CN110004154B - 茶树CsJAZ1基因的应用 - Google Patents
茶树CsJAZ1基因的应用 Download PDFInfo
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- CN110004154B CN110004154B CN201910222384.4A CN201910222384A CN110004154B CN 110004154 B CN110004154 B CN 110004154B CN 201910222384 A CN201910222384 A CN 201910222384A CN 110004154 B CN110004154 B CN 110004154B
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- csjaz1
- tea tree
- arabidopsis thaliana
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Abstract
本发明涉及茶树CsJAZ1基因的应用,以茶树创伤应答反应转录组组装序列为模板设计引物,扩增出了茶树JAZ1基因CsJAZ1的全长序列1267 bp,如SEQ ID NO:1所示,将CsJAZ1基因转到拟南芥中进行异位表达,可使转基因拟南芥植株提早2~3天抽薹开花。依据转录组数据对CsJAZ1基因在茶树不同组织器官中的表达量变化进行分析,推测CsJAZ1基因在茶树中可能主要影响芽的分化,促进茶树营养生长向生殖生长过程的转变,由此可能促进茶树花期提前。该基因在茶树的良种培育中将有着较大的潜在应用价值。
Description
技术领域
本发明属于基因工程技术领域,具体涉及茶树 CsJAZ1基因的应用。
背景技术
茉莉酸途径的抑制因子 JAZ(Jasmonate ZIM domain,JAZ)蛋白(JAZs)是一类参与多个信号通路的阻遏蛋白,它可以响应茉莉酸(JA)的刺激,释放其结合的MYC2转录因子,从而启动JA应答基因的转录。JAZs还可以同其它转录因子和共阻遏蛋白相互结合,把茉莉酸信号通路与其他信号通路相关联起来,从而调节更多的生理过程。研究表明,JAZs蛋白可以调控花青素积累、表皮毛发育、植物体雄性不育、植物的生长与防御等过程。不过茶树中该基因的功能尚不明确。
发明内容
为了分析茶树JAZ1基因的功能,本发明将茶树JAZ1基因CsJAZ1转到拟南芥中进行异位表达。异位表达结果显示,该基因可使转基因拟南芥植株提早2~3天抽薹开花,而且开花早晚与该基因的表达量大小呈正相关关系。
本发明提供以下技术方案:
以茶树创伤应答反应转录组(登录号:SRX1223890)组装序列comp59845_c0_seq2为模板设计引物,根据茶树cDNA文库,运用3’和5’ RACE(cDNA末端快速扩增法)策略扩增出了茶树JAZ1基因的全长序列1267 bp,如SEQ ID NO:1所示,命名为CsJAZ1基因。CsJAZ1基因与JAZ1 [Maesa lanceolata](272 aa)162/268(60%),gap 46/268(17%)。与Jasmonate-zim-domain protein 1, putative [Theobroma cacao](274 aa)144/263(55%),gap 36/263(13%)。
本发明将CsJAZ1基因转到拟南芥中进行异位表达,可使转基因拟南芥植株提早2~3天抽薹开花,因此,可将CsJAZ1基因应用于促进拟南芥提前开花中。
CsJAZ1基因编码294 个AA,如SEQ ID NO:2所示。
将CsJAZ1基因转到拟南芥中进行异位表达,包括以下步骤:
步骤1:设计带有双酶切位点的、用于扩增该基因ORF的异位表达引物对,用异位表达引物对扩增出CsJAZ1基因的ORF序列;
步骤2:将扩增产物和质粒分别用双酶消化后,连接,并将连接产物转入大肠杆菌感受态细胞中,培养,挑菌,检测,测序;经测序验证序列完全正确后,提取该重组质粒;
步骤3:将重组质粒转入农杆菌感受态细胞中,培养获得含有重组表达载体的农杆菌菌液;
步骤4:用含有重组表达载体的农杆菌菌液浸泡拟南芥的花序,转化拟南芥,收获拟南芥新形成的T0种子;
步骤5:筛选阳性植株。
用于扩增CsJAZ1基因ORF的异位表达引物对,正向引物CsJAZ1-left:5’-GCTCTAG AGCATGTCGAGTTCGTCTGGTTCTGCTGA-3’,如SEQ ID NO:3所示,横线所示序列为XbaI酶切位点,反向引物CsJAZ1-right:5’-CGAGCTCCTACAAACGGTGCTCAAACTGCACCGGAGA-3’,如SEQ IDNO:4所示,横线所示序列为SacI酶切位点。
