CN114149997B - 一种拟南芥rna加工因子dcp5基因及其应用 - Google Patents
一种拟南芥rna加工因子dcp5基因及其应用 Download PDFInfo
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Abstract
本发明公开了一种拟南芥RNA加工因子DCP5基因及其应用，涉及基因工程技术领域，拟南芥RNA加工因子DCP5基因，其抑制FLC的表达，且具有如SEQ ID NO.1所示的核苷酸序列。本发明拟南芥RNA加工因子DCP5基因在植物开花时间的调控作用，该基因属于P‑body的组分之一，能够与拟南芥中的另一重要的自主途径的开花调节基因SSF相互作用形成蛋白复合体。DCP5基因在拟南芥中起到促进营养生长到生殖生长的转变，通过对FLC前体mRNA的调控抑制FLC的表达,进而调节拟南芥开花。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及一种拟南芥RNA加工因子DCP5基因及其应用。
背景技术
植物开花是高等植物由营养生长向生殖生长的转变，对于植物繁殖和适应至关重要。调控植物花期有多条途径，主要包括光周期途径、自主途径、年龄途径和赤霉素途径等。而在自主途径中，开花抑制因子FLC在拟南芥花期调控网络中起着关键作用。FLC的表达受许多来自环境和生长发育的信号调控，主要包括：FRI依赖途径、自主途径和春化作用途径。找出这些调控途径中的关键基因，对进一步了解植物开花的遗传调控机理具有重要意义，为通过基因工程指导遗传育种奠定理论依据。因此，提出一种拟南芥RNA加工因子DCP5基因及其应用。
发明内容
本发明的目的在于克服现有技术的不足，提供了一种拟南芥RNA加工因子DCP5基因及其应用。
本发明通过以下技术方案来实现上述目的：
本发明提供了一种拟南芥RNA加工因子DCP5基因，其抑制FLC的表达，且具有如SEQID NO.1所示的核苷酸序列。
本发明还提供了一种上述拟南芥RNA加工因子DCP5基因在促进植物开花时间中的应用。
进一步改进在于，所述加工因子DCP5基因与开花调节基因SSF相互作用形成蛋白复合体，通过抑制开花抑制因子FLC基因的表达进而调控拟南芥花期。
本发明还提供了一种由上述拟南芥RNA加工因子DCP5基因编码的蛋白，该编码蛋白具有如SEQ ID NO.2所示的氨基酸序列。
本发明还提供了一种植物表达载体，所述植物表达载体为通过在pGreenII0179载体上插入上述拟南芥RNA加工因子DCP5基因的核苷酸序列获得。
本发明还提供了一种遗传工程化的宿主细胞，所述宿主细胞为含有如权利要求1所述基因的表达载体的农杆菌细胞，或为基因组中整合有如权利要求1所述基因的农杆菌细胞。
本发明还提供了一种拟南芥RNA加工因子DCP5基因的获得方法，以DCP5基因的核苷酸序列为模板设计特异性扩增引物，以拟南芥野生型Col-0品种为材料，提取RNA并反转录成cDNA，通过PCR扩增技术获得拟南芥DCP5基因。
进一步改进在于，所述PCR扩增的特异性扩增引物为：
DCP5基因-F：5'>ATGGCGGCTGATAATACGG<3'；
DCP5基因-R：5'>TTAGGTAGTACGATTTGATACGCCTCT<3'。
本发明提具有如下有益效果：
本发明提供了一种拟南芥RNA加工因子DCP5基因在植物开花时间的调控作用，该基因属于P-body的组分之一，能够与拟南芥中的另一重要的自主途径的开花调节基因SSF相互作用形成蛋白复合体。DCP5基因在拟南芥中起到促进营养生长到生殖生长的转变，通过对FLC前体mRNA的调控抑制FLC的表达，进而调节拟南芥开花。
附图说明
图1为DCP5基因全长扩增电泳图，图中，泳道1-2为DCP5基因片段，M为Marker(Trans2K)；
图2为酵母双杂交实验四缺培养基筛选鉴定；
图3为DCP5基因突变体植株花期统计；
图4为转基因阳性植株花期统计；
图5为转基因株系主根中DCP5基因亚细胞定位。
具体实施方式
下面结合附图对本申请作进一步详细描述，有必要在此指出的是，以下具体实施方式只用于对本申请进行进一步的说明，不能理解为对本申请保护范围的限制，该领域的技术人员可以根据上述申请内容对本申请作出一些非本质的改进和调整。
1、材料
本实施例所用方法如无特别说明均为本领域技术人员所知晓的常规方法，所用试剂等，如无特别说明，均为市售购买产品。
2、方法
2.1拟南芥DCP5基因的克隆
以拟南芥野生型Col-0品种为材料，提取RNA，并将提取的RNA进行反转录生成cDNA第一链，作为PCR扩增的模板。
用设计的特异性引物：
DCP5基因-F：(5'>ATGGCGGCTGATAATACGG<3')
