CN113881696A - 一个大孢子母细胞特异诱导表达系统 - Google Patents

一个大孢子母细胞特异诱导表达系统 Download PDF

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CN113881696A
CN113881696A CN202111178767.XA CN202111178767A CN113881696A CN 113881696 A CN113881696 A CN 113881696A CN 202111178767 A CN202111178767 A CN 202111178767A CN 113881696 A CN113881696 A CN 113881696A
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程焱
秦源
林志斌
刘金典
熊俊杰
杨攀
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Fujian Agriculture and Forestry University
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Abstract

本发明构建了一个大孢子母细胞特异性诱导表达的系统:ipKNU‑Venus，方法为：构建含有pKNU：：VGE‑5×GAL4 DNA：：Venus表达盒的载体，转化至拟南芥中，获得阳性苗。该系统只有在诱导剂（Methoxyfenozide）诱导作用之后才表达，为研究大孢子母细胞的起源与分化提供了一种新方法。

Description

一个大孢子母细胞特异诱导表达系统
技术领域
本发明涉及植物生物技术领域，具体涉及一个大孢子母细胞特异诱导表达系统。
背景技术
高等植物雌配子体发育一般包括大孢子发生和雌配子体发生两个阶段。大孢子发生一般是从胚珠原基顶端的1个亚表皮细胞分化形成大孢子母细胞开始的，而大孢子母细胞经过一次减数分裂形成4个单倍体大孢子，随着发育进行，处在珠孔端的3个大孢子退化，仅剩合点端的一个大孢子即功能大孢子幸存并参与雌配子体发生；雌配子体发生是从功能大孢子的形成开始，其经过三次连续核有丝分裂形成合胞体，再经过细胞化，最终形成“七胞八核”的雌配子体（也称之为胚囊）的过程。
高等植物雌配子发育的过程起始于MMC，在研究MMC的精准调控的过程中，需要对特定的基因进行功能分析。传统的功能分析中需要进行基因的超表达。过去的超表达系统包括两类：1）以35S等为代表的的组成性启动子驱动目的基因表达的超表达系统；2）以MMC特异表达基因启动子驱动的细胞特异表达系统。第一类系统对目标基因的表达是组成型的表达，第二类系统尽管是在MMC中表达，但是其表达不能受到人为的控制。本研究发明了一种新的适合在MMC中诱导性表达的系统，其目标基因能够在MMC中特异表达，而且，这种特异表达受到人为的控制，即人工诱导后表达。本系统可以用于在MMC中可诱导地表达目标基因。
发明内容
本发明的目的在于提供可以用于在MMC中可诱导地表达目标基因的系统: ipKNU- Venus，其特征是该系统只有在诱导剂（Methoxyfenozide）诱导作用之后才表达，为研究大孢子母细胞的分化发育提供了一种新方法。具体流程：载体构建，转化农杆菌，侵染拟南芥，筛选与鉴定阳性植株，诱导表达，荧光观察。
为实现上述目的，本发明采用如下技术方案。
一个大孢子母细胞特异性诱导表达的系统ipKNU-Venus，构建含有pKNU：：VGE-5× GAL4 DNA：：Venus表达盒的载体，转化至拟南芥中，获得阳性苗。
所述载体构建方法包括以下步骤：
（1）提取AT5G14010基因的全基因组序列，根据序列设计扩增片段的引物，从拟南芥基因组DNA扩增启动子片段，并回收；引物序列为：
Forward Primer：ctagGCGATCGCTGGTAGATTTGTTCTGTGCATCCTATC；
Reversed Primer: gatcCGTCGACGGCATTTTTGAGAGGTTCTTAAGCTACAG；
（2）载体序列为GL3载体，用限制性内切酶SfaAI与SgrDI分别酶切启动子片段与载体质粒，37℃水浴锅中酶切反应2h，电泳检测并切胶回收；GL3载体序列如SEQ ID NO.1所示；
（3）酶切后的启动子片段与GL3载体按照浓度3：1的比例，用T4-DNA连接酶进行连接反应，放入16℃连接仪中反应1h；转化大肠杆菌DB3.1，挑选阳性克隆，提取质粒，pKNU- GL3载体；
（4）pKNU-GL3载体与pENTER-Venus载体LR交换反应，转化大肠杆菌DH5α，挑选阳性克隆，提取质粒，得到pKNU：：VGE-5×GAL4 DNA：：Venus。
所述pENTER-Venus载体的构建方法包括以下步骤：
（1）人工合成cacc-Vennus序列，如SEQ ID NO.2所示；
（2）采用pENTR™ Directional TOPO® Cloning Kits（Catalog numbers K2400-20）进行连接；
（3）转化大肠杆菌DH5α，挑选阳性克隆并提取质粒，得到pENTER-Venus载体入门载体。
一种诱导方法，利用权利要求1所述的诱导表达系统ipKNU-Venus，在昆虫蜕皮激素（Methoxyfenozide）存在的条件下进行诱导。
所述诱导方法具体包括以下步骤：
（1）将权利要求1所述载体导入农杆菌并转化拟南芥，提取T1代植株DNA，进行PCR鉴定，取阳性苗植株诱导；
（2）诱导前对目标植株停止浇水三天，让栽培基质适当干燥；
（3）配置浓度为200 ul/L 昆虫蜕皮激素（Methoxyfenozide）作为诱导剂，将目标植株地上部分放在诱导液中浸泡60S，然后用诱导液浇灌栽培基质致其吸水饱和，2小时后用诱导液对植株的花序进行喷雾至叶片上水珠饱和；
（4）诱导后，24小时取胚珠观察荧光情况。
荧光诱导表达观察：
使用Leica TCS SP8X DLS激光扫描共聚焦显微镜对诱导后植株的胚珠进行荧光观察，如图1所示，在MMC中特异发现绿色荧光信号，说明Vennus在MMC中被特异诱导表达。