依据转录组数据对JAZ1基因在茶树不同组织器官中的表达量变化进行了分析,结果显示,JAZ1基因在茶树的芽、第一片叶和嫩根中的表达量高,在花、果实、枝条和较老的叶中表达量较低。因此推测JAZ1基因在茶树中可能主要影响芽的分化,促进茶树营养生长向生殖生长过程的转变,由此可能促进茶树花期提前。
本发明的有益效果在于:
1、本发明首次发现了茶树的CsJAZ1基因在拟南芥中异位表达,可使其提前开花。
2、由于茶树是叶用作物,其经济价值主要取决于其营养生长。但是,茶树从花芽形成到种子成熟要经历约一年半的时间,一株成龄茶树每年所开花数往往达上千朵,且花期长,由此造成在茶树的生命周期中不挂有花果的时间很短。花果的生长发育需要消耗大量养分,势必影响营养生长,早在1970年代,巴赫达兹研究组就发现茶树的孕蕾程度与茶叶产量间呈中度负相关。因此,生殖生长不利于叶用作物茶树的经济效益。若减少茶树的生殖生长就会促进营养生长,可能会有利于茶叶新芽的生长和新梢对低温等胁迫耐受能力的增强,这可能是提高茶叶产量、品质和经济效益的一条途径,该CsJAZ1基因在茶树的良种培育中将有着较大的潜在应用价值。
附图说明
图1为CsJAZ1基因转拟南芥植株在培养基中的阳性筛选图。
图2为野生型拟南芥和转基因株系的开花情况,图中植株自左边起为WT、株系7、株系14、株系4、株系21、株系22、株系8。
图3为转CsJAZ1基因拟南芥不同株系的基因组PCR验证图,图中M表示Marker,wt表示野生型拟南芥,L7、L14、L4、L21、L22、L8表示株系7、株系14、株系4、株系21、株系22、株系8。
图4为CsJAZ1基因在15个转该基因的拟南芥株系中的表达量图。图中wt指野生型,1、4、7、8、9、13、14、15、16、17、18、21、22、23、25为15个转基因拟南芥株系。
具体实施方式
以下实施例是为了更好地说明本发明的技术方案,而非用来限制本发明的保护范围。
实施例1CsJAZ1基因的克隆
以茶树创伤应答反应转录组(登录号:SRX1223890)组装序列comp59845_c0_seq2为模板设计了一对引物,正向引物5’-TCCGTGGAAGAGTCCCTCA-3’,如SEQ ID NO:5所示,反向引物5’-TGATTCCCACTAGCTCAGCGAGTGC-3’,如SEQ ID NO:6所示,依托发明人所在实验室构建的茶树cDNA文库(构建过程参考1. Wang L., Li X., Zhao Q. et al.:Identification of genes induced in response to low- temperature treatment intea leaves. – Plant Molecular Biology Reporter 27: 257-265, 2009. 2. Li X.,Lin Y., Zhao S., Zhao X., Geng Z., Yuan Z. Transcriptome changes and itseffect on physiological and metabolic processes in tea plant duringmechanical damage. Forest Pathology, 2018; e12432, DOI: 10.1111/efp.12432.),运用3’和5’ RACE(cDNA末端快速扩增法)策略扩增出了全长序列1267 bp的CsJAZ1基因。经序列比对显示,比原组装序列短了539 bp。在读码框(ORF)区仅有2个核苷酸的差别,但在两端的差别要大一些。发明人于2016-8-19将该基因序列提交到NCBI,Accession:KX759630。
实施例2pBI121- CsJAZ1表达载体的构建及转化
(1)根据CsJAZ1基因设计带有XbaI和SacI双酶切位点的引物对:CsJAZ1-left:5’-GCTCTAGAGCATGTCGAGTTCGTCTGGTTCTGCTGA-3’,如SEQ ID NO:3所示,CsJAZ1-right:5’-CG AGCTCCTACAAACGGTGCTCAAACTGCACCGGAGA-3’,如SEQ ID NO:4所示,用此引物对扩增出CsJAZ1基因的ORF序列;
(2)将扩增产物和pBI121质粒分别用XbaI/SacI双酶消化;
(3)将带有目的基因的酶切产物和酶切后的pBI121进行连接;
(4)将连接产物转入大肠杆菌感受态细胞中,培养,挑菌,检测,测序。经测序验证序列完全正确后,提取该重组质粒;
(5)将重组质粒pBI121-CsJAZ1加入农杆菌GV3010感受态细胞中,轻轻混匀;
(6)将混合液移至电转杯中,进行电击,然后立即加入800 µL的LB中,28℃,220rpm培养1 h;
(7)12,000 rpm离心1 min,弃上清,将剩余的菌液混匀,涂布在LB(含Kan和Rif)平板上,28℃培养2 d,获得含有pBI121- CsJAZ1表达载体的农杆菌菌液。