DCP5基因-R：(5'>TTAGGTAGTACGATTTGATACGCCTCT<3')
进行扩增，得到1836bp的基因片段，如图1所示，连接到T克隆载体pEASY-BluntZero Cloning Kit上，获得T-DCP5基因质粒转化进入大肠杆菌，挑取阳性克隆并测序，测序结果与NCBI序列一致，获得如SEQ ID NO.1所示的核苷酸序列。
对上述扩增获得的DCP5基因及其编码蛋白进行生物信息学分析，发现该DCP5基因是进行拟南芥RNA加工的基因，属于P-body的重要组分之一，参与mRNA前加工的多个步骤，包括mRNA去帽、mRNA降解、去腺苷酸化、RNA介导的转录后基因沉默和无义介导的衰变。
2.2酵母双杂交验证该基因与SSF的蛋白相互作用
根据上述获得的拟南芥品种的DCP5基因和TAIR网站公布的SSF(AT2G47310)的CDS序列，设计特异引物：
DCP5基因-F1：5'>CATATGATGGCGGCTGATAATACGG<3'；
DCP5基因-R1：5'>CCCGGGGGTAGTACGATTTGATACGCCTCT<3'，获得带有酶切位点的DCP5基因片段。
SSF-F:5'>CATATGATGGAGAGACGCGCCCCAA<3'
SSF-R：5'>CTGCAGTTATGAACATGTTGTTTCAACAGCTA<3'，获得带有酶切位点的SSF基因片段。
将测序正确的带有酶切位点SSF和DCP5基因编码区及酵母双杂交系统(
Gold Yeast Two-Hybrid System)构建的载体pGBK-T7、pGAD-T7分别经Nde I和Xma I双酶切，回收目的片段与载体。并与T4连接酶混合后，22℃过夜连接，经热激法转入大肠杆菌感受态细胞DH5α，采用菌液PCR方法筛选阳性克隆并进行测序分析，最终构建成酵母双杂交实验表达载体pGBK-SSF和pGAD-DCP5基因。
在5mL YPDA培养液中30℃，250rpm培养酵母细胞(AH109)，过夜培养后吸取800μL扩大至30mL YPDA培养液培养。三至四个小时后菌液浓度达到OD600 0.4-0.6，使用低温离心的方法，制作酵母感受态细胞，热激法转化重组质粒pGBK-SSF和pGAD DCP5基因，并涂布在Leu/Trp缺陷固体培养基中，30℃培养。三天后待酵母长出挑取单菌落使用5-7mL Leu/Trp缺陷液体培养基(SD-Leu/-Trp)30℃，250rpm过夜培养酵母细胞。第二天通过离心法清洗重悬酵母细胞，并涂布在Leu/Trp/His/Ade缺陷型固体培养基上。
通过在Leu/Trp/His/Ade缺陷型固体培养基上筛选鉴定，获得图2，图2中可以看出，DCP5基因能够在酵母中与SSF相互作用。
2.3拟南芥突变体和转基因鉴定该基因的功能
根据DCP5基因序列，从英国诺丁汉拟南芥种子中心(Nottingham ArabidopsisStock Center)订购一个独立的T-DNA插入突变体Salk_008881(DCP5基因-1)，根据TAIR网站提供的T-DNA引物鉴定方法合成鉴定引物：
Salk_008881(DCP5基因-1)：
LP:5'>CCATCAGCAGAGGATGAAGAG<3'
RP:5'>GTCCCAAAATTCAAGGCCTAG<3'
LBb1.3:5'>ATTTTGCCGATTTCGGAAC<3'
通过旁侧序列测序结果分析，该T-DNA插入突变体Salk_008881(DCP5基因-1)插入DCP5基因3端非翻译区上导致功能缺陷。图3可以看出突变体植株在长日照条件下表现出明显的晚花和高FLC表达表型。说明该基因功能是通过影响FLC表达从而调节植物早花。
根据获得的上述拟南芥品种的DCP5基因的基因组DNA序列，设计特异引物构建亚细胞定位及互补载体(横线为酶切位点，斜体小写字母为mini-linker)：
DCP5基因-F2：5'>GGTACCCCATCCCGTCAGATGCAAG<3'
DCP5基因-R2：5'>GTCGACGGTAGTACGATTTGATACGCCTC<3'
DCP5基因-F3：5'>ACTAGTACTGTTCTGCTAGAAAGGGCTCTAA<3'
DCP5基因-R3：5'>GCGGCCGCAATCTTGGAAAGCATCTAGTTCTT<3'
GFP-R:5'>GGATCCTTACTTGTACAGCTCGTCCATGCC<3'
获得带有酶切位点的DCP5基因编码区及GFP片段。
将测序正确带有酶切位点的DCP5基因编码区及GFP片段和构建载体pGreenII0179(http://www.pgreen.ac.uk/JIT/pG0179.htm)分别经Kpn I和Sal I、Spe I和Not I双酶切，切胶回收目的片段与载体。利用T4连接酶，22℃过夜连接，使用热激法转入大肠杆菌感受态细胞DH5α，37℃过夜培养，挑取单菌落采用菌液PCR方法筛选阳性克隆并进行测序分析，最终构建成植物表达载体pGreen-DCP5基因-GFP。
