CN110669785B - 番茄SlLOB40蛋白及其编码基因在调控植物抗旱性中的应用 - Google Patents
番茄SlLOB40蛋白及其编码基因在调控植物抗旱性中的应用 Download PDFInfo
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- CN110669785B CN110669785B CN201911102351.2A CN201911102351A CN110669785B CN 110669785 B CN110669785 B CN 110669785B CN 201911102351 A CN201911102351 A CN 201911102351A CN 110669785 B CN110669785 B CN 110669785B
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Abstract
本发明涉及生物技术领域，具体涉及番茄SlLOB40蛋白及其编码基因在调控植物抗旱性中的应用。本发明发现番茄SlLOB40基因能够负调控植物抗旱性，通过降低SlLOB40基因的表达量，能够有效提高植物抗旱性。SlLOB40基因的抗旱性调控功能的发现为培育高抗旱性的植物新品种提供了宝贵的基因资源和新的方法。本发明利用CRISPR‑Cas9基因组定点编辑系统突变番茄SlLOB40基因，创制了抗旱番茄株系，该番茄株系的抗旱能力显著提高，具有重要的应用价值。
Description
技术领域
本发明涉及生物技术领域，尤其涉及番茄SlLOB40蛋白及其编码基因在调控植物抗旱性中的应用。
背景技术
番茄作为一种重要的园艺作物其生长发育易受到水分亏缺的影响，在水资源日益匮乏的今天，干旱胁迫已经成为影响番茄产量和品质的重要因素之一。
研究表明，LOB基因家族生物学功能多样，其不仅具有转录调控能力，同时可以通过与其他蛋白互作从而发挥功能。LOB基因最初被发现在植物侧生器官中特异表达，与植物的形态建成和生长发育有着密切联系。研究表明，水稻中的ALR1基因编码LOB蛋白，控制着水稻不定根原基发育的起始(Liu et al.2005)；拟南芥中的AS2基因编码LOBmx蛋白可以和与AS1(MYB基因家族成员)蛋白互作形成功能复合体从而在转录水平影响叶片发育(Xu etal.2003)。LOB基因与植物激素的合成与代谢也密切相关。据报道，过表达拟南芥AtASL4基因可以激活下游BAS1基因从而调控油菜素甾醇的积累，拟南芥LOB家族基因DDA1可以响应生长素信号，其转录水平可被外源生长素诱导下调(Mangeon et al.2010)。
目前研究人员通过多种方法来提高番茄的抗旱性。研究报道，番茄中过表达SlWD6基因能够显著增强番茄对干旱和盐胁迫的耐受性(杨述章等，2015)。过量表达SlWRKY39的番茄能够通过提高抗逆基因SlRD22和SlDREB2A的表达，从而使番茄在干旱胁迫下积累更多的脯氨酸和更低含量的MDA，进而提高其抗旱性(SUN et al.,2015)。另外，有研究证明外源ABA喷施可以提高番茄品种Moneymaker的苗期抗旱性(许向阳等，2018)。提高番茄作物对干旱胁迫的耐受性，对于番茄产业的发展将会有很大的帮助。迄今为止，番茄LOB家族转录因子对干旱胁迫耐受性方面的影响机制还没有报道。
发明内容
为解决现有技术中存在的技术问题，本发明的目的在于提供番茄SlLOB40蛋白及其编码基因在调控番茄抗旱性中的应用。
为实现上述目的，本发明对番茄野生型材料进行干旱处理，提取RNA，利用荧光定量PCR筛选能够明显响应干旱的基因，分析后获得基因SlLOB40。该基因的CDS序列如SEQ IDNO.2所示，编码而成的SlLOB40蛋白的氨基酸序列如SEQ ID NO.1所示。
具体地，本发明的技术方案如下：
第一方面，本发明提供番茄SlLOB40蛋白或其编码基因或番茄SlLOB40蛋白的编码基因的抑制因子在调控植物抗旱性中的应用。
第二方面，本发明提供番茄SlLOB40蛋白或其编码基因或番茄SlLOB40蛋白的编码基因的抑制因子在植物遗传育种或转基因植物制备中的应用。
上述植物遗传育种或转基因植物制备的目的在于抗旱植物的培育。
上述应用中，可以通过降低所述番茄SlLOB40蛋白的编码基因的表达量，提高植物的抗旱性。
优选地，所述降低所述番茄SlLOB40蛋白的编码基因的表达量为使SlLOB40蛋白的编码基因失活。
本发明中，所述番茄SlLOB40蛋白具有如下任一种氨基酸序列：
(1)如SEQ ID NO.1所示的氨基酸序列；
(2)如SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的具有相同功能蛋白的氨基酸序列；