此系统的Vennus可以替换成目标目标蛋白基因，实现目标蛋白在MMC中受诱导的特异性的表达。
本发明的优点在于：
（1）实现MMC诱导性显示的Marker。
（2）实现诱导性的MMC细胞特异性的蛋白表达。
（3）诱导性表达系统中，目标基因已经转入基因组中，但是处于静默状态，只有当诱导剂存在的情况下才启动表达。此系统能够克服组成型表达和细胞特异性表达导致致死无法得到后代的情况，也可以通过诱导表达致死基因，人工控制雌性败育。具有农业生产中的潜在应用价值。
附图说明
图1是诱导后不同时期的胚珠中Vennus信号情况；其中（A）MMC时期；（B）FM时期；（C）FG2时期；（D）FG4时期；（E）FG7时期。
具体实施方式
实施例1 载体构建
载体构建：构建pKNU：：VGE-5×GAL4 DNA：：Venus建构
实施例2 诱导方法：
（1） 构建好的载体导入农杆菌并转化拟南芥，提取T1代植株DNA，进行PCR鉴定，取阳性苗植株诱导；
（2）诱导前，对目标植株进行适当干燥处理；
（3）配置浓度为200 ul/L 昆虫蜕皮激素（Methoxyfenozide）作为诱导剂，将目标植株地上部分放在诱导液中浸泡60S，然后用诱导液浇灌栽培基质致其吸水饱和，2小时后用诱导液对植株的花序进行喷雾至叶片上水珠饱和；
（4）诱导后，24小时取胚珠观察荧光情况，结果如图1所示。
荧光诱导表达观察：
使用Leica TCS SP8X DLS激光扫描共聚焦显微镜对诱导后植株的胚珠进行荧光观察，如图1所示，在MMC中特异发现绿色荧光信号，说明Vennus在MMC中被特异诱导表达。
此系统的Vennus可以替换成目标目标蛋白基因，实现目标蛋白在MMC中受诱导的特异性的表达。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
SEQUENCE LISTING
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<120> 一个大孢子母细胞特异诱导表达系统
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gcccagaacg acgcccggcc gacatccgcc gtgccaccga ggcggacatg ccggcggtct 240
gcaccatcgt caaccactac atcgagacaa gcacggtcaa cttccgtacc gagccgcagg 300
aaccgcagga gtggacggac gacctcgtcc gtctgcggga gcgctatccc tggctcgtcg 360
ccgaggtgga cggcgaggtc gccggcatcg cctacgcggg cccctggaag gcacgcaacg 420
cctacgactg gacggccgag tcgaccgtgt acgtctcccc ccgccaccag cggacgggac 480
tgggctccac gctctacacc cacctgctga agtccctgga ggcacagggc ttcaagagcg 540
tggtcgctgt catcgggctg cccaacgacc cgagcgtgcg catgcacgag gcgctcggat 600
atgccccccg cggcatgctg cgggcggccg gcttcaagca cgggaactgg catgacgtgg 660
gtttctggca gctggacttc agcctgccgg taccgccccg tccggtcctg cccgtcaccg 720
agatctgctc aacaatctag ctagagtttg ctcctatcta tatgtaataa ggtatgctga 780
tatgcactat tcaaatagga gcattagcta tgtttgttaa tgtcacttta tgttatgtgg 840
gtaagtcacc taagacactc cacgtaccta cttgttgtct cttacgcggc tttaataaat 900
cttctgccct tgttccatat ttactaatta tccctttctt cactaaaaga aaattgttat 960
cattaagtat tagtctttag aacatatgag gtctttaatt gggtaggttt tacaaattaa 1020
ctaatataaa atgtcataaa atccacgtgg ttaaacaaat gcagaaaatc gacgtcgtct 1080
attggaccga cagttgctat taatataatg ggccaccata gtagactgac aaataaatta 1140
cctgacaaca tcgtttcaca aaaaaacaaa cacaaaaagg gagtgcattt tccagggcat 1200
ttttgtaata aaaaacagtt aaaagggagt gcaatagaaa tataggggtg tggaaatagt 1260
gatttgagca cgtcttgaag cgaattagct tggcactggc cgtcgtttta caacgtcgtg 1320
actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 1380
gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 1440
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tgctattcgg aagaacggca actaagctgc cgggtttgaa acacggatga tctcgcggag 1740
ggtagcatgt tgattgtaac gatgacagag cgttgctgcc tgtgatcaat tcgggcacga 1800
