CN113563439A - 一种果形发育相关蛋白及其编码基因与应用 - Google Patents
一种果形发育相关蛋白及其编码基因与应用 Download PDFInfo
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- CN113563439A CN113563439A CN202110803562.XA CN202110803562A CN113563439A CN 113563439 A CN113563439 A CN 113563439A CN 202110803562 A CN202110803562 A CN 202110803562A CN 113563439 A CN113563439 A CN 113563439A
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Abstract
本发明公开了一种果形发育相关蛋白及其编码基因与应用。过表达本发明所示的DNA分子(靶基因是LsFS1基因),获得LsFS1基因过表达的转基因番茄植株,转基因番茄植株果实由圆变长,果形指数明显变大且LsFS1基因表达量上调。本发明首次鉴定了与瓠瓜果形发育相关蛋白LsFS1,其编码基因在功能过量表达的条件下会引起番茄果形变长的表型,证明瓠瓜果形发育相关蛋白LsFS1或其基因在控制植物果形发育方面发挥重要作用。本发明不仅为瓠瓜果型改良提供新的基因资源,还可以扩展我们对蔬菜作物果形发育调控方面的认知。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种果形发育相关蛋白及其编码基因与应用。
背景技术
瓠瓜[Lagenaria siceraria(Molina)Standl.](2n=2x=22)又称蒲瓜、长瓜、地蒲、瓠子、葫芦等,为葫芦科葫芦属一生年草本植物,具有重要园艺和药用价值。瓠瓜主要以嫩果作菜用,是我国南方地区重要的特色瓜类蔬菜作物之一,瓠瓜栽培效益较高,亩产值可达1.5-3万元。
瓠瓜果形变异丰富,除长棒形、近圆形、梨形、短圆筒形等葫芦科作物常见果实形状外,还包含有长颈瓶形、葫芦形等特有形状,是研究果实形状的理想材料。近年来,一些在葫芦科作物果形发育中发挥调控作用的基因陆续被克隆和研究,但目前尚无调控瓠瓜果形相关基因报道,开展瓠瓜果形发育相关基因挖掘和调控机制研究,有助于培育外观品质佳的瓠瓜新品种,对瓠瓜产业的提质增效意义重大,同时也可为其他葫芦科作物乃至其他蔬菜作物相关研究提供良好的借鉴。
发明内容
本发明的目的在于克服现有技术缺陷,并提供一种果形发育相关蛋白及其编码基因与应用。
本发明所提供的果形发育相关蛋白,名称为LsFS1,来源于瓠瓜[Lagenariasiceraria (Molina)Standl.]地方品种杭州长瓜的氨基酸序列,其包含以下蛋白质:
a)如SEQ ID NO.1的氨基酸残基序列组成的蛋白质;
或
b)如SEQ ID NO.1的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物果形发育相关由a)衍生的蛋白质。
上述b)蛋白质中发生氨基酸残基取代和/或缺失和/或添加的总数不超过10个。
其中,在某些实施方法中,上述b)中的蛋白质可人工合成,也可先合成其编码基因,再进行生物表达得到。
上述如SEQ ID NO.1所示蛋白质由510个氨基酸残基组成。
通常情况下,所述蛋白质使植物果实形状变长。
而且所述植物包括但不限于番茄。
编码所述LsFS1蛋白的DNA分子也属于本发明的保护范围。
所述DNA分子包含如下(1)或(2)或(3)的DNA分子。
(1)如SEQ ID NO.2所示的DNA分子或者其互补DNA分子;
(2)在严格条件下与1)限定的DNA序列杂交且与植物果形发育相关的DNA 分子;
(3)与(1)或(2)限定的DNA序列至少具有70%、至少具有75%、至少具有 80%、至少具有85%、至少具有90%、至少具有95%、至少具有96%、至少具有97%、至少具有98%或至少具有99%同源性且与植物果形发育相关的DNA分子。
所述严格条件可为在0.1×SSPE(或0.1×SSC),0.1%SDS的溶液中,在65℃下杂交并洗膜。
上述如SEQ ID NO.2所示的DNA分子由1533个核苷酸组成。
含有所述编码基因的重组载体、重组菌或转基因细胞系均属于本发明的保护范围。
所述带有所述编码基因的重组载体可以是过表达载体。
在某些具体实施方法中,所述过表达载体是将如SEQ ID NO.2所示的DNA片段1 分子插入双元表达载体中,所述DNA片段1如SEQ ID NO.2自5’末端第1至1530位核苷酸所示;
上述所述过表达载体具体为pCAMBIA1305.1-GFP载体。
扩增所述基因全长或者其任意片段的引物对也属于本发明保护的范围。
本发明的另一个目的是提供一种培育长果形番茄的方法,为在番茄中过表达瓠瓜LsFS1蛋白编码基因,得到转基因番茄。
上述所述长果形番茄的转基因植物具体体现在得到的转基因番茄果形由圆变长。
上述过表达是通过在所述目的植物中转入过表达载体实现的。
所述蛋白在调节果实发育方面的应用也是本发明的保护范围。
本发明的实验证明,本发明克隆得到LsFS1基因,过表达LsFS1基因的番茄植株出现果实由圆变长表型,证明LsFS1蛋白在植物果形发育过程中发挥重要作用。
本发明具有以下有益效果:可以基于本申请的基因和蛋白质,创制不同形状的植物品种材料,作为作物育种的素材,例如番茄等。
附图说明
图1为LsFS1蛋白所编码基因定位结果。
