CN117126859A - 一种调控拟南芥器官大小的基因及其应用 - Google Patents

一种调控拟南芥器官大小的基因及其应用 Download PDF

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CN117126859A
CN117126859A CN202110792234.4A CN202110792234A CN117126859A CN 117126859 A CN117126859 A CN 117126859A CN 202110792234 A CN202110792234 A CN 202110792234A CN 117126859 A CN117126859 A CN 117126859A
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孔英珍
唐贤丰
刘宇
周功克
柴国华
王丛鹏
孟杰
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Qingdao Agricultural University
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Abstract

本发明请求保护一种调控拟南芥器官大小的基因及其应用，属于基因工程领域，具体的说是一种CCCH锌指蛋白基因C3H15及其应用。CCCH基因AtC3H15的碱基序列如SEQ ID No.1中所示。所述AtC3H15基因作为参与器官大小发育，进而增加植株生物量的调控基因的应用。本发明从拟南芥（col‑0）中克隆得到的AtC3H15基因，具有潜在的应用前景。

Description

一种调控拟南芥器官大小的基因及其应用
技术领域
本发明属于基因工程领域，具体的说是一种CCCH锌指蛋白基因C3H15及其应用。
背景技术
植物器官的大小是影响植物生物量的重要因素，其主要受环境信号及遗传因子的双重调节。拟南芥具有基因组小，易于转化，生长快速等特点，是研究植物器官发育的理想模式植物，但目前为止，报道的影响植物器官大小的基因很少，因此，深入挖掘与解析拟南芥中调控器官大小的相关基因不仅有助于加深对植物形态建成的调控机理的理解，同时也可为其他重要作物在提高粮食产量，品质等方面提供重要的应用指导与借鉴意义。
拟南芥CCCH锌指蛋白基因家族包含68个成员，根据其结构域组成数目和类型分为11个亚家族，参与多种生长发育及胁迫过程。其中，拟南芥AtTZF4、AtTZF6、AtTZF3、AtTZF9分别在种子萌发，心期胚珠形成，ABA信号响应盐胁迫及抗病免疫途径中发挥重要作用。我们通过生物信息学在拟南芥中鉴定了一个CCCH基因AtC3H15，该基因在植株各个组织中均有表达（图2），意味着其可能参与调控植株的器官发育过程。
器官大小是影响生物量的重要因素，而且与作物产量息息相关，对其调控机制的研究意义重大。以往研究报道的关键调控基因十分有限，因此需要挖掘更多参与调控器官大小发育的基因，为增加植物生物量提供更多选择。
发明内容
本发明目的在于提供一种CCCH基因AtC3H15及其应用。
为实现上述目的，本发明采用技术方案为： 一种CCCH基因AtC3H15的碱基序列如SEQ ID No .1中所示。
CCCH基因AtC3H15的编码蛋白的氨基酸序列如SEQ ID No .2中所示。
一种CCCH基因AtC3H15的应用，所述AtC3H15基因作为调控植株器官大小发育，进而为增加植株生物量提供更多选择的调控基因的应用。
一种植物表达载体，载体带有所述CCCH基因SEQ ID No .1核苷酸序列。
所述植物为拟南芥及其它作物。
一种植物表达载体的应用，所述植物表达载体在增加植株生物量中的应用。
本发明所具有的优点： 1.本发明从拟南芥中获得能够在植株各个组织广谱表达的AtC3H15基因； 2.本发明通过获得的具有广谱表达特性的AtC3H15基因，遗传转化产生AtC3H15基因的过表达植株，并鉴定AtC3H15及其同源基因AtC3H14的双突变体。AtC3H15过表达转基因植株相对于对照植株各个器官变小，c3h14c3h15双突变体植株变大，具有较大的研究价值。
附图说明
图1为本发明实施例提供的拟南芥C3H15基因及其在水稻，高粱，玉米和小麦中的同源基因氨基酸序列比对结果，以上四种作物的氨基酸序列如SEQ ID No .3-6中所示。比对结果显示这个基因在几种作物中都含有保守的两个串联的C8C5C3H结构域。
图2为本发明实施例提供的qRT-PCR 分析拟南芥AtC3H15基因的组织表达模式图；拟南芥Actin2 基因用作为内参，结果显示这个基因在拟南芥各个组织中均有表达，但在茎秆中表达相对较高。
