低温胁迫下山葡萄叶片酵母cDNA文库构建

马登辉; 马红义; 李承男; 虎志瑞; 丁小雨; 梁敏; 尹晓

doi:10.19303/j.issn.1008-0384.2022.009.008

低温胁迫下山葡萄叶片酵母cDNA文库构建

doi: 10.19303/j.issn.1008-0384.2022.009.008

宁夏大学农学院，宁夏　银川　750021

基金项目: 宁夏回族自治区自然科学基金项目（2022AAC03010）；宁夏回族自治区重点研发计划项目（2022BBF03004）；宁夏大学大学生创新创业训练计划项目（S202210749076）

详细信息

作者简介:
马登辉（2002−），男，主要从事果树分子育种相关研究（E-mail：denghui0629@sina.com）

通讯作者:
尹晓（1990−），男，博士，讲师，主要从事果树分子育种教学和相关研究（E-mail：yinxiao90@nxu.edu.cn）

中图分类号: S 663.1
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- 被引次数: 0
出版历程
- 收稿日期: 2022-07-22
- 录用日期: 2022-07-22
- 修回日期: 2022-09-03
- 网络出版日期: 2022-10-05
- 刊出日期: 2022-09-30

Construction of Yeast cDNA Library Using Vitis amurensis Leaves Under Low-temperature Stress

College of Agronomy, Ningxia University, Yinchuan, Ningxia　750021, China

摘要

摘要: 目的葡萄栽培过程中，由低温造成的冷害或冻害严重影响葡萄的生长发育、果实品质和产量。山葡萄是葡萄抗寒育种的重要种质资源，本文通过构建低温胁迫下山葡萄叶片酵母cDNA文库，为葡萄抗寒分子机理研究提供物质基础。方法以抗寒山葡萄左山-1（V. amuerensis cv.Zuoshan-1）扦插盆栽苗为材料，提取4 ℃低温处理0、2、4、8、12、24 h后叶片总RNA，采用SMART技术反转录为cDNA，经纯化和短片段去除后克隆至pGADT7三框质粒载体上，经过纯化后获得初级cDNA文库，对初级文库进行扩增、提取文库质粒后转入酵母Y187菌株中，制作扩增的酵母文库，并对文库质量进行鉴定。结果经检测所构建的3个读码框初级文库库容分别为1.7×10⁶ 、2.0×10⁶ 和1.9×10⁶ cfu，重组率为100%，插入的片段长度主要分布在500～2000 bp；电泳检测和测序结果表明，插入片段所对应的编码蛋白类型丰富，具有良好的多态性。最终获得的Y187酵母文库滴度为4.0×10⁸ cfu·mL⁻¹。结论构建的低温胁迫下山葡萄叶片酵母cDNA文库质量较高，为筛选山葡萄低温胁迫下的互作蛋白提供了材料基础。
- 山葡萄 /
- 酵母cDNA文库 /
- 低温 /
- 非生物胁迫
Abstract: Objective A yeast cDNA library was constructed using the leaves from a cold tolerant Amur grape plant under low-temperature stress to facilitate the study of the resistance mechanism. Method As a well-known frost resistant Vitis amurensis Rupr., V. amuerensis cv. Zuoshan-1 was cultured in a pot experiment for a total RNA extraction from the seedling leaves after 0, 2, 4, 8, 12, and 24 h at 4 °C. The extract was then reverse transcribed into cDNA using the SMART technology to be purified. Short fragments were removed and ligated into pGADT7 3-frame plasmid vector to establish a primary cDNA library. After amplification, plasmid was extracted and transformed into yeast Y187 for the library construction, followed by a qualitative verification. Result The capacities of the 3 reading frame primary libraries were tested to be 1.7×10⁶ cfu, 2.0×10⁶ cfu, and 1.9×10⁶ cfu with a recombination rate of 100%. The lengths of the inserted fragments were largely in the 500-2 000 bp region. The electrophoretic detected and sequenced encoded proteins corresponding to the inserted fragments were abundant and highly polymorphic. The titer of the finally obtained Y187 yeast library was approximately 4.0×10⁸ cfu·mL⁻¹. Conclusion A high quality yeast cDNA library using Amur grape leaves under low temperature was successfully constructed for future studies on the interacting proteins under cold stress that could induce injury impairing the growth, development, fruit quality, and yield of grape vines.
- Vitis amurensis Rupr. /
- yeast cDNA library /
- low temperature /
- abiotic stress

