA水平的双重鉴定获得T0代SlDAO2过表达番茄转基因阳性株，T0代番茄阳性株单株收种，获得T1代种子。T1代种子每个株系取30-50粒播种于50孔穴盘，待苗长至两叶一心时取幼嫩真叶进行RT-PCR阳性鉴定，鉴定引物为35S-pCAMIBIA-F和35S-pCAMIBIA-R。统计植株分离比进行卡方检验，符合3:1的株系表明为单拷贝插入株系，将符合3:1株系的阳性株进行单株移栽，单株收获种子，获得T2代种子。T2代种子每个单株取30-50粒播种于1/2Hoagland固体培养基(0.75％agar，pH6.7，含7.5mg/L潮霉素)上，生长7-10d后进行表型鉴定，阳性幼苗表现为子叶大，根系长。筛选出的全抗株系随后移栽于基质中培养，培养7d后取幼嫩叶片提取进行RT-PCR鉴定，所有单株均含有目的条带的株系即为T2代纯合株系。
引物序列为:
35S-pCAMIBIA-F：5'-GAGCAGAAGTTGATTTCAGAAGAAG-3'；
35S-pCAMIBIA-R：5'-GTCATGGTCTTTGTAGTCACGC-3'；
将T2代种子播种在1/2Hoagland固体培养基上生长5d，收集下胚轴。利用RNApreppure植物总RNA提取试剂盒(DP432，天根)提取总RNA，通过1.2％凝胶电泳检测RNA的完整性及质量，利用NanoDrop One超微量紫外分光光度计(Thermo Fisher Scientific，USA)测定样品RNA浓度。采用 All-in-One First-Strand cDNA Synthesis Kit(AT341，全式金)进行反转录，采用/> Top Green qPCR SuperMix荧光染料试剂盒(AQ131，全式金)进行实时荧光定量PCR(qRT-PCR)分析，qRT-PCR使用Light />96实时PCR仪(Roche，瑞士)进行，qRT-PCR反应程序采用三步法：95℃5s，58℃10s，72℃10s，50个循环。以番茄看家基因Actin-41作为内参基因，基因相对表达量采用2-ΔΔCt法计算，所用引物为qSlDAO2-F和qSlDAO2-R，选取SlDAO2表达量显著高于野生型的株系。
引物序列为：
qSlDAO2-F：5'-TGAATGCGTTGGTGGTCTCG-3'；
qSlDAO2-R：5'-TGCTCCATACCGTGCCTATG-3'。
选取SlDAO2基因表达水平是野生型WT的4倍以上的两个过表达株系作为研究材料(图3)。图3中，WT代表未转基因的野生型番茄AC，SlDAO2-oe#3和SlDAO2-oe#23分别代表SlDAO2的一个过表达株系。
六、SlDAO2基因编辑转基因株系的筛选与鉴定
利用卡那霉素抗性筛选(出芽时50mg/L、生根时30mg/L)获得T0代抗性；取T0代抗性株幼嫩叶片，采用高效植物基因组DNA提取试剂盒(DP350-02，天根)提取基因组DNA，采用Cas9特异引物Cas9-F和Cas9-R，以基因组DNA为模板，进行PCR鉴定，得到T0代抗性株系中含Cas9的阳性株系。
所用引物序列为：
Cas9-F：5'-CACTATCCTTCGCAAGACCC-3'；
Cas9-R：5'-GAGATTCCCGAACAAGCCG-3'。
在靶点1的上下游400bp内设计特异性引物T1-check-F和T1-check-R，在靶点2的上下游400bp内设计特异性引物T2-check-F和T2-check-R。以基因组DNA为模板，进行PCR扩增，扩增产物纯化后进行测序，获得每个靶点的序列信息，将测序结果与野生型AC中SlDAO2基因序列进行比对，并利用网站DSDecodeM(skl.scau.edu.cn/dsdecode/)分析敲除类型，选取纯合体进行进一步分析。
所用引物序列为：
T1-check-F：5'-AGGTAAGAGCAAGAGCAAAAACAC-3'；
T1-check-R：5'-AGGTTACACCAATGAGCTAAACA-3'；
T2-check-F：5'-GGGCATGTTTGGGTAAAGTGTA-3'；
T2-check-R：5'-CCAGGGGTGAGGGAGTAGGA-3'。
取靶位点发生有意突变的、敲除类型为纯合体的T0代，繁种得T1代，T1代植株仅发生Cas9和Cas9-Free分离。取T1代幼苗的幼嫩叶片提取基因组DNA，利用Cas9载体特异引物Cas9-F和Cas9-R，进行PCR扩增，选取无PCR产物的株系即为Cas9-Free株系，取Cas9-Free的株系进一步利用靶点特异性引物T1-check-F、T1-check-R和T2-check-F、T2-check-R进行PCR扩增，将PCR扩增产物纯化后进行测序，进一步确认其为突变体纯合株系，将该株系移栽、收获种子，即为SlDAO2基因编辑株系dao2(图4)。