图3为本发明实施例提供的拟南芥AtC3H15基因过表达构建示意图和c3h14c3h15双突变体的T-DNA插入系。
图4为本发明实施例提供的qRT-PCR 分析AtC3H15及其同源基因AtC3H14在AtC3H15过表达植株和c3h14c3h15双突变中的表达水平示意图。拟南芥Actin2 基因用作内参。
图5为本发明实施例提供的拟南芥AtC3H15转基因植株的表型分析图。col-0, 作为对照的野生型；C3H15OE过表达转基因株系；c3h14c3h15双突变体株系。
具体实施方式
实验所涉及的药品均购自Sigma 公司、Fermentas 公司、Thermo Fisher公司、北京天根公司。具体实验操作依据《分子克隆》手册。
实施例1 拟南芥CCCH基因AtC3H15的分子克隆
1. 拟南芥AtC3H15基因的克隆
取培养室生长1.5个月左右的拟南芥茎秆，利用TRIZOL法提取其总RNA，利用RT-PCR技术扩增AtC3H15基因的全长cDNA，将其连入pMD18-T中测序（参见SEQ ID No .1）。扩增条件为：95℃ 5 min预变性；94℃ 30s, 55℃ 40s,72℃ 1min,35个循环；72℃ 8min 片段延伸。扩增引物为：
AtC3H15cDNAF: 5’-ATGGAAAACAAAATCGCGC-3’；
AtC3H15cDNAR: 5’-TCATGTGATCAGCTTGAGGGAT-3’；
2. 拟南芥AtC3H15基因的序列信息与特性分析
AtC3H15基因包含927bp的编码区（参见SEQ ID No.1），编码蛋白的分子量为34.2kDa，C端有一个由25个氨基酸组成的CCCH保守结构域，是典型的CCCH锌指蛋白；
3. 拟南芥AtC3H15基因的表达模式分析以培养室生长1.5个月的拟南芥为材料，选取其果荚、根、莲座叶、嫩叶、基部茎、上部茎、芽、花，利用TRIZOL法分别提取其总RNA，反转录得到第一
链cDNA。以AtC3H15qPCR(5’-CGCGCCGTTTAGTTACAGCG-3’; 5’-CACAACGCCTCGCGAT
TCACC-3’)为引物，利用现有的qRT-PCR方法检测拟南芥AtC3H15基因在这七个组织中的表达情况。扩增条件为：95℃ 3min 预变性；94℃ 10s, 60℃ 20s,72℃ 20s , 40个循环；72℃ 5 min 片段延伸。拟南芥Actin2基因用作内参。结果表明AtC3H15基因在植株各个组织中均有表达（参见图2）。
实施例2拟南芥CCCH基因AtC3H15的转基因应用。
1. 拟南芥CCCH基因AtC3H15过表达植株和c3h14c3h15双突变体的获得
将拟南芥CCCH基因全长cDNA正向序列连接到pCAMBIAL1300载体中，测序正确后得到含有AtC3H15基因的过表达载体。将C3H14和C3H15的T-DNA插入纯合突变体进行杂交,PCR检测后获得双突变体（参见图3）。
2.过表达载体通过农杆菌介导转化拟南芥 将过夜培养的5ml pCAMBIAL1300-AtC3H15菌液全部转入300ml LB液体培养基中（含有相应抗生素），28-30℃，220RPM培养过夜，摇至OD=2.0；将过夜培养的300ml菌液离心，室温，6000rpm，离心10min，将上清倒掉，收菌；将收集的农杆菌用150ml转化Buffer重悬（转化Buffer：0.5%蔗糖溶液含有0.02%Silwet），在将重悬好的菌液倒入150ml烧杯中；将拟南芥花序倒置浸入菌液中，尽量使全部的花序都浸入菌液中，反复浸入5-6次，然后将拟南芥倒放于接水盘中，用保鲜膜覆盖，与培养间（22℃）过夜，然后正常培养。转化一周以后，可以再重新转化一次，常规管理至种子成熟。
3.转基因植株分子鉴定
取生长1个月的候选转基因拟南芥叶片，在液氮中利用TRIZOL法提取其总RNA，用DNAase I（Sigma公司）去除可能污染的基因组DNA，然后利用反转录试剂盒（Fermentas公司）反转录成第一链cDNA。以AtC3H15qPCRF/R及AtC3H14qPCRF/R(5’-AGAGTTCGCCGCAAGCTTCGC-3’;5’-CGTAATGCTCGTTGAGTTCGTCGT-3’)为引物，利用qRT-PCR法检测其相对于对照植株（转化空载体）的表达水平。扩增条件为：95℃ 3min 预变性；94℃10 s, 60℃ 20s,72℃ 20s , 40个循环；72℃ 5 min 片段延伸。拟南芥Actin2基因用作内参。结果如图4所示，AtC3H15基因在过表达植株中的表达量要比对照高，而在c3h14c3h15双突变体植株中的表达量要比对照低，且AtC3H14基因表达量在双突变体植株中要比对照低。这些结果表明证实AtC3H15过表达和c3h14c3h15双突变体的有效性。