将pGreen-DCP5基因-GFP和pSoup(http://www.pgreen.ac.uk/a_pls_fr.htm)质粒共同转入感受态农杆菌GV3101中，采用菌液PCR法鉴定阳性克隆。
挑选阳性菌液，加入5mL含有卡那霉素Kan、利福平Rif和四环素Tet抗性的LB液体培养基，28℃200rpm培养两天，然后按体积比1∶100接种于100mL的含有卡那霉素Kan、利福平Rif和四环素Tet抗性的LB液体培养基，28℃200rpm培养至OD600值在0.6～0.8之间。
通过离心法收集清洗菌体，使用花粉介导法转入DCP5基因-1突变体拟南芥中，待种子成熟后(T0)，对种子进行潮霉素抗性筛选，阳性苗(T1)于人工气候室培养至种子成熟。
筛选T3代纯合转基因种子，播种后观察并鉴定表型，结果如图4所示。可以看出，DCP5转基因株系与野生型Col-0开花时间无明显差异，回补了突变体的晚花表型，表明DCP5基因在调控花期发挥作用。
2.4转基因观察该基因的亚细胞定位
将上述已获得的pGreen-DCP5基因-GFP纯合转基因种子进行播种，取其生长三天的根使用4',6-二脒基-2-苯基吲哚(DAPI，solarbio)进行染色后在激光共聚焦下观察，并将GFP场和DAPI场进行合并得到合并场(Merged)。如图5所示，GFP和DAPI能够在细胞核内重合，表明DCP5基因在细胞核和细胞质中都存在。
2.5结论
DCP5基因是拟南芥一种RNA加工因子，通过抑制FLC的表达进而调控拟南芥花期，DCP5基因能与拟南芥中自主途径中的一个开花时间调控基因SSF相互作用，调节拟南芥开花，起到促进营养生长向生殖生长的转变。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。
序列表
<110> 安徽农业大学
<120> 一种拟南芥RNA加工因子DCP5及其应用
<141> 2021-12-10
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1836
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
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actgatatca aggatttgca agtcaaagct tctccaccag ttcagcctcc tgcatctact 300
ataaataatg accctgctat aattcagtct cactacccta gcccgatgcc aacatctggt 360
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cccaatggcc ttccccaatt gcatcagcag tctctgattc gaccccctca tgggctgcca 600
atgcccaact ctcttcagca acccctgcaa tatcctaact ttaatacccc tcctccacct 660
actgggtcat catctttgca ggggtcaagc ttgccagaag caccctcttc attgtttcct 720
tttagtacca gttctcaaat gttagctcct agttcattgc ctttccctgg tctaccacct 780
gtgacgctct cgtctagcct gcagtctaca ctgcaatcag caccatctcc ttcactagct 840
tcagagatgg cacctccgtt gctgtcaaac aaagctccta ttactgctcc tcctacccta 900
cctcaggata ctaatttgct ttcgttttct ctatctacta cgagagccac agaagcaagt 960
actggccttc cactgtctaa caagcctagt gtagtaactg gtccaatttc tccgccgcag 1020
acaacaccat taacatctgc tcctgttgcg ggagtttcta gttccattag ccaagataag 1080
ccaaaacctt tgttggttac tcctggtcaa ctgctgcagt ctggatcttc tgcagtctct 1140