acccagtgga cataagcctg ttcggttcgt aagctgtaat gcaagtagcg tatgcgctca 1860
cgcaactggt ccagaacctt gaccgaacgc agcggtggta acggcgcagt ggcggttttc 1920
atggcttgtt atgactgttt ttttggggta cagtctatgc ctcgggcatc caagcagcaa 1980
gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca gcaacgatgt tacgcagcag 2040
ggcagtcgcc ctaaaacaaa gttaaacatc atgggggaag cggtgatcgc cgaagtatcg 2100
actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc 2160
gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga tattgatttg 2220
ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat caacgacctt 2280
ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga agtcaccatt 2340
gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact gcaatttgga 2400
gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat cgacattgat 2460
ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg tccagcggcg 2520
gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa tgaaacctta 2580
acgctatgga actcgccgcc cgactgggct ggcgatgagc gaaatgtagt gcttacgttg 2640
tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac 2700
tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc tagacaggct 2760
tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc 2820
cactacgtga aaggcgagat caccaaggta gtcggcaaat aatgtctagc tagaaattcg 2880
ttcaagccga cgccgcttcg cggcgcggct taactcaagc gttagatgca ctaagcacat 2940
aattgctcac agccaaacta tcaggtcaag tctgctttta ttatttttaa gcgtgcataa 3000
taagccctac acaaattggg agatatatca tgcatgacca aaatccctta acgtgagttt 3060
tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 3120
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gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 3360
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ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 3480
agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 3540
aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 3600
aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 3660
ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 3720
cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat 3780
tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 3840
accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc 3900
cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct 3960
gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg 4020
cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat 4080
ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt 4140
catcaccgaa acgcgcgagg cagggtgcct tgatgtgggc gccggcggtc gagtggcgac 4200
ggcgcggctt gtccgcgccc tggtagattg cctggccgta ggccagccat ttttgagcgg 4260
ccagcggccg cgataggccg acgcgaagcg gcggggcgta gggagcgcag cgaccgaagg 4320