(3)与如SEQ ID NO.1所示的氨基酸序列具有至少80％同源性的氨基酸序列；优选地，所述同源性为至少90％；更优选为95％。
如SEQ ID NO.1所示的氨基酸序列为番茄SlLOB40蛋白的氨基酸序列，本领域技术人员可根据番茄SlLOB40蛋白的氨基酸序列以及氨基酸的保守性替换等本领域常规技术手段，在不影响其活性的前提下，取代、缺失和/或增加一个或几个氨基酸，得到与番茄SlLOB40蛋白具有相同功能的SlLOB40蛋白的突变体。
本发明中，所述番茄SlLOB40蛋白的编码基因的CDS序列具有如下任一种核苷酸序列：
(1)如SEQ ID NO.2所示的核苷酸；
(2)如SEQ ID NO.2所示的核苷酸序列经一个或多个核苷酸的替换、插入或缺失得到的具有相同功能蛋白的核苷酸序列。
上述如SEQ ID NO.2所示的核苷酸序列为番茄中SlLOB40蛋白的CDS序列。考虑到密码子的简并性，所有编码所述番茄SlLOB40蛋白的核苷酸序列均在本发明的保护范围内。
本发明中，所述番茄SlLOB40蛋白的编码基因的抑制因子包括能够抑制番茄SlLOB40蛋白的编码基因表达的gRNA或干扰RNA。
优选地，所述gRNA的靶序列为所述番茄SlLOB40蛋白的编码基因的第一个外显子的第59-78位。
更优选地，所述gRNA包含如SEQ ID NO.3所示的核苷酸序列。
上述gRNA的靶位点为本发明通过大量筛选获得，利用上述gRNA能够实现高效的番茄SlLOB40的敲除，使得番茄SlLOB40失活。
第三方面，本发明提供一种编辑番茄SlLOB40基因的gRNA，所述gRNA的靶序列包含所述番茄SlLOB40基因的第一个外显子的第59-78位。
上述gRNA可与CRISPR/Cas9基因编辑系统配合作用，实现番茄SlLOB40基因的高效敲除。
优选地，所述gRNA包含如SEQ ID NO.3所示的核苷酸序列。
第四方面，本发明提供包含所述用于编辑番茄SlLOB40基因的gRNA的生物材料，所述生物材料包括表达盒、载体、转基因细胞或工程菌。
所述表达盒可为含有AtU6启动子和所述gRNA的表达盒。
所述载体可为含有AtU6启动子、所述gRNA以及Cas9表达盒的CRISPR-Cas9基因编辑载体。
所述工程菌可为含有所述CRISPR-Cas9基因编辑载体的大肠杆菌或农杆菌。
第五方面，本发明提供一种调控植物抗旱性的方法，其包括：调控植物中番茄SlLOB40基因的表达量；
所述番茄SlLOB40基因的编码蛋白具有如下任一种氨基酸序列：
(1)如SEQ ID NO.1所示的氨基酸序列；
(2)如SEQ ID NO.1所示的氨基酸序列经一个或多个氨基酸的替换、插入或缺失得到的具有相同功能蛋白的氨基酸序列；
(3)与如SEQ ID NO.1所示的氨基酸序列具有至少80％同源性的氨基酸序列；优选地，所述同源性为至少90％；更优选为95％。
优选地，上述方法中，通过降低所述植物中番茄SlLOB40基因的表达量，提高所述植物的抗旱性；或者，通过将番茄SlLOB40基因的失活突变株系与其他株系杂交，培育抗旱性株系。
上述降低所述植物中番茄SlLOB40基因的表达量可通过本领域常规技术手段实现。
优选地，降低所述植物中番茄SlLOB40基因的表达量为利用CRISPR/Cas9系统实现，所述CRISPR/Cas9系统使用的gRNA的靶序列包含所述番茄SlLOB40基因第一个外显子上含有NGG序列特征的序列，具体为SlLOB40基因的第一个外显子的第59-78位。
作为本发明的优选实施方式，所述CRISPR-Cas9系统的构建方法如下：将包含AtU6启动子和如SEQ ID NO.3所示的gRNA序列的AtU6-26-sgRNA-SK质粒克隆到pCAMBIA1300-pYAO:Cas9载体中。
本发明中，所述植物为单子叶植物或双子叶植物。所述植物包括但不限于番茄、水稻、拟南芥、葡萄、大豆、黄瓜、小麦、玉米等。
本发明提供番茄SlLOB40蛋白及其编码基因在调控植物抗旱性中的应用，具备如下有益效果：
本发明通过筛选分析发现番茄SlLOB40基因能够负调控植物抗旱性，通过降低SlLOB40基因的表达量，可以能够有效提高植物抗旱性。SlLOB40基因的抗旱性调控功能的发现为培育高抗旱性的植物新品种提供了宝贵的基因资源和新的方法：一方面可通过将栽培番茄的SlLOB40基因定点突变，获得抗旱性更强的番茄品系；另一方面，也可利用本发明提供的SlLOB40基因失活植株(LOB-KO-3和LOB-KO-5)与其他番茄品种杂交，培育抗旱品系，丰富番茄抗旱种质资源。SlLOB40基因及其抑制因子在番茄抗旱性育种中具有重大的应用价值。
附图说明
图1为本发明实施例1中SlLOB40基因的基因结构以及纯合突变体植株的LOB基因靶序列突变情况，其中，KO-3和KO-5代表两个纯合的敲除株系LOB-KO-3和LOB-KO-5，WT为野生型；-表示碱基缺失；
