香蕉<i>MaAQP1</i>启动子诱饵载体及干旱胁迫酵母单杂交cDNA文库的构建

许奕; 李羽佳; 魏卿; 王安邦; 王笑一; 宋顺; 李敬阳

doi:10.19303/j.issn.1008-0384.2020.10.005

香蕉MaAQP1启动子诱饵载体及干旱胁迫酵母单杂交cDNA文库的构建

doi: 10.19303/j.issn.1008-0384.2020.10.005

许奕^{1, 2,},
李羽佳¹,
魏卿¹,
王安邦¹,
王笑一¹,
宋顺^{1, 2},
李敬阳^1, ,

1.
中国热带农业科学院海口实验站/海南省香蕉遗传改良重点实验室，海南　海口　571101
2.
海南省南繁生物安全与分子育种重点实验室，海南　三亚　572000

基金项目: 中国热带农业科学院基本科研业务费专项项目（1630092020002）；国家自然科学基金项目（31501371）；中央级公益性科研院所基本科研业务专项项目（1630092020007）

详细信息

作者简介:
许奕（1985−），女，硕士，研究方向：作物遗传育种（E-mail：lukydog163@163.com）

通讯作者:
李敬阳（1976−），男，硕士，副研究员，研究方向：作物遗传育种（E-mail：jingyanglee@163.com）

中图分类号: S 668.1
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出版历程
- 收稿日期: 2020-06-15
- 修回日期: 2020-07-29
- 刊出日期: 2020-10-28

Constructions of Banana MaAQP1 Bait Vector and Drought-resistance cDNA Library

XU Yi^{1, 2
,},
LI Yujia¹,
WEI Qing¹,
WANG Anbang¹,
WANG Xiaoyi¹,
SONG Shun^{1, 2},
LI Jingyang^{1
, ,}

1.
Haikou Experimental Station, Chinese Academy of Tropical Agricultural Sciences/Hainan Key Laboratory of Banana Genetic Improvement, Haikou, Hainan　571101, China
2.
Hainan Key Laboratory for Biosafety Monitoring and Molecular Breeding in Off-Season Reproduction Regions, Sanya, Hainan　572000, China

摘要

摘要: 目的香蕉MaAQP1能够提高植物的耐旱性，研究香蕉MaAQP1的相关特性可为了解其干旱胁迫响应机制奠定基础。方法通过克隆MaAQP1的启动子，将启动子构建到pHIS2诱饵质粒上，转化酵母菌构建诱饵表达载体，同时构建干旱胁迫的香蕉（Musa acuminat L. AAA group cv. Brazilian）cDNA文库。结果克隆获得1 362 bp的启动子序列，通过分析其顺式作用元件，结果显示启动子序列中共有72个顺式作用元件，包括了TATA-box和CAAT-box核心元件，ABA响应元件、MYB元件、MYC元件、ERE元件、MeJA响应元件、光响应元件以及分生组织响应元件等；成功构建了MaAQP1诱饵载体和干旱胁迫条件下的cDNA文库，文库库容为1.25×10⁷ CFU，插入片段平均在1 200 bp左右。结论本研究克隆获得了香蕉MaAQP1启动子并构建了干旱胁迫酵母单杂交cDNA文库，为下一步运用酵母单杂交筛选MaAQP1互作的转录因子、解析MaAQP1响应干旱胁迫的作用机制奠定了基础。
- 香蕉 /
- 干旱 /
- MaAQP1 /
- 诱饵载体 /
- cDNA文库
Abstract: Objective To clone MaAQP1 from a drought stressed banana plant using a constructed bait vector, and establish a cDNA library of the transformed drought-resistance gene from single-hybrids yeast cells. Method The promoter of MaAQP1 from a banana plant (Musa acuminat L. AAA group cv. Brazilian) under draught-stress was constructed on the pHIS2 plasmid as the bait to be transformed into yeast cells for the gene cloning. A cDNA library of MaAQP1 in the yeast was subsequently established for the study. Result The MaAQP1 bait vector was successfully constructed. The sequence of the 1 362 bp promoter was cloned and analyzed to show 72 cis-acting TATA-box and CAAT-box core elements as well as elements of MYB, MYC, ERE, and MeJA as well as those of ABA, light, and meristem responses, etc. A drought-resistance cDNA library of 1.25×10⁷ CFU in capacity with an average insert size of about 1 200 bp was established. Conclusion The results provided a basis for screening transcription factors of the cloned MaAQP1 in the yeast single-hybrids to decipher the mechanism of MaAQP1 in response to drought for improving the stress resistance of plants.
- Banana /
- drought /
- MaAQP1 /
- bait vector /
- cDNA library

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图 1 MaAQP1启动子的PCR鉴定

注：泳道1–2分别为随机挑取的2个启动子的PCR扩增产物，M为DNA Marker。

Figure 1. PCR identification of MaAQP1 promoter

Note：Lanes 1–2 are the PCR amplification products of 2 promoters picked at random, M is DNA Marker.

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图 2 诱饵质粒pHIS2-MaAQP1菌落PCR鉴定

注：M为DNA Marker，泳道1～6分别为随机挑取的6个pHIS2-A启菌落的扩增产物。

Figure 2. PCR results of bait plasmid pHIS2-MaAQP1 colony

Note：M is DNA Marker, Lanes 1–6 are the amplification products of 6 randomLy selected pHIS2-A starter colonies.