gtaggcgctt tttgcagctc ttcggctgtg cgctggccag acagttatgc acaggccagg 4380
cgggttttaa gagttttaat aagttttaaa gagttttagg cggaaaaatc gccttttttc 4440
tcttttatat cagtcactta catgtgtgac cggttcccaa tgtacggctt tgggttccca 4500
atgtacgggt tccggttccc aatgtacggc tttgggttcc caatgtacgt gctatccaca 4560
ggaaagagac cttttcgacc tttttcccct gctagggcaa tttgccctag catctgctcc 4620
gtacattagg aaccggcgga tgcttcgccc tcgatcaggt tgcggtagcg catgactagg 4680
atcgggccag cctgccccgc ctcctccttc aaatcgtact ccggcaggtc atttgacccg 4740
atcagcttgc gcacggtgaa acagaacttc ttgaactctc cggcgctgcc actgcgttcg 4800
tagatcgtct tgaacaacca tctggcttct gccttgcctg cggcgcggcg tgccaggcgg 4860
tagagaaaac ggccgatgcc gggatcgatc aaaaagtaat cggggtgaac cgtcagcacg 4920
tccgggttct tgccttctgt gatctcgcgg tacatccaat cagctagctc gatctcgatg 4980
tactccggcc gcccggtttc gctctttacg atcttgtagc ggctaatcaa ggcttcaccc 5040
tcggataccg tcaccaggcg gccgttcttg gccttcttcg tacgctgcat ggcaacgtgc 5100
gtggtgttta accgaatgca ggtttctacc aggtcgtctt tctgctttcc gccatcggct 5160
cgccggcaga acttgagtac gtccgcaacg tgtggacgga acacgcggcc gggcttgtct 5220
cccttccctt cccggtatcg gttcatggat tcggttagat gggaaaccgc catcagtacc 5280
aggtcgtaat cccacacact ggccatgccg gccggccctg cggaaacctc tacgtgcccg 5340
tctggaagct cgtagcggat cacctcgcca gctcgtcggt cacgcttcga cagacggaaa 5400
acggccacgt ccatgatgct gcgactatcg cgggtgccca cgtcatagag catcggaacg 5460
aaaaaatctg gttgctcgtc gcccttgggc ggcttcctaa tcgacggcgc accggctgcc 5520
ggcggttgcc gggattcttt gcggattcga tcagcggccg cttgccacga ttcaccgggg 5580
cgtgcttctg cctcgatgcg ttgccgctgg gcggcctgcg cggccttcaa cttctccacc 5640
aggtcatcac ccagcgccgc gccgatttgt accgggccgg atggtttgcg accgctcacg 5700
ccgattcctc gggcttgggg gttccagtgc cattgcaggg ccggcagaca acccagccgc 5760
ttacgcctgg ccaaccgccc gttcctccac acatggggca ttccacggcg tcggtgcctg 5820
gttgttcttg attttccatg ccgcctcctt tagccgctaa aattcatcta ctcatttatt 5880
catttgctca tttactctgg tagctgcgcg atgtattcag atagcagctc ggtaatggtc 5940
ttgccttggc gtaccgcgta catcttcagc ttggtgtgat cctccgccgg caactgaaag 6000
ttgacccgct tcatggctgg cgtgtctgcc aggctggcca acgttgcagc cttgctgctg 6060
cgtgcgctcg gacggccggc acttagcgtg tttgtgcttt tgctcatttt ctctttacct 6120
cattaactca aatgagtttt gatttaattt cagcggccag cgcctggacc tcgcgggcag 6180
cgtcgccctc gggttctgat tcaagaacgg ttgtgccggc ggcggcagtg cctgggtagc 6240
tcacgcgctg cgtgatacgg gactcaagaa tgggcagctc gtacccggcc agcgcctcgg 6300
caacctcacc gccgatgcgc gtgcctttga tcgcccgcga cacgacaaag gccgcttgta 6360
gccttccatc cgtgacctca atgcgctgct taaccagctc caccaggtcg gcggtggccc 6420
atatgtcgta agggcttggc tgcaccggaa tcagcacgaa gtcggctgcc ttgatcgcgg 6480
acacagccaa gtccgccgcc tggggcgctc cgtcgatcac tacgaagtcg cgccggccga 6540