实施例3 转CsJAZ1基因拟南芥的培育
(1)浸花法转化:将野生型拟南芥Col-1(Arabidopsis thaliana L.)的种子于4℃条件下春化3 d,播种于栽培基质中(所述栽培基质中蛭石:营养土:珍珠岩=9:3:1),并置于拟南芥培养温室培养,拟南芥培养温室的温度22~25℃,光周期为14 h光照/ 10 h 黑暗。培养30 d左右至花期,用含有pBI121- CsJAZ1表达载体的农杆菌菌液浸泡拟南芥花序,转化拟南芥,收获拟南芥新形成的T0种子;
(2) 阳性植株筛选:
①培养基上筛选培养阳性植株:将所收获的新形成的拟南芥T0种子消毒灭菌后,分别放在含有卡那霉素的1/2MS培养基上萌发,温度22~25℃,光周期为14 h光照/10 h黑暗,从而获得拟阳性植株,如图1所示。
②对获得的拟阳性植株进行DNA提取及PCR检测:在含有卡那霉素的1/2MS培养基上萌发15 d左右,将健壮的幼苗转移至栽培基质中培育;之后10 ~15d采集野生型拟南芥DNA及不同转基因拟南芥株系植株的嫩叶,采用CTAB法提取其DNA,以提取的DNA为模板和特异性引物对CsJAZ1-left 和CsJAZ1-raight进行PCR扩增,电泳检测扩增产物。PCR扩增体系为:20µL反应体系:双蒸水7 µL,CsJAZ1-left 1 µL,CsJAZ1-raight 1 µL,Premix Taq 10µL ,DNA模板 1 µL,每管总体积20 µL。PCR扩增程序为:98℃预热10 s;30个循环:98℃变性10 s,56℃退火30 s,72℃延伸1 min;最后72℃保温5 min。
通过阳性苗筛选,获得了20个转CsJAZ1基因的拟南芥T2代株系,图3为所获转CsJAZ1基因拟南芥中的6个不同株系(株系7、株系14、株系4、株系21、株系22、株系8)的基因组DNA的PCR验证的电泳图谱(其他株系的结果未展示),且以野生型拟南芥DNA或水为模板做对照时,均未扩出目的条带。
(3)为了了解目的基因在转基因植株中的表达量高低,提取所获转CsJAZ1基因拟南芥15个不同株系T2代植株的RNA,对CsJAZ1基因在转基因植株中的表达量进行了qRT-PCR分析,正向引物JAZq1:5’-CACTCCTCCAGAGTGCCCGAGA-3’,如SEQ ID NO:7所示,
正向引物JAZq2:5’-CGTGAGGTTTCTCGTCACCGGAGT-3’,如SEQ ID NO:8所示。qRT-PCR体系为:DDW(双蒸水)7.0 µL,JAZq1 /JAZq2(N-Actin-1/2 )各0.8 µL,SYBR 10.0 µL,ROX 0.4 µL,cDNA模板1 µL,每管总体积20 µL。qRT-PCR扩增程序为:Stage1:95℃ 2 min;Stage2(40个循环):95℃ 15 s, 56℃ 30 s,72℃ 30 s;Stage3:熔解曲线程序:95℃ 15s—60℃ 30 s—95℃ 15 s。CsJAZ1基因在15个转该基因的拟南芥株系中的表达量结果如图4所示。株系7中CsJAZ1基因表达量最高,株系14次之。
(4)比较转CsJAZ1基因拟南芥不同株系与野生型植株的生长情况,结果表明,转基因拟南芥植株抽薹开花明显提前,如图2所示。但是,在花的结构、果荚的大小和所结种子的大小与数量未见明显差别。依据转录组数据(数据来源:TEA030190.1,安徽农大茶树不同器官转录组数据)对JAZ1基因在茶树不同组织器官中的表达量变化进行了分析(表1),结果显示,JAZ1基因在茶树的芽、第一片叶(Leaf1)和嫩根(Root)中的表达量高(表中的星号所示),在花、果实、枝条和较老的叶(Leaf2和3)中表达量较低。因此推测JAZ1基因在茶树中可能主要影响芽的分化,促进茶树营养生长向生殖生长过程的转变,由此可能促进茶树花期提前。若是选育该基因表达量低的个体用于育种,使其花期推迟至冬季,借助低温抑制花芽的形成及花的授粉,就有可能培育出开花结果少、营养生长旺盛的优良茶树品种。
表1. JAZ1基因在茶树不同组织器官中的表达量(数据来源:TEA030190.1)
不同器官 | Bud | Flower | Fruit | Leaf1 | Leaf2 | Leaf3 | Root | Shoot |
RPKM | 1250.9575 | 143.3700 | 189.6417 | 788.3548 | 140.9829 | 83.4837 | 893.5520 | 273.1034 |
★ | ★ | ★ |
注:1. RPKM:RPKM是Reads Per Kilobase per Million mapped reads的缩写,代表每百万reads中来自于某基因每千碱基长度的reads数。2. Leaf1:茶树新生的第一片叶,Leaf2/3则指第二和第三片叶。3. ★:指示表达量高器官。