HTML全文

图 1 低温处理下山葡萄左山-1叶片RNA电泳检测

M：DNA Marker 5 000；1：总RNA。

Figure 1. Agarose gel electrophoresis of RNA extracted from V. amurensis cv. Zuoshan-1

M: DNA marker 5 000; 1: Total RNA.

下载: 全尺寸图片幻灯片

图 2 cDNA的合成与纯化

A中M为DNA Marker 5 000，1为均一化后的cDNA，B中M为DNA Marker 5 000，1为过柱纯化后的cDNA。

Figure 2. Synthesis and purification of cDNA

A: M-DNA marker 5 000; 1: Normalized cDNA; B: M-DNA marker 5 000; 1: Purified cDNA.

下载: 全尺寸图片幻灯片

图 3 初级文库库容检测

A：读码框1，B：读码框2，C：读码框3。

Figure 3. Determination of primary library capacity

A: Reading frame 1; B: Reading frame 2; C: Reading frame 3.

下载: 全尺寸图片幻灯片

图 4 PCR检测初级文库插入片段

M：DNA Marker 5 000，1～16：初级文库插入片段。

Figure 4. PCR detection of inserted fragments from primary library

M: DNA marker 5 000; 1–16: Inserted fragments from primary library.

下载: 全尺寸图片幻灯片

图 5 cDNA文库质粒扩增

M：DNAMarker 15 000；1：扩增的cDNA文库质粒。

Figure 5. Amplification of plasmid cDNA library

M: DNA marker 15 000; 1: Amplified plasmid cDNA library.

下载: 全尺寸图片幻灯片

图 6 SD/-Leu平板上酵母cDNA文库滴度检测

Figure 6. Detection of yeast cDNA library titer on SD/-Leu plate

下载: 全尺寸图片幻灯片

表 1 文库中插入片段功能分析

Table 1. Functional characterization on inserted fragments from cDNA library

序号 Number	候选蛋白 Candidate proteins	蛋白登录号 Accession number
1	剪接因子U2af Splicing factor U2af	XP_010649167.1
2	MOB激酶激活因子1A MOB kinase activator-like 1A	XP_003631858.1
3	KAKU4蛋白 Protein KAKU4	XP_019075105.1
4	未知蛋白 Uncharacterized protein	XP_002265235.1
5	类韧皮部蛋白2 Phloem protein 2-like	XP_002277133.1
6	60S核糖体蛋白L10a-1 60S ribosomal protein L10a-1	XP_002282641.1
7	psbP 结构域蛋白-6 psbP domain-containing protein 6	XP_019076972.1
8	组氨酸磷酸转移蛋白1 Histidine-containing phosphotransfer protein 1	XP_002278411.1
9	谷胱甘肽S-转移酶 DHAR3 Glutathione S-transferase DHAR3	XP_002266106.1
10	腺苷酸激酶 Adenylate kinase	XP_003634590.1
11	E3类泛素蛋白UFM1连接酶1 E3 UFM1-protein ligase 1	XP_002284524.1
12	nudix水解酶10 nudix hydrolase 10	XP_003634765.1
13	分泌载体相关膜蛋白3 Secretory carrier-associated membrane protein 3	XP_010646776.1
14	超敏诱导反应蛋白4 Hypersensitive-induced response protein 4	XP_010662382.1
15	L-抗坏血酸过氧化酶3 L-ascorbate peroxidase 3	XP_002278281.1
16	镁离子转运蛋白MRS2-F Magnesium transporter MRS2-F	XP_010652955.1

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