tggccttcac gtcgcggtca atcgtcgggc ggtcgatgcc gacaacggtt agcggttgat 6600
cttcccgcac ggccgcccaa tcgcgggcac tgccctgggg atcggaatcg actaacagaa 6660
catcggcccc ggcgagttgc agggcgcggg ctagatgggt tgcgatggtc gtcttgcctg 6720
acccgccttt ctggttaagt acagcgataa ccttcatgcg ttccccttgc gtatttgttt 6780
atttactcat cgcatcatat acgcagcgac cgcatgacgc aagctgtttt actcaaatac 6840
acatcacctt tttagacggc ggcgctcggt ttcttcagcg gccaagctgg ccggccaggc 6900
cgccagcttg gcatcagaca aaccggccag gatttcatgc agccgcacgg ttgagacgtg 6960
cgcgggcggc tcgaacacgt acccggccgc gatcatctcc gcctcgatct cttcggtaat 7020
gaaaaacggt tcgtcctggc cgtcctggtg cggtttcatg cttgttcctc ttggcgttca 7080
ttctcggcgg ccgccagggc gtcggcctcg gtcaatgcgt cctcacggaa ggcaccgcgc 7140
cgcctggcct cggtgggcgt cacttcctcg ctgcgctcaa gtgcgcggta cagggtcgag 7200
cgatgcacgc caagcagtgc agccgcctct ttcacggtgc ggccttcctg gtcgatcagc 7260
tcgcgggcgt gcgcgatctg tgccggggtg agggtagggc gggggccaaa cttcacgcct 7320
cgggccttgg cggcctcgcg cccgctccgg gtgcggtcga tgattaggga acgctcgaac 7380
tcggcaatgc cggcgaacac ggtcaacacc atgcggccgg ccggcgtggt ggtgtcggcc 7440
cacggctctg ccaggctacg caggcccgcg ccggcctcct ggatgcgctc ggcaatgtcc 7500
agtaggtcgc gggtgctgcg ggccaggcgg tctagcctgg tcactgtcac aacgtcgcca 7560
gggcgtaggt ggtcaagcat cctggccagc tccgggcggt cgcgcctggt gccggtgatc 7620
ttctcggaaa acagcttggt gcagccggcc gcgtgcagtt cggcccgttg gttggtcaag 7680
tcctggtcgt cggtgctgac gcgggcatag cccagcaggc cagcggcggc gctcttgttc 7740
atggcgtaat gtctccggtt ctagtcgcaa gtattctact ttatgcgact aaaacacgcg 7800
acaagaaaac gccaggaaaa gggcagggcg gcagcctgtc gcgtaactta ggacttgtgc 7860
gacatgtcgt tttcagaaga cggctgcact gaacgtcaga agccgactgc actatagcag 7920
cggaggggtt ggatcaaagt actttaaagt actttaaagt actttaaagt actttgatcc 7980
cgaggggaac cctgtggttg gcatgcacat acaaatggac gaacggataa accttttcac 8040
gcccttttaa atatccgatt attctaataa acgctctttt ctcttaggtt tacccgccaa 8100
tatatcctgt caaacactga tagtttaaac tgaaggcggg aaacgacaat ctgatccaag 8160
ctcaagctcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 8220
ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 8280
agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 8340
gaattgtgag cggataacaa tttcacacag gaaacagcta tgacatgatt acgaattcga 8400
gctcggtacc cgggagatct ggcgcgccgc aagacgtcca gaaggtaatt atccaagatg 8460
tagcatcaag aatccaatgt ttacgggaaa aactatggaa gtattatgtg agctcagcaa 8520
gaagcagatc aatatgcggc acatatgcaa cctatgttca aaaatgaaga atgtacagat 8580
acaagatcct atactgccag aatacgaaga agaatacgta gaaattgaaa aagaagaacc 8640
aggcgaagaa aagaatcttg aagacgtaag cactgacgac aacaatgaaa agaagaagat 8700
aaggtcggtg attgtgaaag agacatagag gacacatgta aggtggaaaa tgtaagggcg 8760
gaaagtaacc ttatcacaaa ggaatcttat cccccactac ttatcctttt atatttttcc 8820