图2为本发明实施例1中PCR筛选含有Cas9基因的转基因植株的电泳检测结果图，其中，第1泳道为转基因植株LOB-KO-3，第2泳道为转基因植株LOB-KO-5，M为DNA Mark，条带大小由上向下依次为2000bp、1000bp、750bp；
图3为本发明实施例1中PCR扩增转基因植株中SlLOB40基因的电泳检测结果图，其中，第1泳道为LOB-KO-3，第2泳道为LOB-KO-5，M为DNA Mark，条带大小由上向下依次为2000bp、1000bp、750bp；
图4为本发明实施例2中提供的SlLOB40基因敲除株系(LOB-KO-3和LOB-KO-5)和野生型(WT)材料干旱胁迫处理前后的生长状态对照图；
图5为本发明实施例2中提供的SlLOB40基因敲除株系(LOB-KO-3和LOB-KO-5)和野生型(WT)材料叶片在烘干过程中的失水率比较图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
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1基于CRISPR-Cas9系统的番茄SlLOB40基因定点突变
(1)取一定数量的番茄种子Micro Tom(南京丰硕园艺有限公司，货号3558)，将种子用2.5％的次氯酸钠浸泡20-25分钟，后用无菌水洗涤7-8次，每瓶30-40粒种子密度播于MS培养基上。在暗室培养3-4天，后光下培养3-4天。随后将番茄移至无菌水中，裁剪5*5mm的叶片方块，置于预培养培养基上，培养基上提前铺好滤纸，子叶背面朝上放置，摆放间隔以5-10mm，进行暗培养。
(2)gRNA靶点的选择
登录NCBI网站(https://www.ncbi.nlm.nih.gov/)，查询番茄SlLOB40基因的序列(CDS序列如SEQ ID NO.2所示，编码蛋白序列如SEQ ID NO.1所示)。根据SlLOB40基因序列可知，其含有两个外显子(图1)，基于CRISPR-Cas9的sgRNA序列特点，本发明通过筛选和分析选择位于SlLOB40基因的第一个外显子正义链上含有NGG序列特征的序列，具体为第59位至第78位：5’-GCATTAGGCCTTGTCTTCAA-3′(SEQ ID NO.3)，经转录后形成的RNA链可特异结合SlLOB40基因并指导CRISPR-Cas9系统对番茄SlLOB40基因进行高效的定点突变。
(3)sgRNA片段的克隆
根据选定的sgRNA靶点序列，合成引物LOB-gRNA-S(SEQ ID NO.4)和LOB-gRNA-A(SEQ ID NO.5)，将引物LOB-gRNA-S和LOB-gRNA-A通过退火方法合成双链DNA gRNA-LOB,并将该双链DNA电泳后回收。
将AtU6-26-sgRNA质粒经BsaI酶切后与DNA gRNA-LOB连接，使双链靶序列连接到质粒AtU6-26-sgRNA-SK载体上，通过T4连接的方法将包含AtU6启动子和sgRNA序列的AtU6-26-sgRNA-SK的质粒克隆到pCAMBIA1300-pYAO:Cas9载体上，获得重组载体YAO-gRNA-Cas9-LOB。
所述载体AtU6-26-sgRNA-SK，pCAMBIA1300-pYAO:Cas9来源于中国科学院遗传与发育生物学研究所。
(4)重组根癌农杆菌的获得
将步骤(3)中的重组载体YAO-gRNA-Cas9-LOB通过热激方法转化进入农杆菌GV3101菌株中，获得含有重组表达载体YAO-gRNA-Ca s9-LOB的重组农杆菌，命名为GV3101-YAO-gRNA-Cas9-LOB。
(5)农杆菌介导的番茄转化
将重组根癌农杆菌GV3101-YAO-gRNA-Cas9-LOB侵染番茄品种Micro Tom幼嫩子叶，在含除草剂抗生素培养基上成功再生及生根的植株为转基因阳性植株LOB-KO-3和LOB-KO-5。
(6)转基因番茄鉴定及突变位点检测
提取再生植株叶片总DNA，分别以1300-gRNA检测引物1300-gRNA-S(SEQ ID NO.6)和1300-gRNA-A(SEQ ID NO.7)PCR扩增1300-gRNA部分序列。再以1300-gRNA阳性植株基因组为模板运用引物SlLOB40-S(SEQ ID NO.8)和SlLOB40-A(SEQ ID NO.9)扩增SlLOB40基因全序列，送PCR产物测序，通过测序序列判断靶位点是否发生突变。