图2为LsFS1过表达植株的表型。
图3为过表达阳性植株果形数据统计和基因转录水平检测。
具体实施方式
下面结合具体实施例对本发明进行进一步的详细描述,给出的实施例仅为了更好的阐明本发明,而不限定本发明。
下述实施例中的实验方法,如无特殊说明,均为常规方法。
下述实施例中所用的材料、生化试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中的瓠瓜[Lagenaria siceraria(Molina)Standl.]地方品种杭州长瓜,公众可从浙江省农业科学院蔬菜研究所获得,该生物材料只为重复本发明的相关实验所用,不可作为其它用途使用。
农杆菌为根癌农杆菌GV3101,公众可从商业途径或浙江省农业科学院蔬菜研究所获得。
实施例1.LsFS1基因的获得
1.LsFS1基因的定位
以长棒形瓠瓜和近圆形瓠瓜为亲本,通过杂交获得了一个包含150个株系的F2分离群体,利用复合区间作图(CIM)、BSA、功能作图(GM-FM)等三种方法,在第6染色体检测到一个高LOD值的主效QTL,如图1所示。在此区间中共有13 个基因,无已知果形基因同源基因。我们以两亲本为模板,对13个基因全部测序,发现仅在HG_GLEAN_10009435基因外显子上两亲本存在4个碱基的差异并导致了蛋白翻译提前终止,故把该基因编码蛋白作为新的瓠瓜果形发育相关蛋白,命名为 LsFS1蛋白,其序列如SEQ ID NO.1所示,其对应的编码基因命名为LsFS1基因,其开放阅读框CDS序列如SEQ ID NO.2所示。
2.LsFS1基因的克隆
根据瓠瓜数据库GourdBase(http://www.gourdbase.cn/),设计引物LsFS1-CDS-F和LsFS1-CDS-R。
LsFS1-CDS-F:ATGGGAAAATTAGAATGGC
LsFS1-CDS-R:GAAATTATGAAAATGAGAACAT
用植物总RNA提取试剂盒(购于北京天根生化科技有限公司),提取瓠瓜杭州长瓜的开花前7天左右的幼嫩子房总RNA,反转成cDNA。以此为模板,以 LsFS1-CDS-F和LsFS1-CDS-R为引物进行扩增,扩增的PCR产物对应的基因命名为LsFS1,该基因的编码区为SEQ IDNO.2所示的核苷酸;该基因编码的蛋白命名为LsFS1,氨基酸序列为如SEQ ID NO.1所示。
实施例2.LsFS1过表达载体的构建
1.LsFS1基因加接头片段的获得
以实例1中扩增的PCR产物为模板,用LsFS1-GFP-InXbaI-InF和 LsFS1-GFP-InBamHI-InR引物进行扩增,得到片段2。
LsFS1-GFP-InXbaI-InF:CGGAGCTAGCTCTAGAATGGGAAAATTAGAATGGC
LsFS1-GFP-InBamHI-InR:TGCTCACCATGGATCCGAAATTATGAAAATGAGAAC
2.LsFS1过表达载体的构建(重组载体pCAMBIA1305.1-GFP-LsFS1)
用限制性内切酶XbaI和BamHI双酶切过表达载体pCAMBIA1305.1-GFP,得到线性表达载体,回收该线性片段。将片段2采用同源重组的方法重组到该线性表达载体上(具体方法参考clontech infusion kit说明书),挑取单克隆,测序正确后得到重组载体pCAMBIA1305.1-GFP-LsFS1。
实施例3.过表达植株的获得与鉴定
一.过表达转基因植株的获得
1.将重组载体pCAMBIA1305.1-GFP-LsFS1用热激法转入根癌农杆菌GV3101感受态中,得到的含有重组载体的根癌农杆菌命名为 GV3101/pCAMBIA1305.1-GFP-LsFS1。
2.将GV3101/pCAMBIA1305.1-GFP-LsFS1农杆菌侵染番茄品种micro-Tom的愈伤组织。经过无菌苗获得、外植体制备、农杆菌扩繁、农杆菌侵染和共培养、芽诱导分化以及生根及抗性植株筛选等一系列过程,待植株地上高度长至10cm左右时,开瓶口历练1天,然后清洗掉培养基,移栽至光照培养箱培养,即为T0代转基因植株。 T0代转基因植株自交收获种子并种植,得到稳定的T1代转基因植株。
二.过表达转基因植株的PCR鉴定
采用CTAB法提取上述获得的T1代转基因植株和野生型番茄品种micro-Tom植株的幼苗(以下简称为WT)的基因组DNA,分别以待测T1代转基因植株、WT、超纯水、pCAMBIA1305.1-GFP-LsFS1质粒为模板,以LsFS1-检测-F (5’-CACTATCCTTCGCAAGACCCT-3’)和LsFS1-检测-R (5’-GTGCCAACAGTTCAGTAGTCGTT-3’)为引物,进行PCR扩增,WT、超纯水扩增不出,待测T1代转基因植株扩增片段产物大小为1384bp且测序正确的植株为阳性苗。
三.过表达转基因植株的表型鉴定
分别将T1代转基因植株阳性苗和WT植株种植于人工智能气候室中(28℃光照14h,24℃黑暗10h,湿度70%),待长至盛果期,观察果实形状表型是否有差异。如图2所示,与WT植株果实相比,T1代转基因植株阳性苗植株果实由圆变长。而且,通过测量LsFS1的基因表达量以及果形指数可知,随着表达量的增加,果形指数明显变大,如图3所示。其中,果形指数是指果实纵径与横径的比值。从而证明了LsFS1蛋白影响植物果形发育,过表达其编码基因,番茄植株出现果实由圆变长表型。
四.过表达转基因植株的LsFS1基因表达水平的鉴定