4. AtC3H15过表达植株矮小，而c3h14c3h15双突变体植株高大 观察温室培养1.5个月 AtC3H15转基因植株的表型，相对于对照（转化空载体），过表达植株生长受到抑制，各个器官包括花，果荚，叶等均变小而双突变体植株生长得到促进，各个器官均变大，生物量增加（参见图5）。
序列表
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<211> 352
<212> PRT
<213> 玉米(Zea mays)
<400> 5
Met Gln Glu Ala Leu Val Ser Pro Ala Asn Ala Gly Arg Leu Arg Ser
1 5 10 15
Ala Leu Glu Leu Lys Pro Phe Ala Phe Gly Asp Gln Arg Leu Ala Ser
20 25 30
Pro Arg Tyr Leu Asn Leu Ala Ser Val Ala Gly Gly Gly Gly Gly Asp
35 40 45
Asp Ala Leu Phe Arg Cys Ser Ser Pro Phe Ser Pro Ser Phe Gly Phe
50 55 60
Ser Ser Pro Ser Pro Leu Ala Thr Ser Ser Thr Ile Ser Leu Ser Pro
65 70 75 80
Ser Ser Ser Ser Ser Leu Val Gly Asp Cys Asp Cys Asp Asp Asp Ala
85 90 95
Asp Ala Asp Ala Ala Thr Gly His Arg Leu Gln Leu Ala Arg Leu Ala
100 105 110
Leu Gln Tyr Gln Glu Val Ala Asp Arg Tyr Glu Leu Cys Leu Ala Arg
115 120 125
Leu Ala Asp Ala Ala Asp Glu Ala Ala Ala Leu Arg Arg Glu Asn Ala
130 135 140
Glu Leu Arg Val Ala Asn Gly Asp Leu Thr Arg Arg Leu Ala Leu Leu
145 150 155 160
Ser Gly Ile Gly Lys Gln Ala Ala Ala Ala Ala Ile Ala Asp Glu Val
165 170 175
Arg Arg Leu Arg Phe Gly Glu Gln Lys Ala Ala Lys Glu Arg Ala Pro
180 185 190
Glu Lys Pro Ala Val Leu Pro Lys Ser Ile Ser Val Arg Ser Asn Asp
195 200 205
Tyr Leu Lys Met Asn Gln Pro Gln Gln Val Gln Ala Pro Ala Thr Pro
210 215 220
Ala Glu Asp Asn Arg Lys Pro Arg Ala Ser Asn Pro Thr Thr Asn Arg
225 230 235 240
Ser Ser Gln Arg Val Tyr Lys Gly Asn Gly Gly Asp Lys Arg Ser Glu
245 250 255
Glu Pro Lys Glu His Arg Thr Ala Gly Gly Val Glu Leu Glu Val Tyr
260 265 270
Asn Gln Gly Met Leu Lys Thr Glu Leu Cys Asn Lys Trp Glu Glu Thr
275 280 285
Gly Ala Cys Pro Tyr Gly Asp Gln Cys Gln Phe Ala His Gly Val Ala
290 295 300
Glu Leu Arg Pro Val Ile Arg His Pro Arg Tyr Lys Thr Gln Val Cys
305 310 315 320
Arg Met Val Leu Ala Gly Glu Val Cys Pro Tyr Gly His Arg Cys His
325 330 335
Phe Arg His Thr Leu Thr Pro Ala Glu Arg Leu His Leu Pro Arg Pro
340 345 350
<210> 6
<211> 335
<212> PRT
<213> 小麦(Triticum aestivum)
<400> 6
Met Gln Glu Ala Leu Val Ser Pro Ala Asn Ala Asp Arg Leu Arg Ser