PCR扩增的电泳检测结果见图2和图3。将上述SlLOB40基因的PCR片段送测序，测序引物为SlLOB40-A，测序结果见图1。结果显示，两个转基因植株的SlLOB40基因均在PAM位点(即TGG序列)附近出现缺失突变，且两条染色体同时发生突变。LOB-KO-3植株两染色体上的SlLOB40基因第一个外显子75位发生突变，缺失一个碱基，缺失后的DNA序列如SEQ IDNO.10所示，氨基酸序列如SEQ ID NO.11所示；LOB-KO-5植株的染色体上的SlLOB40基因第一个外显子第71到76位发生突变，缺失六个碱基，缺失后的DNA序列如SEQ ID NO.12所示，氨基酸序列如SEQ ID NO.13所示。由于碱基的缺失，造成PAM位点后序列翻译错位，蛋白翻译提前终止，功能改变。
实施例2、转基因株系番茄抗旱性检测
(1)选取经测序验证为纯合的敲除株系LOB-KO-4和LOB-KO-5，对其进行干旱处理，进而分析SlLOB40在调节番茄植株抗旱中的功能。
挑选四周龄长势一致的野生型番茄株系，基因敲除株系LOB-KO-3，LOB-KO-5的幼苗。干旱处理前拍照作为对照。停止浇水后，一直观察各株系的生长状况，直到各株系之间生长情况出现较明显的差异后，拍照，结果如图4所示，与WT相比，LOB-KO-3，LOB-KO-5在干旱处理后生长状态更好。
(2)另选取经测序验证为纯合的敲除株系LOB-KO-3和LOB-KO-5，和野生型(WT)材料共同播种，待苗子长一个月左右，每个株系选取3株材料，每株选4片叶子。取下叶子后立即称重叶子的鲜重。然后将叶片放到50℃烘箱中失水。前3个小时内，每30分钟称重一次。然后在24小时时称重一次。最后烘干至重量不再变化，称量叶片的干重。叶片含水量＝鲜重-干重，每个时间点的失水率＝(叶片含水量-每个时间的叶片重量)/叶片含水量。
得到如图5所示结果，与WT相比，LOB-KO-3，LOB-KO-5在烘干过程中失水率更低，保水能力更强。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
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<400> 10
atgcgtatga gttgcaatgg ttgtagagtt cttcgtaaag gttgcagcga aagttgtagc 60
attaggcctt gtctcaatgg atcaaaaccc cggattctca gtccaacgcc actgtttttc 120
tggcaaagtt ttacggccgt gctggtctga tgaatctcat taacgctggc cctgatcatc 180
ttcgtcctgc gatatttagg tcattgcttt atgaggcttg tggaagaata gtgaacccga 240
tttatggatc ggtagggttg ttatggtcag ggaattggca gctttgtcaa aatgctgtgg 300
aggcggtact caaaggaact ccaattactc ccatagcttc tgaaattgct gtaaacaaca 360
acggtcctcc tttgaaattg ccttacgata ttaggcatat taacaaagat gaaaactcta 420
ccaagtccag cgagctgcat cgggtcagga cccgatgtcg attcaagcgc tcaggtgcta 480
acacaaaagc aaaaaactcg aatccggtat gttccgggtc gggtgatgaa ttggcccatg 540
agaaaatgaa cgggtctacg agccatgagt cttcgttgag tcaccagtct gaagaagaag 600
ctgcggcggc ggcggctgtg gctatgaatg tggaatgcga tagctcgggt atggctgaag 660
tggaagattc agctaaagat gtcgaattgg aactgacttt aggtttttcg tctttaggaa 720
ctgttgacag taaaccgaag gaaactaaac ggaataaaga tgttcagttg gtgaacggcg 780
ccggcgaatg taaaatagag cttcgacttc attga 815
<210> 11
<211> 49
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Arg Met Ser Cys Asn Gly Cys Arg Val Leu Arg Lys Gly Cys Ser
1 5 10 15
Glu Ser Cys Ser Ile Arg Pro Cys Leu Asn Gly Ser Lys Pro Arg Ile
20 25 30
Leu Ser Pro Thr Pro Leu Phe Phe Trp Gln Ser Phe Thr Ala Val Leu
35 40 45
Val
<210> 12
<211> 810