ttgtccccac cctcgacaaa tgctgataaa gatgttgagg tggttcaagt atcgtcatca 1200
gctgggttgg aacaatctgt tccagttaca tccgaagccc aacctccaat actgccattg 1260
ccttcctctg caagaccaac tcagaagcca aacgggcatt ctttcccaaa tcataatggt 1320
tacaggggac gtggaagagg acgaggaaga ggagcaggga gatcacacca agtaatgaaa 1380
ttcactgaag attttgattt cacagcaatg aacgaaaagt tcaataagga tgaagtatgg 1440
ggtcatcttg ggaagagtac taccttagat ggtgatgaag atgatgattc cccgactgta 1500
gatgaggctg agcttcctaa gatcgaggcc aaacctgttt acaacaaaga tgactttttc 1560
gatagcctat catcaaacac cattgaccga gagtcccaaa attcaaggcc taggttctca 1620
gagcaacgga aactagatac tgagacattt ggtgaattct cgagattccg aggaggtcga 1680
ggagggcgtg gtggttatgg acgaaacaac ggttactcac gtggtggtta tggagggcgt 1740
ggttatggcg gttatggtgg ccgtggcggc ggcggcggtg gctacgggta tggtggacgt 1800
ggccagggga gaggcgtatc aaatcgtact acctaa 1836
<210> 2
<211> 611
<212> PRT
<213> 拟南芥(Arabidopsis thaliana)
<400> 2
Met Ala Ala Ala Ala Thr Gly Ser Leu Ser Ser Ser Ala Ala Ala Ser
1 5 10 15
Thr Val Gly Ser Leu Ile Ser Leu Thr Ser Leu Ser Gly Ile Ala Thr
20 25 30
Gly Gly Ile Leu Thr Ala Ile Ala Thr Ala Gly Ser Ser Ile Gly Leu
35 40 45
Gly Ala Val Ala Ser Pro Gly Thr Gly Gly Ala Leu Leu Ala Gly Pro
50 55 60
Gly Val Pro Pro Ser Ala Leu Val Thr Gly Thr Ile Leu Pro Ala Gly
65 70 75 80
Thr Ala Ile Leu Ala Leu Gly Val Leu Ala Ser Pro Pro Val Gly Pro
85 90 95
Pro Ala Ser Thr Ile Ala Ala Ala Pro Ala Ile Ile Gly Ser His Thr
100 105 110
Pro Ser Pro Met Pro Thr Ser Gly Ser Leu Pro Ser Thr Ala Ser Gly
115 120 125
Ser Leu Pro Ala Ile Ser Ser His Ala Gly Gly Pro Gly Gly His Gly
130 135 140
Met Gly Pro Gly Ala Ala Met Pro Leu Thr Gly Pro Gly Gly Ala Leu
145 150 155 160
Gly Ser Thr Gly Ala Ser Pro Gly Pro Pro Met Thr Thr Gly Gly Pro
165 170 175
Thr Thr Pro Pro Pro Ala Gly Leu Pro Gly Leu His Gly Gly Ser Leu
180 185 190
Ile Ala Pro Pro His Gly Leu Pro Met Pro Ala Ser Leu Gly Gly Pro
195 200 205
Leu Gly Thr Pro Ala Pro Ala Thr Pro Pro Pro Pro Thr Gly Ser Ser
210 215 220
Ser Leu Gly Gly Ser Ser Leu Pro Gly Ala Pro Ser Ser Leu Pro Pro
