CN117646030A - SlRAV1基因在番茄株型改良育种中的应用 - Google Patents
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Abstract
本发明提供了SlRAV1基因在番茄株型改良育种中的应用；该SlRAV1基因的核苷酸序列如SEQ ID NO.1所示；SlRAV1基因编码的蛋白质的氨基酸序列如SEQ ID NO.2所示。本发明通过生物技术和组织培养技术构建番茄SlRAV1过表达植株，提高SlRAV1基因的表达水平，发现过表达SlRAV1基因对番茄株型发育具有显著影响，即过表达植株与野生型相比，侧枝生长显著被抑制，叶夹角的开度变小，株型更紧凑。
Description
技术领域
本发明涉及植物基因工程技术领域，尤其涉及SlRAV1基因在番茄株型改良育种中的应用。
背景技术
番茄(Solanum lycopersicum L.)是世界上重要的蔬菜作物之一，在我国及世界范围内广泛种植。近年来，设施园艺的面积快速增加，鲜食番茄生产从露地栽培逐渐转向设施栽培为主。在设施番茄栽培中除了扩大番茄种植规模和改善栽培条件外，设施栽培专用的理想株型番茄品种选育工作正在广泛开展。番茄作为喜光蔬菜作物，株型对其群体光合作用的影响尤为显著。良好的株型不仅可以改善设施番茄受光面积，而且便于管理，有利于养分的合理分配，提高肥料和水分的利用效率等。
株型作为植物的形态特征，主要包含植株的高度、叶片大小、叶夹角、以及侧枝发育等植物学性状。侧枝是植物株型的重要方面，侧枝发育受到自身遗传因子，内源激素如生长素、细胞分裂素、独脚金内酯和油菜素内酯以及环境等诸多因素影响。随着设施生产环境的不断改善，番茄侧枝生长旺盛，需要人工整枝调整番茄株型，才能实现光合产物的合理分配。但是当前农业劳动力短缺，番茄人工整枝显著提高生产成本。因此，亟待创制侧枝少的番茄新种质。
叶夹角作为株型的构成因素主要影响株幅，通常叶夹角越小，株幅越小，有利于通过提高种植密度增加群体光合作用，从而提升单位面积番茄产量。目前株型研究集中在小麦、水稻和玉米等大田作物上。但是番茄株型方面的研究较少。佟友丽(佟友丽.番茄株型特性的研究[博士学位论文].沈阳农业大学，2001)筛选出叶形差异较大的番茄品系，并对其进行研究。结果表明，上举型番茄的株型紧凑，能更好地适应环境，同时果实产量也随之提高。
RAV(Related to ABI3/VP1)是植物特有的转录因子，属于AP2/ERF转录因子家族中的亚家族。已在玉米和水稻中报道RAV转录因子具有调控株型发育的功能。玉米RAVL1基因敲除株系的叶夹角减小，株型紧凑，有利于提高玉米产量(Tian J,Wang C,Xia J,etal.Teosinte ligule allele narrows plant architecture and enhances high-density maize yields.Science,2019,365:658-664.)。水稻RAVL1基因敲除株系的叶夹角变小，植株半矮化；过表达株系的叶夹角则变大(Je BI,Piao HL,Park SJ,et al.RAV-Like1 maintains brassinosteroid homeostasis via the coordinated activation ofBRI1 and biosynthetic genes in rice.Plant Cell,2010,22:1777-1791.)。而拟南芥RAV1基因过表达株系的侧根和莲座叶数量明显减少(Hu YX,Wang YX,Liu XF,etal.Arabidopsis RAV1 is down-regulated by brassinosteroid and may act as anegative regulator during plant development.Cell Research,2004,14:8-15.)。
截至目前，SlRAV1基因所属的RAV亚家族在调控番茄株型方面仍未有研究报道。
发明内容
本发明提供了一种SlRAV1基因在番茄株型改良育种中的新应用，丰富了番茄侧枝生长发育的分子调控理论，为番茄种质资源创新，改良番茄株型，实现高产优产提供参考依据。
具体技术方案如下：
本发明提供了SlRAV1基因在番茄株型改良育种中的应用，所述SlRAV1基因的核苷酸序列如SEQ ID NO.1所示。