gtgtcatttt tgcccttgag ttttcctata taaggaacca agttcggcat ttgtgaaaac 8880
aagaaaaaat ttggtgtaag ctattttctt tgaagtactg aggatacaac ttcagagaaa 8940
tttgtaagtt tggatccgcc accatggccc ccccgaccga tgtcagcctg ggggacgaac 9000
tccacttaga cggcgaggac gtggcgatgg cgcatgccga cgcgctagac gatttcgatc 9060
tggacatgtt gggggacggg gattccccag gtccgggatt taccccccac gactccgccc 9120
cctacggcgc tctggatatg gccgacttcg agtttgagca gatgtttacc gatgcccttg 9180
gaattgacga gtacggtggg aagcttctag gtacctccag aagaatatca ggcggggaat 9240
tcggcgggat gaagctactg tcttctatcg aacaagcatg cgatatttgc cgacttaaaa 9300
agctcaagtg ctccaaagaa aaaccgaagt gcgccaagtg tctgaagaac aactgggagt 9360
gtcgctactc tcccaaaacc aaaaggtctc cgctgactag ggcacatctg acagaagtgg 9420
aatcaaggct agaaagactg gaacagctat ttctactgat ttttcctcga gaagaccttg 9480
acatgatttt gaaaatggat tctttacagg atataaaagc attgttaaca ggattatttg 9540
tacaagataa tgtgaataaa gatgccgtca cagatagatt ggcttcagtg gagactgata 9600
tgcctctaac attgagacag catagaataa gtgcgacatc atcatcggaa gagagtagta 9660
acaaaggtca aagacagttg actgtatcgg gcgggtctag aagaatatcg tggcctgagt 9720
gcgtagtacc cgagactcag tgcgccatga agcggaaaga gaagaaagca cagaaggaga 9780
aggacaaact gcctgtcagc acgacgacgg tggacgacca catgccgccc attatgcagt 9840
gtgaacctcc acctcctgaa gcagcaagga ttcacgaagt ggtcccaagg tttctctccg 9900
acaagctgtt ggagacaaac cggcagaaaa acatccccca gttgacagcc aaccagcagt 9960
tccttatcgc caggctcatc tggtaccagg acgggtacga gcagccttct gatgaagatt 10020
tgaagaggat tacgcagacg tggcagcaag cggacgatga aaacgaagag tctgacactc 10080
ccttccgcca gatcacagag atgactatcc tcacggtcca acttatcgtg gagttcgcga 10140
agggattgcc agggttcgcc aagatctcgc agcctgatca aattacgcta cttaaggctt 10200
tctcaagtga ggtaatgatg ctccgagtcg cgcgacgata cgatgcggcc tcagacagtg 10260
ttctgttcgc gaacaaccaa gcgtacactc gcgacaacta ccgcaaggct ggcatggcct 10320
acgtcatcga ggatctactg cacttctgcc ggtgcatgta ctctatggcg ttggacaaca 10380
tccattacgc gctgctcacg gctgtcgtca tcttttctga ccggccaggg ttggagcagc 10440
cgcaactggt ggaagagatc cagcggtact acctgaatac gctccgcatc tatatcctga 10500
accagctgag cgggtcggcg cgttcgtccg tcatatacgg caagatcctc tcaatcctct 10560
ctgagctacg cacgctcggc atgcaaaact ccaacatgtg catctccctc aagctcaaga 10620
acagaaagct gccgcctttc ctcgaggaga tctgggatgt ggcggacatg tcgcacaccc 10680
aaccgccgcc tatcctcgag tcccccacga atctctagta accagtgtgc tggatctacg 10740
cgttaagctt aattcccgat cgttcaaaca tttggcaata aagtttctta agattgaatc 10800
ctgttgccgg tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa 10860
taattaacat gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc 10920
aattatacat ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat 10980