225 230 235 240
Pro Ser Thr Ser Ser Gly Met Leu Ala Pro Ser Ser Leu Pro Pro Pro
245 250 255
Gly Leu Pro Pro Val Thr Leu Ser Ser Ser Leu Gly Ser Thr Leu Gly
260 265 270
Ser Ala Pro Ser Pro Ser Leu Ala Ser Gly Met Ala Pro Pro Leu Leu
275 280 285
Ser Ala Leu Ala Pro Ile Thr Ala Pro Pro Thr Leu Pro Gly Ala Thr
290 295 300
Ala Leu Leu Ser Pro Ser Leu Ser Thr Thr Ala Ala Thr Gly Ala Ser
305 310 315 320
Thr Gly Leu Pro Leu Ser Ala Leu Pro Ser Val Val Thr Gly Pro Ile
325 330 335
Ser Pro Pro Gly Thr Thr Pro Leu Thr Ser Ala Pro Val Ala Gly Val
340 345 350
Ser Ser Ser Ile Ser Gly Ala Leu Pro Leu Pro Leu Leu Val Thr Pro
355 360 365
Gly Gly Leu Leu Gly Ser Gly Ser Ser Ala Val Ser Leu Ser Pro Pro
370 375 380
Ser Thr Ala Ala Ala Leu Ala Val Gly Val Val Gly Val Ser Ser Ser
385 390 395 400
Ala Gly Leu Gly Gly Ser Val Pro Val Thr Ser Gly Ala Gly Pro Pro
405 410 415
Ile Leu Pro Leu Pro Ser Ser Ala Ala Pro Thr Gly Leu Pro Ala Gly
420 425 430
His Ser Pro Pro Ala His Ala Gly Thr Ala Gly Ala Gly Ala Gly Ala
435 440 445
Gly Ala Gly Ala Gly Ala Ser His Gly Val Met Leu Pro Thr Gly Ala
450 455 460
Pro Ala Pro Thr Ala Met Ala Gly Leu Pro Ala Leu Ala Gly Val Thr
465 470 475 480
Gly His Leu Gly Leu Ser Thr Thr Leu Ala Gly Ala Gly Ala Ala Ala
485 490 495
Ser Pro Thr Val Ala Gly Ala Gly Leu Pro Leu Ile Gly Ala Leu Pro
500 505 510
Val Thr Ala Leu Ala Ala Pro Pro Ala Ser Leu Ser Ser Ala Thr Ile
515 520 525
Ala Ala Gly Ser Gly Ala Ser Ala Pro Ala Pro Ser Gly Gly Ala Leu
530 535 540
Leu Ala Thr Gly Thr Pro Gly Gly Pro Ser Ala Pro Ala Gly Gly Ala
545 550 555 560
Gly Gly Ala Gly Gly Thr Gly Ala Ala Ala Gly Thr Ser Ala Gly Gly
565 570 575
Thr Gly Gly Ala Gly Thr Gly Gly Thr Gly Gly Ala Gly Gly Gly Gly
580 585 590
Gly Gly Thr Gly Thr Gly Gly Ala Gly Gly Gly Ala Gly Val Ser Ala
595 600 605
Ala Thr Thr
610
Claims (2)
1.一种拟南芥RNA加工因子DCP5基因在促进植物早花中的应用，其特征在于，所述DCP5基因为如SEQ ID NO.1所示的核苷酸序列，或为与SEQ ID NO.1所示序列互补的核苷酸序列。
2.根据权利要求1所述的应用，其特征在于，所述加工因子DCP5基因通过抑制开花抑制因子FLC基因的表达进而使拟南芥花期提前。
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