日本结缕草衰老叶片全长cDNA文库构建及酵母单杂交筛选验证

doi:10.11733/j.issn.1007-0435.2019.06.004

草地学报 ›› 2019, Vol. 27 ›› Issue (6): 1486-1493.DOI: 10.11733/j.issn.1007-0435.2019.06.004

日本结缕草衰老叶片全长cDNA文库构建及酵母单杂交筛选验证

滕珂¹, 檀鹏辉², 范希峰¹, 岳跃森¹, 姜红岩², 武菊英¹

1. 北京市农林科学院, 北京草业与环境研究发展中心, 北京 100097;
2. 北京林业大学, 草业与草原学院, 北京 100083

收稿日期:2019-06-26 修回日期:2019-08-21 出版日期:2019-12-15 发布日期:2019-12-31
通讯作者: 武菊英,E-mail:wujuying@grass-env.com
作者简介:滕珂(1989-),男,汉族,山东淄博人,助理研究员,研究方向为草坪草分子生物学,E-mail:tengke.123@163.com
基金资助:
国家自然科学青年基金（31901397）；北京市农林科学院科技创新能力建设专项（KJCX20180101，20180706）；北京市科技计划项目（No.171100007217001）资助

Generating of a Full Length cDNA Library Using Senescence Leaves of Zoysia japonica and Yeast One-hybrid Screening Validation

TENG Ke¹, TAN Peng-hui², FAN Xi-feng¹, YUE Yue-sen¹, JIANG Hong-yan², WU Ju-ying¹

1. Beijing Research and Development Center for Grass and Environment, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China;
2. College of Grassland Science, Beijing Forestry University, Beijing 100083, China

Received:2019-06-26 Revised:2019-08-21 Online:2019-12-15 Published:2019-12-31

摘要/Abstract

摘要： 脱镁叶绿素脱镁叶绿酸水解酶（Pheophytin pheophorbide hydrolase，PPH）是叶绿素降解过程中的一个关键酶，目前有关日本结缕草（Zoysia japonica）PPH基因的上游调控机制仍不是十分清楚，筛选日本结缕草ZjPPH1基因上游转录因子可以为延长绿期延缓衰老提供理论依据。本研究利用Clontech的SMRT cDNA合成技术构建一个日本结缕草衰老叶片的高质量全长cDNA文库，其滴度为2.0×10⁶ cfu·mL^-1，插入片段平均长度大于1 kb，cDNA片段插入重组率为100%。该文库的完整性较高，可涵盖绝大多数基因信息，为酵母单杂交筛选奠定了基础。利用酵母单杂交技术，筛选到可与ZjPPH1基因启动子片段结合的候选菌落42个，并对20个候选基因进行测序和生物信息学分析，初步筛选到6个参与细胞生长和衰老过程的重要候选基因，为进一步探索ZjPPH1基因在日本结缕草衰老和叶绿素降解中的上游调控机制提供了理论支撑。

关键词: 日本结缕草, 全长cDNA文库, 酵母单杂交, 脱镁叶绿素脱镁叶绿酸水解酶基因

Abstract: Heophytin pheophorbide hydrolase (PPH) is a key enzyme in chlorophyll degradation. Currently,the upstream regulating mechanism of ZjPPH1 has not been well illustrated in Zoysia japonica. Screening the upstream transcription factors of Zoysia japonica ZjPPH1 gene will provide theoretical basis for prolonging its green period and delaying senescence. In this study,a high quality full length cDNA library of Zoysia japonica senescence leaves was constructed using SMRT cDNA synthesis method of Clonetch company. Its titer was 2.0×10⁶ cfu·mL^-1. The average length of the insert segments was more than 1 kb,and the recombination rate was 100%. This cDNA library could cover the most transcriptional information and provide qualified material for further yeast one-hybrid screening. We found 42 candidate clones which could be interacted with the promoter segment of ZjPPH1 through yeast one-hybrid screening. Then 20 clones were selected to be sequenced and analyzed using bio-informatics methods. 6 important candidate genes involved in cell growth and senescence processes were screened initially. This study paves the way to further investigate the upstream regulating mechanism of ZjPPH1 in senescence processes and chlorophyll degradation of Zoysia japonica.

Key words: Zoysia japonica, Full length cDNA library, Yeast one-hybrid screening, PPH

中图分类号:

S543.9

滕珂, 檀鹏辉, 范希峰, 岳跃森, 姜红岩, 武菊英. 日本结缕草衰老叶片全长cDNA文库构建及酵母单杂交筛选验证[J]. 草地学报, 2019, 27(6): 1486-1493.

TENG Ke, TAN Peng-hui, FAN Xi-feng, YUE Yue-sen, JIANG Hong-yan, WU Ju-ying. Generating of a Full Length cDNA Library Using Senescence Leaves of Zoysia japonica and Yeast One-hybrid Screening Validation[J]. Acta Agrestia Sinica, 2019, 27(6): 1486-1493.

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日本结缕草衰老叶片全长cDNA文库构建及酵母单杂交筛选验证

Generating of a Full Length cDNA Library Using Senescence Leaves of Zoysia japonica and Yeast One-hybrid Screening Validation

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