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图 3 诱饵自激活检测结果

Figure 3. Detection by self-activation bait

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图 4 RNA提取结果

注：M为DNA Marker，泳道1～2分别为提取的RNA扩增产物。

Figure 4. Results of RNA extraction

Note: M is DNA Marker; Lanes 1-2 are the RNA amplification products.

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图 5 电泳检测mRNA条带

Figure 5. Electrophoretic detection of mRNA bands

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图 6 cDNA文库插入片段电泳图

Figure 6. Electrophoresis of insert fragment in cDNA library

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表 1 酵母转化反应

Table 1. Yeast transformation reaction

反应 Reaction	AD质粒 AD plasmid	BD质粒 BD plasmid	转化平板 Conversion plate	检测平板 Detection plate	检测内容 Test content
1	——	pHIS2-pMaAQP1	SD-T	SD-TH+3AT	自激活检测 Self-activation detection
2	pGAD53m	pHIS2	SD-TL	SD-TLH+3AT	阴性对照 Negative control
3	pGAD53m	pHIS2-p53	SD-TL	SD-TLH+3AT	阳性对照 Positive control

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表 2 MaAQP1启动子元件分析

Table 2. Element analysis on MaAQP1 promoter

序号 Number	结合位点 TFBs	元件功能 Function of the motif	器官 Organism	序列 Sequence	位置 Position
1	AAGAA-motif	LRE	燕麦 Avena sativa	GAAAGAA/gGTAAAGAAA	−1 172,−1 178
2	ABRE	脱落酸响应 Abscisic acid responsiveness	拟南芥 Arabidopsis thaliana	ACGTG	−55,−863
3	AT～TATA-box	LRE	拟南芥 Arabidopsis thaliana	TATATA	−319,−343,−331,−355,−327,−353,−339,−363, −323,−347,−335,−359,−329,−351,−341,−321, −345,−333,−325,−337,−357,−349,−361
4	Box II	光响应元件 light responsive element	马铃薯 Solanum tuberosum	TGGTAATAA	−32
5	CAAT-box	核心启动子 Core promoter	烟草/拟南芥/豌豆 Nicotiana glutinosa/Arabidopsis thaliana/Pisum sativum	CAAT	−43,−459,−315,−316,−125,−583,−1 197, −208,−725,−421,−471,−158
6	CAT-box	分生组织表达 meristem expression	拟南芥 Arabidopsis thaliana	GCCACT	−254
7	CCAAT-box	结合位点 MYBHv1 binding site MYBHv1	大麦 Hordeum vulgare	CAACGG	−935
8	CGTCA-motif	响应 MeJA-responsiveness MeJA	大麦 Hordeum vulgare	CGTCA	−1 088
9	ERE	烯响应元件 ethylene-responsive element	烟草 Nicotiana glutinos	ATTTTAAA	−377,−775
11	GT1-motif	乙烯响应元件 light responsive element	燕麦 Avena sativa	GGTTAAT	−65
12	I-box	光响应元件 light responsive element	小麦/棉花 Triticum aestivum/ Gossypium hirsutum	AAGATAAGGCT/AGATAAGG	−611,−612
13	MYB	SRE	拟南芥 Arabidopsis thaliana	CAACCA	−151
14	MYB recognition site	SRE	拟南芥 Arabidopsis thaliana	CCGTTG	−878
15	MYC	SRE	拟南芥 Arabidopsis thaliana	CATTTG/CATGTG	−111,−264
16	TATA-box	核心启动子元件 Core promoter element	甘蓝/拟南芥 Brassica napus/ Arabidopsis thaliana	ATATAT/TATATA/taTATAAAtc/ATTATA	−318,−326,−323,−320,−328,−324,−332,−319, −327,−322,−321,−325,−330,−329,−333,−334, −335,−336,−343,−344,−345,−346,−347,−348, −349,−350,−351,−352,−353,−354,−355, −356,−357,−358,−359,−363,−364,−365, −389,−391,−837,−838
17	TGACG-motif	MeJA响应 MeJA-responsiveness	大麦 Hordeum vulgare	TGACG	−58,−226
18	Unnamed__1	未知 unknown	玉米 Zea mays	CGTGG	−864,−1 274
19	Unnamed__4	未知 unknown	欧芹 Petroselinum hortense	CTCC	−28,−1 016,−1 009,−1 353,−913,−1 255, −1 013,−1 261,−922
20	as-1	LRE	拟南芥 Arabidopsis thaliana	TGACG	−58,−226

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表 3 自激活检测结果

Table 3. Results of self-activation test

反应 Reaction	转化平板 Conversion plate	检测平板 Detection plate	检测内容 Test content	3AT/（mmol·L⁻¹）
				0	25		50
				生长数目 Growth number	生长数目 Growth number	25/0 生长比例 25/0 Growth ratio	生长数目 Growth number	50/0 生长比例 50/0 Growth ratio
1	SD-T	SD-TH	自激活检测 Self-activation detection	3 424	408	11.9%	68	1.9%
2	SD-TL	SD-TLH	阴性对照 Negative control	3 968	1 376	34.6%	31	0.7%
3	SD-TL	SD-TLH	阳性对照 Positive control	5 024	5 136	102.2%	4 160	82.8%

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