分别提取3株T1代转基因植株阳性苗和WT植株叶片的RNA,以番茄actin为内参,设计内参引物actin-RT-F和actin-RT-R,以及LsFS1基因特异性定量引物 LsFS1-RT-F和LsFS1-RT-R进行荧光定量PCR。结果显示,与WT植株相比,阳性 T1代转基因植株中LsFS1基因表达水平显著上调,如图3所示。上述引物如下:
actin-RT-F:5’-TCCCTGGTATTGCTGATAGG-3’
actin-RT-R:5’-TGGAATGTGCTGAGAGAGG-3’
LsFS1-RT-F:5’-CAATCGCTATCCGTCCTCCT-3’
LsFS1-RT-R:5’-CAAGCTGTTCCTTGGTGCTT-3’。
序列表
<110> 浙江省农业科学院
<120> 一种果形发育相关蛋白及其编码基因与应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 510
<212> PRT
<213> 瓠瓜(Lagenaria siceraria)
<400> 1
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Ala Thr Val Asp His Ser Arg Arg His Arg Arg Pro Ser Leu Pro Ser
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Cys Met Thr Thr Leu Phe His Phe Phe Asp Phe Arg Ser Ser Arg Phe
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Thr Cys Ile Val Phe Asp Asn Arg Tyr Pro Ser Ser Phe Asp Leu Ser
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Pro His Pro Pro Ser Leu Pro Lys Ala Ser Tyr His Gly Val Glu Ala
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Pro Arg Asn Ser Leu Glu Leu Asp Gly Ala Ser Ile Pro Cys Leu Arg
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Ile Lys Glu Glu Asn Leu Gln Leu Gln Met Gly Leu Gln Ile Lys Thr
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Arg Asn Gly Cys Thr Lys Pro Lys Ala Ser Glu Gln Gln Leu Pro Asn
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Asn Asp His Ile Ile Ala Leu Glu Ser Pro Ser Ala Lys Arg Pro Asn
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Leu Leu Ala Arg Leu Met Gly Leu Asp Ile Leu Pro Gln Thr Ser Ser
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Ser Ser Ser Asn Ser His Gly Pro Asn Tyr Gly Thr Arg Ser Leu Pro
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Glu Ser Pro Arg Ile Ser Ser Ala Arg Leu Ser Asp Val Asp Tyr His
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Arg Arg Leu Ser Leu Gln Ile Ile Pro Glu Lys Glu Asn Ile Glu Ile
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Cys Thr Glu Glu Ile Lys Gln Glu Lys Glu Lys Val Arg Arg Lys Val
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Ala Leu Val Asp Ile Thr Asn Asn Asn Lys Lys Thr Glu Phe Gly Lys
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Gln Glu Val Gly Ile Ser Gln Ser Lys Val Glu Ile Lys Ser Asn Lys
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atgggaaaat tagaatggca ctttggagga agatcatttt cacgtcgagc caccgtcgat 60
cactcccgac gacaccgccg cccttctctt ccgagctgta tgaccaccct ctttcacttt 120
tttgattttc gttcctctcg ttttacttgc attgtcttcg acaatcgcta tccgtcctcc 180