cgcgcgcggt gtcatctatg ttactagatc ggggaattaa ccattccaaa acagatcacc 11040
tttattatta agtttcagca ttgcgattta aatggctagg ctatgaaaga aagagttatt 11100
cgttctattt tctcgggttt tgagagcata ccgttgagcc aaacctttcc tgtagatagt 11160
ttgtgatgaa gaccaaaggc acctactcaa acacattggt tggtcagaat atgagattag 11220
agtttagtaa aaatcgactg cggccgcttg gatccgctcg gaggacagta ctccgctcgg 11280
aggacagtac tccgctcgga ggacagtact ccgctcgagg acagtactcc gctcggagga 11340
cagtactccg atccgtagat ctgcaagacc cttcctctat ataaggaagt tcatttcatt 11400
tggagaggac acgctgaacg atatcacaag tttgtacaaa aaagctgaac gagaaacgta 11460
aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata 11520
ctgtaaaaca caacatatcc agtcactatg gcggccgcat taggcacccc aggctttaca 11580
ctttatgctt ccggctcgta taatgtgtgg attttgagtt aggatccgtc gagattttca 11640
ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc 11700
caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac 11760
cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag 11820
ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt 11880
atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt 11940
ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg 12000
cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc 12060
cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc 12120
agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc 12180
aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc 12240
gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag 12300
tggcagggcg gggcgtaaac gcgtggatcc ggcttactaa aagccagata acagtatgcg 12360
tatttgcgcg ctgatttttg cggtataaga atatatactg atatgtatac ccgaagtatg 12420
tcaaaaagag gtatgctatg aagcagcgta ttacagtgac agttgacagc gacagctatc 12480
agttgctcaa ggcatatatg atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa 12540
tgaagcccgt cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag ggatggctga 12600
ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac gagaacaggg gctggtgaaa 12660
tgcagtttaa ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac 12720
agagtgatat tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc 12780
tgctgtcaga taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct 12840
ggcgcatgat gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg 12900
ctgatctcag ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa 12960
tataaatgtc aggctccctt atacacagcc agtctgcagg tcgaccatag tgactggata 13020
tgttgtgttt tacagtatta tgtagtctgt tttttatgca aaatctaatt taatatattg 13080
atatttatat cattttacgt ttctcgttca gctttcttgt acaaagtggt gatatcttaa 13140
ttaagctagc actagttcta gagtccgcaa aaatcaccag tctctctcta caaatctatc 13200
tctctctatt tttctccaga ataatgtgtg agtagttccc agataaggga attagggttc 13260