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atgcgtatga gttgcaatgg ttgtagagtt cttcgtaaag gttgcagcga aagttgtagc 60
attaggcctt aatggatcaa aaccccggat tctcagtcca acgccactgt ttttctggca 120
aagttttacg gccgtgctgg tctgatgaat ctcattaacg ctggccctga tcatcttcgt 180
cctgcgatat ttaggtcatt gctttatgag gcttgtggaa gaatagtgaa cccgatttat 240
ggatcggtag ggttgttatg gtcagggaat tggcagcttt gtcaaaatgc tgtggaggcg 300
gtactcaaag gaactccaat tactcccata gcttctgaaa ttgctgtaaa caacaacggt 360
cctcctttga aattgcctta cgatattagg catattaaca aagatgaaaa ctctaccaag 420
tccagcgagc tgcatcgggt caggacccga tgtcgattca agcgctcagg tgctaacaca 480
aaagcaaaaa actcgaatcc ggtatgttcc gggtcgggtg atgaattggc ccatgagaaa 540
atgaacgggt ctacgagcca tgagtcttcg ttgagtcacc agtctgaaga agaagctgcg 600
gcggcggcgg ctgtggctat gaatgtggaa tgcgatagct cgggtatggc tgaagtggaa 660
gattcagcta aagatgtcga attggaactg actttaggtt tttcgtcttt aggaactgtt 720
gacagtaaac cgaaggaaac taaacggaat aaagatgttc agttggtgaa cggcgccggc 780
gaatgtaaaa tagagcttcg acttcattga 810
<210> 13
<211> 23
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Arg Met Ser Cys Asn Gly Cys Arg Val Leu Arg Lys Gly Cys Ser
1 5 10 15
Glu Ser Cys Ser Ile Arg Pro
20
Claims (2)
1.一种用于编辑番茄SlLOB40基因的gRNA在提高番茄抗旱性中的作用；
所述gRNA的核苷酸序列如SEQ ID NO.3所示；
所述番茄SlLOB40基因的编码蛋白的氨基酸序列如SEQ ID NO.1所示。
2.一种用于编辑番茄SlLOB40基因的gRNA在抗旱番茄遗传育种或抗旱转基因番茄制备中的应用；
所述gRNA的核苷酸序列如SEQ ID NO.3所示；
所述番茄SlLOB40基因的编码蛋白的氨基酸序列如SEQ ID NO.1所示。
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| CN112876551B (zh) * | 2021-04-09 | 2022-09-09 | 新疆农业科学院园艺作物研究所 | 一种调控番茄耐旱性的转录因子SpbHLH89及其应用 |
| CN114457106B (zh) * | 2021-04-23 | 2023-06-20 | 山东农业大学 | 番茄基因SlCIPK7在调控植物抗旱性中的应用 |
| CN113563439B (zh) * | 2021-07-15 | 2024-06-18 | 浙江省农业科学院 | 一种果形发育相关蛋白及其编码基因与应用 |
| CN114149984A (zh) * | 2021-11-19 | 2022-03-08 | 东北农业大学 | 一种番茄抗逆基因及其编码的蛋白质和应用 |
| CN115772536B (zh) * | 2022-07-06 | 2023-07-21 | 南通大学 | 一种SsRMT4基因在调控植物抗旱性中的应用 |
| CN117088957B (zh) * | 2023-10-17 | 2024-01-02 | 山东省烟台市农业科学研究院(山东省农业科学院烟台市分院) | 番茄SlMYB13蛋白及其编码基因在调控植物耐盐、耐旱性中的应用 |
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