ttcgacctct cgcctcatcc tccttctcta cccaaagctt cctatcatgg tgttgaagca 240
ccaaggaaca gcttggaatt agatggagct tcaattcctt gcttaagaat taaagaagaa 300
aatttgcaac ttcaaatggg acttcaaatc aaaactagaa atggttgcac aaaaccaaaa 360
gcaagtgagc aacaacttcc aaataatgat cacattattg cattagaatc tccaagtgca 420
aagagaccaa atctcttggc aagattaatg ggtcttgata ttctccctca aacctcctct 480
tcttcttcca attctcacgg accaaattac ggaacccgtt ctctcccaga gagcccgaga 540
atatcgtcag caagattatc agacgtcgac tatcatcgtc gtctctcact ccaaattatt 600
cctgaaaaag agaatatcga aatttgtact gaagagatta agcaagaaaa agaaaaagtg 660
agaaggaaag ttgcactcgt tgatatcact aataataaca agaaaacaga attcggaaaa 720
caagaagttg gtattagtca aagtaaagtc gagatcaagt ctaataagaa acttaagaag 780
atggtagctg atgaatcaag ttgttcaaaa atcgtgctta gaaatcgaga ggttatgatt 840
tccaagaagc aaaagctaat atcgatgtcg atgcaaaaac ggaagccaag ggctcgagaa 900
ggtgaaacat ttgattgtcc aacaagtaat aaccttctta acaacgtcaa tcattcaact 960
atttttccag taaagaaaga gccttctcct ccggcgacca aggtccctcg tgaacagccg 1020
tgtaggtact caaagggcaa ggcgaagccg gcgggcagag acggcggaga aagaaacgcc 1080
ttgaacctta ccaccaccac agacggcgga tcagccgagt tcaaatacat caaaagaata 1140
ctaaccaatc accgcaattc aaacttgatc atctcaccct ctaataaccc aatgaacccc 1200
tcaatcttcc accacctaga aaccgcggcc gccgccgccg tggaggacca gcaatggaac 1260
aaacgactac tgaactgttg gcacgtgcga agaggaatga agagatggga attgggtgag 1320
gaagtgaggg agagagtgaa gaaagagtac ttcccacgtg tgaaatatga agtagtggaa 1380
aatatggata ccttaataat caacaagaga atggcggagg aaatagaagg gattgtgaag 1440
gtggttgagc ttcacatttt ggattccctt ttacgagaaa ctattgctct aatttcttcc 1500
ctaccaaaat gttctcattt tcataatttc taa 1533
Claims (9)
1.一种果形发育相关蛋白,其特征在于,包含:
a)如SEQ ID NO.1的氨基酸残基序列组成的蛋白质;
或
b)如SEQ ID NO.1的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加所得到的衍生蛋白质,且仍具有a)所示蛋白质的活性;
其中,所述b)中氨基酸残基的取代和/或缺失和/或添加的总数不超过10个。
2.如权利要求1所述的果形发育相关蛋白,其特征在于,所述蛋白质使植物果实形状变长。
3.如权利要求1所述的果形发育相关蛋白,其特征在于,所述植物包括但不限于番茄。
4.一种如权利要求1-3任一所述果形发育相关蛋白的编码基因。
5.根据权利要求4所述果形发育相关蛋白的编码基因,其特征在于,包含以下任一种DNA分子:
(1)如SEQ ID NO.2所示的DNA分子或其互补DNA分子;
(2)在严格条件下与1)限定的DNA序列杂交且与植物果形发育相关的DNA分子;
(3)与(1)或(2)限定的DNA序列至少具有70%同源性且与植物果形发育相关的DNA分子;
所述严格条件为在0.1×SSPE(或0.1×SSC),0.1%SDS的溶液中,在65℃下杂交并洗膜。
6.一种含有如权利要求4或5所述编码基因的重组载体,其特征在于,所述重组载体为重组过表达载体。
7.一种含有如权利要求4或5所述编码基因或含有如权利要求6所述重组载体的重组菌或转基因细胞系。
8.一种培育长果形番茄的方法,其特征在于,在番茄中过表达如权利要求4或5所述的编码基因,得到转基因番茄。
9.如权利要求8所述的培育长果形番茄的方法,其特征在于,所述过表达是指在番茄中转入所述过表达载体实现。
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