如图4所示，SlDAO2基因编辑株系dao2中，SlDAO2基因的靶位点2中缺失了4-bp，导致SlDAO2基因翻译提前终止，蛋白长度仅为192aa(图5)，SlDAO2的蛋白功能受到影响。
七、野生型和SlDAO2转基因株系的种子萌发率测定
取番茄野生型WT、过量表达转基因株系SlDAO2-oe#3、SlDAO2-oe#23和SlDAO2基因编辑株系dao2的种子，置于无菌水中浸种6h，3％NaClO消毒25min，用无菌水冲洗6遍，将种子平铺在装有浸湿的灭菌滤纸的培养皿中，置于28℃恒温进行萌发，每隔12h统计萌发种子数量，按下列公式计算种子萌发率。以胚根突出种孔0.5mm以上的种子为萌发种子。共设3次重复，每重复包括50粒种子。
结果如图6所示，SlDAO2过量表达的转基因株系SlDAO2-oe#3和SlDAO2-oe#23的种子萌发显著快于WT，种子萌发整齐度提高；SlDAO2编辑株系dao2的种子萌发最慢，且dao2种子最终的萌发率也显著低于WT(图6)。
八、野生型和SlDAO2转基因株系的株高测定
取番茄野生型WT、过量表达转基因株系SlDAO2-oe#3、SlDAO2-oe#23和SlDAO2基因编辑株系dao2的种子，置于无菌水中浸种6h，3％NaClO消毒25min，用无菌水冲洗6遍，将种子平铺在装有浸湿的灭菌滤纸的培养皿中，置于28℃恒温催芽，选取出芽整齐一致(胚根长约1cm)的种子播种于装有复合栽培基质(草炭:蛭石:珍珠岩＝3:1:1)的营养钵中，放置于中国农业科学院蔬菜花卉研究所温室中进行培养，温室培养环境条件为光照强度200μmol/m2/s,温度28±2℃/20±2℃，空气相对湿度80％～85％，自然光周期。分别于播种后14d和44d拍照，用直尺测定从基质表面到植株顶端生长点之间的长度，即为株高。
结果如图7所示，与野生型WT相比，SlDAO2过量表达株系的株高显著降低，表现出矮化表型。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
序列表
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<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gggcatgttt gggtaaagtg ta 22
<210> 18
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ccaggggtga gggagtagga 20
Claims (4)
1.一种促进番茄种子萌发的方法，包括使番茄中SEQ ID No.2所示蛋白质的表达被提高，或使番茄中SEQ ID No.1所示DNA分子的表达被提高。
2.根据权利要求1所述的方法，包括将含有SEQ ID No.1所示DNA分子的过量表达载体导入出发番茄中，得到转基因番茄。
3.过表达SEQ ID No.2所示蛋白质在促进番茄种子萌发中的应用。
4.过表达SEQ ID No.1所示DNA分子在促进番茄种子萌发中的应用。
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Non-Patent Citations (4)
| Title |
|---|
| NONE.PREDICTED: Solanum lycopersicum 2-oxoglutarate-dependent dioxygenase DAO(LOC101268251), mRNA，ACCESSION NO：XM_004233228.4.GenBank.2018,CDS、ORIGIN部分. * |
| PREDICTED: Solanum lycopersicum 2-oxoglutarate-dependent dioxygenase DAO(LOC101268251), mRNA，ACCESSION NO：XM_004233228.4;NONE;GenBank;CDS、ORIGIN部分 * |
| 光质配比和营养液耦合对番茄生长的影响;蔡华等;北方园艺(第10期);第12页左栏第1段 * |
| 小麦突变体dms的表型、遗传与差异表达基因分析;沈椿才;中国优秀硕士学位论文全文数据库 农业科技辑(第5期);第5页第2段 * |
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