1 5 10 15
Ala Phe Glu Leu Lys Pro Tyr Ala Phe Gly Asp Gln Arg Leu Ser Ser
20 25 30
Ser Pro Arg Tyr Leu Pro Gly Gly Asp Asp Gly Leu Tyr Arg Cys Ser
35 40 45
Ser Pro Phe Ser Pro Ser Leu Gly Phe Ser Ser Pro Ser Pro Leu Thr
50 55 60
Thr Ser Val Ser Leu Ser Pro Ser Ser Ser Gly Ser Leu Val Asp Asp
65 70 75 80
Gly Asp Tyr Asp Gly Ala Ala Ala Ala Asp Ala Thr Gly His Arg Leu
85 90 95
Gln Leu Ala Arg Leu Ala Leu Gln Tyr Gln Glu Val Ala Asp Arg Tyr
100 105 110
Glu Leu Cys Leu Ser His Leu Ala Glu Ala Val Asp Glu Ala Ala Ile
115 120 125
Leu Arg Arg Glu Asn Ala Glu Leu Arg Val Ala Asn Asn Asp Leu Ala
130 135 140
Arg Arg Val Ala Val Leu Gly Gly Lys Glu Thr Ala Ala Val Val Ile
145 150 155 160
Ala Asp Glu Ile Arg Arg Phe Arg Leu Gly Glu His Lys Gly Ala Ser
165 170 175
Lys Gln Arg Ala Pro Glu Lys Leu Ala Val Leu Pro Lys Ser Ile Ser
180 185 190
Val Arg Ser Asn Asp Tyr Leu Lys Met Asn Gln Ala Ala Pro Ala Ala
195 200 205
Ala Thr Pro Ala Ala Tyr Asn Arg Lys Pro Ser Ser His Leu Asp Arg
210 215 220
Gln Gln Arg Ala Tyr Ala Gly Leu Gly Ala Asp Gly Gly Lys Lys Gly
225 230 235 240
Glu Glu Gln Lys Thr Lys Gln Asp Ala Ala Gly Glu Leu Asp Val Tyr
245 250 255
Asn Gln Gly Met Phe Lys Thr Glu Leu Cys Asn Lys Trp Glu Glu Thr
260 265 270
Gly Ala Cys Pro Tyr Cys Asp Gln Cys Gln Phe Ala His Gly Val Ser
275 280 285
Glu Leu Arg Pro Val Ile Arg His Pro Arg Tyr Lys Thr Glu Val Cys
290 295 300
Arg Met Val Leu Asn Gly Glu Val Cys Pro Tyr Gly His Arg Cys His
305 310 315 320
Phe Arg His Ser Leu Thr Ala Ala Glu Arg Leu Leu Arg Ser Arg
325 330 335

Claims (6)

1.一种CCCH基因AtC3H15，其特征在于：AtC3H15的碱基序列如SEQ ID No .1中所示。
2.如权利要求1所述的CCCH基因AtC3H15的编码蛋白，其特征在于：AtC3H15的编码蛋白的氨基酸序列如SEQ ID No .2中所示。
3.一种权利要求1所述的CCCH基因AtC3H15的应用，其特征在于：所述AtC3H15基因作为参与器官大小发育，进而增加植株生物量的调控基因的应用。
4.一种植物表达载体，其特征在于：载体带有所述CCCH基因SEQ ID No .1核苷酸序列。
5.如权利要求4所述的植物表达载体，其特征在于：所述植物为拟南芥及作物。
6.一种权利要求4所述的植物表达载体的应用，其特征在于：所述植物表达载体在调控器官大小发育及植株生物量中的应用。
CN202110792234.4A 2021-07-14 2021-07-14 一种调控拟南芥器官大小的基因及其应用 Pending CN117126859A (zh)

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