进一步地，所述SlRAV1基因编码的蛋白质的氨基酸序列如SEQ ID NO.2所示。本发明通过构建SlRAV1基因过表达及基因编辑突变体，发现SlRAV1基因具有调控番茄侧枝生长发育的作用。
具体的，所述调控的方式为以下之一：
(i)通过过表达SlRAV1基因，减小番茄叶夹角的开度，抑制番茄侧枝的形成，从而获得侧枝较少、株型较为紧凑的株系；
(ii)通过敲除SlRAV1基因，番茄侧枝长度增加，番茄叶夹角的开度变大，株型较为松散。
其中，构建过表达SlRAV1基因的突变体的方法为：
(1)构建含有番茄SlRAV1基因CDS序列的过表达载体，CDS序列如SEQ ID NO.3所示；
(2)将所述载体转入农杆菌感受态细胞中，获得含SlRAV1基因过表达载体的农杆菌；
(3)利用所述农杆菌侵染普通野生型番茄的子叶，通过组织培养重新获得幼苗，筛选获得过表达SlRAV1基因的阳性转基因植株。
进一步地，所述载体为具有35S启动子的pAC007-FLAG；宿主细胞为农杆菌GV3101。
其中，敲除番茄SlRAV1基因的方法为：
(1)设计SlRAV1基因的两个靶序列sgRNA，构建番茄SlRAV1基因编辑的CRISPR/Cas9载体；
(2)将所述载体转入农杆菌感受态细胞中，获得CRISPR/Cas9载体的农杆菌；
(3)利用含SlRAV1基因编辑载体的农杆菌侵染液侵染普通野生型番茄的子叶，通过组织培养重新获得幼苗，筛选获得SlRAV1基因缺失的突变体植株。
进一步地，步骤(1)中，所述靶序列sgRNA如SEQ ID NO.4和SEQ ID NO.5所示。
与现有技术相比，本发明具有以下有益效果：
本发明通过生物技术和组织培养技术构建番茄SlRAV1过表达植株，提高SlRAV1基因的表达水平，发现过表达SlRAV1基因对番茄株型生长具有显著影响，即过表达植株与野生型相比，侧枝生长显著被抑制，叶夹角的开度变小，株型更紧凑。
附图说明
图1为实施例1中SlRAV1基因纯合突变体植株基因编辑位点及氨基酸变化；
其中，WT为普通野生型番茄品种Condine Red，rav1-1#和rav1-2#为SlRAV1基因敲除突变体的两个株系，rav1-1#株系在基因编辑靶点一处发生了1个碱基的插入并在靶点二处发生了4个碱基的缺失，导致翻译提前终止；rav1-2#株系在基因编辑靶点一处发生了1个碱基的插入并在靶点二处发生了7个碱基的缺失，也导致翻译提前终止。
图2为实施例2中SlRAV1基因过表达番茄株系中转基因蛋白的Western Blot检测结果；
其中，WT为野生型番茄(Solanum lycopersicum cv Condine Red)；OE-RAV1-1#和OE-RAV1-2#为SlRAV1过表达植株；anti-FLAG为检测FLAG标签的特异性抗体；anti-ACTIN为检测ACTIN内参的特异性抗体，作为控制蛋白上样量的标准。
图3为实施例3中野生型番茄植株、SlRAV1基因敲除突变体和SlRAV1基因过表达植株的叶夹角表型；
其中，a为植株叶夹角表型；b为第一节位叶夹角开度的统计；c为第二节位叶夹角开度的统计；WT为野生型番茄；rav1-1#和rav1-2#为SlRAV1基因敲除突变体；OE-RAV1-1#和OE-RAV1-2#为SlRAV1过表达植株。
图4为实施例3中SlRAV1基因敲除突变体和SlRAV1基因过表达植株的侧枝长度；
其中，a为植株侧枝表型；b为植株自下而上五个节位的侧枝总长度。
具体实施方式
下面结合具体实施例对本发明作进一步描述，以下列举的仅是本发明的具体实施例，但本发明的保护范围不仅限于此。应理解，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。
SEQ ID NO.1：
ATGGAGGTAAGTTGCATAGATCAAGAAATTAGCACAACTAGTGACTCCCCATCAACCATTTCCCCGGCGGGGTCTCCCCGCCAGATGGGAAGTGGAGTCAGCGTTGTAACAGACGCTGACTCCACAAACGGTGTAGAAGCAGAGTCGAAAAATACTACTATTACTAATAATAACAACAATAAGCTTCCTTCTTCAAAGTTCAAAGGTGTTGTCCCTCAACCAAATGGTCGATGGGGTGCACAAATCTACGAAAAACATCAAAGGATTTGGCTTGGTACGTTCAATGGCGAAGAAGAAGCAGCAAGAGCTTATGATATCGCGGCACAGAGATTCCGTGGTAGAGATGCCGTAACAAATTTCAAACCCCTGTTTGATTTTCAAACAGAGGAAATCGAGATCTCATTTTTGAACTCTCGTTCTAAGGTTGAGATAGTTGAAATGTTACGTAAACACACGTATAATGACGAACTTCAACATGCCAAGAAAGTATATAACAACAATACTTTCGAGATGAATGGAAGGAGCACATGCCATACTAATACTAATATCGCGACTTTTTCATTAGATACTAATGAAAAAGTCGCGAATATAGCTAGTGAACTGCTTTTTGAAAAAGTAGTTACACCGAGCGACGTTGGAAAACTTAATAGGCTTGTTATTCCAAAACAACACGCAGAGAGATATTTCCCTTTGGTTGCCAAAGTAAATAAAAATGATAATACTTCTAAAGGGGTATTATTAAATTTCGAAGATATGAATGGGAAAATGTGGCGATTTCGATATTCGTATTGGAATAGTAGTCAAAGTTACGTATTAACTAAAGGATGGAGTCGTTACGTTAAGGAAAAAAAGTTGAAGGCCGGAGATATAGTGAGTTTCAAACGATGTTCTGGAGTTGAAATCGAAGATAAACTTCTTATTGATTCGAAGAACAGAATAATACGAGGCGAACAAGTAAAAATGGTTAGATTATTTGGAGTGAATATATGTAAGGTTCAAGATGTTAGTAATAATGTTGTTGTTGAGAAAAATATGAGTGTTGGCAAAAGAATGAGGGAGATGGAGTTGTTGGCATTTGAGTGTAGCAAGAAACAAAGGGTAATAATTGATGCCTTGTGA
SEQ ID NO.2：
MEVSCIDQEISTTSDSPSTISPAGSPRQMGSGVSVVTDADSTNGVEAESKNTTITNNNNNKLPSSKFKGVVPQPNGRWGAQIYEKHQRIWLGTFNGEEEAARAYDIAAQRFRGRDAVTNFKPLFDFQTEEIEISFLNSRSKVEIVEMLRKHTYNDELQHAKKVYNNNTFEMNGRSTCHTNTNIATFSLDTNEKVANIASELLFEKVVTPSDVGKLNRLVIPKQHAERYFPLVAKVNKNDNTSKGVLLNFEDMNGKMWRFRYSYWNSSQSYVLTKGWSRYVKEKKLKAGDIVSFKRCSGVEIEDKLLIDSKNRIIRGEQVKMVRLFGVNICKVQDVSNNVVVEKNMSVGKRMREMELLAFECSKKQRVIIDAL*
SEQ ID NO.3：
ATGGAGGTAAGTTGCATAGATCAAGAAATTAGCACAACTAGTGACTCCCCATCAACCATTTCCCCGGCGGGGTCTCCCCGCCAGATGGGAAGTGGAGTCAGCGTTGTAACAGACGCTGACTCCACAAACGGTGTAGAAGCAGAGTCGAAAAATACTACTATTACTAATAATAACAACAATAAGCTTCCTTCTTCAAAGTTCAAAGGTGTTGTCCCTCAACCAAATGGTCGATGGGGTGCACAAATCTACGAAAAACATCAAAGGATTTGGCTTGGTACGTTCAATGGCGAAGAAGAAGCAGCAAGAGCTTATGATATCGCGGCACAGAGATTCCGTGGTAGAGATGCCGTAACAAATTTCAAACCCCTGTTTGATTTTCAAACAGAGGAAATCGAGATCTCATTTTTGAACTCTCGTTCTAAGGTTGAGATAGTTGAAATGTTACGTAAACACACGTATAATGACGAACTTCAACATGCCAAGAAAGTATATAACAACAATACTTTCGAGATGAATGGAAGGAGCACATGCCATACTAATACTAATATCGCGACTTTTTCATTAGATACTAATGAAAAAGTCGCGAATATAGCTAGTGAACTGCTTTTTGAAAAAGTAGTTACACCGAGCGACGTTGGAAAACTTAATAGGCTTGTTATTCCAAAACAACACGCAGAGAGATATTTCCCTTTGGTTGCCAAAGTAAATAAAAATGATAATACTTCTAAAGGGGTATTATTAAATTTCGAAGATATGAATGGGAAAATGTGGCGATTTCGATATTCGTATTGGAATAGTAGTCAAAGTTACGTATTAACTAAAGGATGGAGTCGTTACGTTAAGGAAAAAAAGTTGAAGGCCGGAGATATAGTGAGTTTCAAACGATGTTCTGGAGTTGAAATCGAAGATAAACTTCTTATTGATTCGAAGAACAGAATAATACGAGGCGAACAAGTAAAAATGGTTAGATTATTTGGAGTGAATATATGTAAGGTTCAAGATGTTAGTAATAATGTTGTTGTTGAGAAAAATATGAGTGTTGGCAAAAGAATGAGGGAGATGGAGTTGTTGGCATTTGAGTGTAGCAAGAAACAAAGGGTAATAATTGATGCCTTGTGA
实施例1 SlRAV1基因敲除植株的获得
1、SlRAV1基因CRISPR/Cas9基因编辑载体的构建
从网站NCBI(http://www.ncbi.nlm.nih.gov/)数据库中搜索获得番茄SlRAV1的基因序列，使用CRISPR-P(https://www.genome.arizona.edu/crispr/CRISPRsearch.html)设计SlRAV1基因sgRNA(small guide RNA)；
靶点一：CTCCCCATCAACCATTTCCC(SEQ ID NO.4)，靶点二：ACAGACGCTGACTCCACAAA(SEQ ID NO.5)。
图1为两个sgRNA在番茄SlRAV1基因上的位置和序列。
载体具体构建过程：首先设计分别包含靶点一和靶点二的PCR扩增引物，并以质粒Y0014472-1为模板分别进行两个靶点的PCR片段扩增，具体引物序列见表1。
扩增后的两个PCR产物通过Golden Gate组装试剂盒(BsaI-HFv2)(NEB，E1601)连接到含有内切酶BbsI位点的pAEE401质粒上。所得载体pAEE401-SlRAV1经测序确认是否构建成功。
表1构建CRISPR/Cas9载体的PCR引物序列
2、SlRAV1基因CRISPR/Cas9基因编辑植株的获取
通过电击法获得转入重组质粒pAEE401-SlRAV1的农杆菌，农杆菌感受态为GV3101，通过农杆菌侵染野生型番茄子叶，侵染后的子叶再经过植物组织培养发育成为完整的转基因番茄植株。
随后根据靶点位置设计验证引物，分别为cri-rav1-F：ACAATACCCACATCTCAATATCTTC(SEQ ID NO.10)和cri-rav1-R：CCAATACGAATATCGAAATCGCC(SEQ ID NO.11)。
提取转基因植株DNA，利用PCR技术扩增，并进行测序验证。所获得的突变类型分别为插入1个碱基和缺失4个碱基的rav1-1#、插入1个碱基和缺失7个碱基的rav1-2#的杂合T0代植株，之后自交得到T1代纯合rav1-1#、rav1-2#植株(图1)。
实施例2 SlRAV1基因过表达植株的获得
1、SlRAV1基因过表达载体的构建
为创制侧枝少、株型紧凑的番茄新种质，探究SlRAV1基因在番茄株型调控上的功能，从番茄基因组数据库中下载了SlRAV1基因序列，其代码为Solyc04g007000。
根据SlRAV1编码区序列、过表达载体pAC007-FLAG的多克隆位点及其附近序列，选取限制性酶切位点(AscI和BamHI)并设计同源臂，使用软件CE DesignV1.03生成特异性引物SlRAV1-F(ttacaattaccatggggcgcgccatggaggtaagttgcatagatcaaga)和SlRAV1-R(gtccttatagtccatggatcccaaggcatcaattattaccctttg)，序列分别如SEQ ID NO.12和SEQ IDNO.13所示。
用KOD高保真酶PCR扩增SlRAV1片段并纯化回收PCR产物，对pAC007-FLAG载体进行双酶切并纯化回收酶切后线性载体，然后用诺唯赞同源重组酶Exnase II将SlRAV1片段连接到pAC007-FLAG上，得到植物过表达载体pAC007-SlRAV1-FLAG。该表达载体在植物体内成功表达后会产生SlRAV1-FLAG融合蛋白。将上述重组质粒送到公司测序确认，所得基因SlRAV1的核苷酸序列如SEQ ID NO.1所示；该基因编码蛋白质的氨基酸序列如SEQ ID NO.2所示。