以上所述之实施例,只是本发明的较佳实施例而已,仅仅用以解释本发明,并非限制本发明实施范围,对于本技术领域的技术人员来说,当然可根据本说明书中所公开的技术内容,通过置换或改变的方式轻易做出其它的实施方式,故凡在本发明的原理及工艺条件所做的变化和改进等,均应包括于本发明申请专利范围内。
SEQUENCE LISTING
<110>信阳师范学院
<120>茶树 CsJAZ1基因的应用
<130>无
<160> 8
<170>PatentIn version 3.5
<210> 1
<211> 1267
<212> DNA
<213> 茶树(Camellia sinensis)
<400> 1
aatggccattacggccggggaaccaaaccaaagcgataagccttaccctattactcagca 60
cctatacatatacacacagtgctattacttccagagagagaaaacaaaaaccataaggta 120
gagagagagagagagaaggcagtttaagtttttggttgatttctttgatcctctgatcga 180
ttttgagtttgtttgctggaaactatgtcgagttcgtctggttctgctgattccgggagg 240
ttttccggccatagacactcctccagagtgcccgagaagtcggcgtcgtcgagcttctct 300
cagacgtgtagtttgttgagccagtacctgaaggagaagaaaggcacattcggagatctc 360
agtctcggcatgccaaccgcaacgacaaccatgaatttgttccccgttgctatgaaatcg 420
agtgaggtttcccgaaatggaactccggtgacgagaaacctcacgtctatggacttattt 480
ccacagcagtctggttttggttccaacatttccgtggaagagtccctcaacaaggttgat 540
tcgagtgccataaacaagtcagagccagaaactgcacaaatgaccatattctatgccggg 600
caagtgattgtgtttaatgatttcccggctgataaggcaaaggaaatcatgctcttagct 660
agcaagggaagttcccatcacaaccccggcacgcttgcttcaacaccggtccaaaaacca 720
atcgaacccactaatttgattcccactagctcagcgagtgctattgtccctaattcaggc 780
aacaatacgatctcaatcgaacccattagtttgattcccactacctcgagtgttgctgcc 840
cctaattttagcaacaatatgattcaagaacgtgtccaggtccaacgggcttctcaaccc 900
attgctaccgatctaccaattgctagaaaagcgtcgctcacccggttcttggagaagaga 960
aaagataggatcacatcaagagcaccataccaaataagcagctcaacttctcctcccaag 1020
ccgactgaaagcaagtcatggctcggcttggcagctcaatctccggtgcagtttgagcac 1080
cgtttgtagtcattttggcagcatcagttatattcagcagtgttatgttgtattttttac 1140
tatatgtttcggcttattttggttattgaatctacaaagccagccattagatctaactgt 1200
ttcgatcttacttttagtatgtgtaattagattaatgggaaagtcttcattataaaaaaa 1260
aaaaaaa
1267
<210> 2
<211> 294
<212> PRT
<213> 茶树(Camellia sinensis)
<400> 2
Met Ser Ser Ser Ser Gly Ser Ala Asp Ser Gly Arg Phe Ser Gly His
1 5 10 15
Arg His Ser Ser Arg Val Pro Glu Lys Ser Ala Ser Ser Ser Phe Ser
20 25 30
Gln Thr Cys Ser Leu Leu Ser Gln Tyr Leu Lys Glu Lys Lys Gly Thr
35 40 45
Phe Gly Asp Leu Ser Leu Gly Met Pro Thr Ala Thr Thr Thr Met Asn
50 55 60
Leu Phe Pro Val Ala Met Lys Ser Ser Glu Val Ser Arg Asn Gly Thr
65 70 75 80
Pro Val Thr Arg Asn Leu Thr Ser Met Asp Leu Phe Pro Gln Gln Ser
85 90 95
Gly Phe Gly Ser Asn Ile Ser Val Glu Glu Ser Leu Asn Lys Val Asp
100 105 110
Ser Ser Ala Ile Asn Lys Ser Glu Pro Glu Thr Ala Gln Met Thr Ile
115 120 125
Phe Tyr Ala Gly Gln Val Ile Val Phe Asn Asp Phe Pro Ala Asp Lys
130 135 140
Ala Lys Glu Ile Met Leu Leu Ala Ser Lys Gly Ser Ser His His Asn
145 150 155 160
Pro Gly Thr Leu Ala Ser Thr Pro Val Gln Lys Pro Ile Glu Pro Thr
165 170 175
Asn Leu Ile Pro Thr Ser Ser Ala Ser Ala Ile Val Pro Asn Ser Gly
180 185 190
Asn Asn Thr Ile Ser Ile Glu Pro Ile Ser Leu Ile Pro Thr Thr Ser
195 200 205
Ser Val Ala Ala Pro Asn Phe Ser Asn Asn Met Ile Gln Glu Arg Val
210 215 220
Gln Val Gln Arg Ala Ser Gln Pro Ile Ala Thr Asp Leu Pro Ile Ala
225 230 235 240
Arg Lys Ala Ser Leu Thr Arg Phe Leu Glu Lys Arg Lys Asp Arg Ile
245 250 255
Thr Ser Arg Ala Pro Tyr Gln Ile Ser Ser Ser Thr Ser Pro Pro Lys
260 265 270
Pro Thr Glu Ser Lys Ser Trp Leu Gly Leu Ala Ala Gln Ser Pro Val
275 280 285
Gln Phe Glu His Arg Leu
290
<210> 3
<211> 36
<212> DNA
<213>人工序列(Artificial Sequence)
<400> 3
gctctagagcatgtcgagttcgtctggttctgctga 36
<210> 4
<211> 37
<212> DNA
<213>人工序列(Artificial Sequence)
<400> 4
cgagctcctacaaacggtgctcaaactgcaccggaga 37
<210> 5
<211> 19
<212> DNA
<213>人工序列(Artificial Sequence)
<400> 5
tccgtggaagagtccctca 19
<210> 6
<211> 25
<212> DNA
<213>人工序列(Artificial Sequence)
<400> 6
tgattcccactagctcagcgagtgc 25
<210> 7
<211> 22
<212> DNA
<213>人工序列(Artificial Sequence)
<400> 7
cactcctccagagtgcccgaga 22
<210> 8
<211> 24
<212> DNA
<213>人工序列(Artificial Sequence)
<400> 8
cgtgaggtttctcgtcaccggagt 24
Claims (3)
1.茶树 CsJAZ1基因在促进拟南芥提前开花中的应用,其特征在于,所述CsJAZ1基因的序列如SEQIDNO:1所示。
2.根据权利要求1所述的应用,其特征在于,所述CsJAZ1基因编码的蛋白的氨基酸序列如SEQIDNO:2所示。
3.根据权利要求1所述的应用,其特征在于,具体为将CsJAZ1基因转到拟南芥中进行异位表达,包括以下步骤:
步骤1:设计带有双酶切位点的、用于扩增CsJAZ1基因ORF的异位表达引物对,用异位表达引物对扩增出CsJAZ1基因的ORF序列;
步骤2:将扩增产物和质粒分别用双酶消化后,连接,并将连接产物转入大肠杆菌感受态细胞中,培养,挑菌,检测,测序;经测序验证序列完全正确后,提取该重组质粒;
步骤3:将重组质粒转入农杆菌感受态细胞中,培养获得含有重组表达载体的农杆菌菌液;
步骤4:用含有重组表达载体的农杆菌菌液浸泡拟南芥的花序,转化拟南芥,收获拟南芥新形成的T0种子;
步骤5:筛选阳性植株。
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