ttatagggtt tcgctcatgt gttgagcata taagaaaccc ttagtatgta tttgtatttg 13320
taaaatactt ctatcaataa aatttctaat tcctaaaacc aaaatccagt gacctgcagg 13380
catgcaagct tatcgatacc gtcgacctcg agggggggcc ctctagactg cagccatggg 13440
cgcgccggat cctctagagt cgacctgcag gcatgcaagc ttgcatgcct gcaggtcaac 13500
atggtggagc acgacacact tgtctactcc aaaaatatca aagatacagt ctcagaagac 13560
caaagggcaa ttgagacttt tcaacaaagg gtaatatccg gaaacctcct cggattccat 13620
tgcccagcta tctgtcactt tattgtgaag atagtggaaa aggaaggtgg ctcctacaaa 13680
tgccatcatt gcgataaagg aaaggccatc gttgaagatg cctctgccga cagtggtccc 13740
aaagatggac ccccacccac gaggagcatc gtggaaaaag aagacgttcc aaccacgtct 13800
tcaaagcaag tggattgatg tgataacatg gtggagcacg acacacttgt ctactccaaa 13860
aatatcaaag atacagtctc agaagaccaa agggcaattg agacttttca acaaagggta 13920
atatccggaa acctcctcgg attccattgc ccagctatct gtcactttat tgtgaagata 13980
gtggaaaagg aaggtggctc ctacaaatgc catcattgcg ataaaggaaa ggccatcgtt 14040
gaagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag gagcatcgtg 14100
gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga tatctccact 14160
gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc tatataa 14217
<210> 2
<211> 720
<212> DNA
<213> 人工序列
<400> 2
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagct gatctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca ccgccgacaa gcagaagaac 480
ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 3
<211> 39
<212> DNA
<213> 人工序列
<400> 3
ctaggcgatc gctggtagat ttgttctgtg catcctatc 39
<210> 4
<211> 40
<212> DNA
<213> 人工序列
<400> 4
gatccgtcga cggcattttt gagaggttct taagctacag 40

Claims (4)

1.一个大孢子母细胞特异性诱导表达的系统ipKNU-Venus，其特征在于，构建含有pKNU：：VGE-5×GAL4 DNA：：Venus表达盒的载体，转化至拟南芥中，获得阳性苗。
2.根据权利要求1所述的大孢子母细胞特异性诱导表达的系统ipKNU-Venus，其特征在于，所述载体构建方法包括以下步骤：
（1）提取AT5G14010基因的全基因组序列，根据序列设计扩增片段的引物，从拟南芥基因组DNA扩增启动子片段，并回收；引物序列为：
Forward Primer：ctagGCGATCGCTGGTAGATTTGTTCTGTGCATCCTATC；
Reversed Primer: gatcCGTCGACGGCATTTTTGAGAGGTTCTTAAGCTACAG；
（2）载体序列为GL3载体，用限制性内切酶SfaAI与SgrDI分别酶切启动子片段与载体质粒，37℃水浴锅中酶切反应2h，电泳检测并切胶回收；
（3）酶切后的启动子片段与GL3载体按照浓度3：1的比例，用T4-DNA连接酶进行连接反应，放入16℃连接仪中反应1 h；转化大肠杆菌DB3.1，挑选阳性克隆，提取质粒，得到pKNU- GL3载体；
（4）pKNU-GL3载体与pENTER-Venus载体LR交换反应，转化大肠杆菌DH5α，挑选阳性克隆，提取质粒，得到pKNU：：VGE-5×GAL4 DNA：：Venus。
3.一种诱导方法，其特征在于，利用权利要求1所述的诱导表达系统ipKNU-Venus，在昆虫蜕皮激素存在的条件下进行诱导。
4.根据权利要求3所述的方法，其特征在于，所述诱导方法具体包括以下步骤：
（1）将权利要求1所述载体导入农杆菌并转化拟南芥，提取T1代植株DNA，进行PCR鉴定，取阳性苗植株诱导；
（2）诱导前对目标植株停止浇水三天，让栽培基质适当干燥；
（3）配置浓度为200 ul/L 昆虫蜕皮激素作为诱导剂，将目标植株地上部分放在诱导液中浸泡60S，然后用诱导液浇灌栽培基质致其吸水饱和，2小时后用诱导液对植株的花序进行喷雾至叶片上水珠饱和；
（4）诱导后，24小时取胚珠观察荧光情况。
CN202111178767.XA 2021-10-11 2021-10-11 一个大孢子母细胞特异诱导表达系统 Pending CN113881696A (zh)

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