结果表明，所克隆的序列与数据库中公布的序列一致，提取阳性质粒备用。
2、SlRAV1基因过表达植株的获取
将阳性过表达载体质粒pAC007-SlRAV1-FLAG电击转化农杆菌GV3101。以普通番茄品种Condine Red作为野生型(WT)，通过子叶侵染法进行农杆菌侵染，然后通过调整激素比例相继诱导愈伤组织、芽分化和生根，最终获得T0代组培苗。利用Western Blot验证SlRAV1过表达阳性转基因植株，结果显示WT没有蛋白条带，而过表达株系有SlRAV1-FLAG条带(图2)。
实施例3
1、SlRAV1基因敲除植株和SlRAV1基因过表达植株的叶夹角
以野生型番茄WT、SlRAV1基因敲除植株和SlRAV1基因过表达植株为实验材料。
将种子在50℃左右恒温浸种15min，之后在28℃恒速摇床(200rpm/min)培养2天左右，期间约12h换水一次，待种子胚根露白后，播种于装有草炭和蛭石3：1混合的72孔穴盘中。穴盘苗生长于植物工厂，其光周期为12h/12h，环境温度为21℃/19℃，相对湿度为75％左右，平均光强为200μmol·m-2s-1，营养液为1/2Hoagland营养液。待幼苗长到三叶一心时，将单株移栽到塑料盆中培养，培养条件同上。待WT植株生长至五叶一心时(要求侧枝未长出)统计叶夹角的表型，每组处理10个生物重复。
结果：SlRAV1基因敲除番茄突变体叶夹角的开度显著大于野生型；与之相反，SlRAV1基因过表达植株叶夹角的开度显著小于野生型(图3)。
2、SlRAV1基因敲除突变体和SlRAV1基因过表达植株的侧枝长度
待野生型植株生长至七叶一心时，统计侧枝的生长表型。侧枝长统计以叶腋处为起始位置，末端为侧枝顶端，共测量从下往上数5个节位，每组处理10个生物重复。
结果：SlRAV1基因过表达番茄植株侧枝生长显著受抑制；然而，SlRAV1基因敲除番茄突变体侧枝长度增加(图4)。
综合以上研究，本发明发现了过量表达SlRAV1基因抑制番茄侧枝发生，并使番茄叶夹角开度变小，有利于创制株型紧凑的番茄新种质。
此外应理解，在阅读了本发明的上述描述内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (7)
1.SlRAV1基因在番茄株型改良育种中的应用，其特征在于，所述SlRAV1基因的核苷酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的应用，其特征在于，所述SlRAV1基因编码的蛋白质的氨基酸序列如SEQ ID NO.2所示。
3.如权利要求1所述的应用，其特征在于，所述调控的方式为以下之一：
(i)通过过表达SlRAV1基因，减小番茄叶夹角的开度，抑制番茄侧枝的形成，从而获得侧枝较少、株型较为紧凑的株系；
(ii)通过敲除SlRAV1基因，番茄侧枝长度增加，番茄叶夹角的开度变大，株型较为松散。
4.如权利要求3所述的应用，其特征在于，构建过表达SlRAV1基因的突变体的方法为：
(1)构建含有番茄SlRAV1基因CDS序列的过表达载体，CDS序列如SEQ ID NO.3所示；
(2)将所述载体转入农杆菌感受态细胞中，获得含SlRAV1基因过表达载体的农杆菌；
(3)利用所述农杆菌侵染普通野生型番茄的子叶，通过组织培养重新获得幼苗，筛选获得过表达SlRAV1基因的阳性转基因植株。
5.如权利要求3所述的应用，其特征在于，敲除番茄SlRAV1基因的方法为：
(1)设计SlRAV1基因的两个靶序列sgRNA，构建番茄SlRAV1基因编辑的CRISPR/Cas9载体；
(2)将所述载体转入农杆菌感受态细胞中，获得CRISPR/Cas9载体的农杆菌；
(3)利用含SlRAV1基因编辑载体的农杆菌侵染液侵染普通野生型番茄的子叶，通过组织培养重新获得幼苗，筛选获得SlRAV1基因缺失的突变体植株。
6.如权利要求5所述的应用，其特征在于，所述载体为具有35S启动子的pAC007-FL AG；宿主细胞为农杆菌GV3101。
7.如权利要求5所述的应用，其特征在于，步骤(1)中，所述两个靶序列sgRNA如SEQ IDNO.4和SEQ ID NO.5所示。
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