遗传 ›› 2007, Vol. 29 ›› Issue (4): 490-490―498.doi: 10.1360/yc-007-0490

• 研究报告 • 上一篇    下一篇

忽地笑二级侧脉凸起突变体相关基因表达差异分析与克隆

高磊1,2,3, 郭素敏1, 崔永兰1,4, 诸葛强1, 黄敏仁1   

  1. 1. 南京林业大学林木遗传和基因工程重点实验室, 南京 210037;
    2. 中国科学院武汉植物园, 武汉 430074;
    3. 中国科学院研究生院, 北京 100049;
    4. 上海师范大学生命科学学院, 上海 200234

  • 收稿日期:2006-07-21 修回日期:2006-10-09 出版日期:2007-04-10 发布日期:2007-04-10
  • 通讯作者: 黄敏仁

The analysis of differential expression and cloning of genes related to raised secondary lateral veins mutant of Lycoris aurea

GAO Lei1,2,3, GUO Su-Min1, CUI Yong-Lan1,4, ZHUGE Qiang1, HUANG Min-Ren1

  

  1. 1. The Key Laboratory of Forest Genetics and Gene Engineering, Nanjing Forestry University, Nanjing 210037, China;
    2. Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China;
    3. Graduate School of Chinese Academy of Sciences, Beijing 100049, China;
    4. College of Life of Shanghai Normal University, Shanghai 200234, China
  • Received:2006-07-21 Revised:2006-10-09 Online:2007-04-10 Published:2007-04-10
  • Contact: HUANG Min-Ren

摘要:

忽地笑Raised Secondary Lateral Veins (RSLV)突变体平行脉间横向二级侧脉显著凸起, 生长不良, 雄蕊完全退化。本研究利用表达型噬菌体载体ZAP构建了野生型和突变体忽地笑叶片cDNA文库, 分别随机挑取cDNA克隆进行5′端测序, 共得到3,122条有效ESTs。利用这些ESTs序列, 选择非冗余基因, 设计512条70 mer的特异性序列为寡核苷酸探针, 制作芯片, 对忽地笑野生型和突变体叶器官进行基因表达谱分析。进一步采用实时定量PCR技术验证芯片实验结果, 最终确定所检测的基因中, 有5个基因在突变型和野生型间表达差异显著, 分别是韧皮部蛋白2 (phloem protein 2, PP2)、铁蛋白(ferritin)、果胶甲酯酶(pectin methyl esterases, PMEs), 以及叶绿素a/b结合蛋白和丙酮酸脱羧酶等。克隆获得了其全长cDNA, 并探讨了这些差异基因与忽地笑突变型形成的可能关系。

关键词: 忽地笑, RSLV突变型, 叶发育, 基因芯片

Abstract:

Lycoris aurea exhibits parallel venation, the main vein with many lateral veins in a longitudinal parallel arrangement. There are secondary lateral veins (SLV) between each longitudinal veins. In general, SLVs are not remarkable. In this paper, the material was one kind of Lycoris aurea mutant called Raised Secondary Lateral Veins mutant (RSLV), because many Raised Secondary Lateral Veins are in abaxial surface of its leaves. Its growing potential is weaker than that of wild type and its blades are very thin. Moreover, the stamens of RSLV degenerate completely. Two cDNA libraries were constructed from RSLV mutant and wild type (WT) leaves. From the libraries, 3,122 ESTs, which are longer than 100 bp each after vector sequence removed, were acquired by single-pass sequencing from the 5′end. Following a multistep

selection, 512 70-mer oligo-DNA probes were designed for attachment on the microarray slide based on the ESTs. The gene expression profile of RSLV mutant and WT leaves was compared through the microarray at transcriptional level. The mi-croarray experiment results were further confirmed by Quantitative Real-Time PCR (QRT-PCR). We identified 5 genes whose expressions changed more than 2-fold between RSLV mutant and WT leaves. They encode phloem protein 2 (PP2), ferritin, pectin methyl esterase (PME), chlorophyll a/b binding protein (CAB protein) and pyruvate decarboxylase (PDC), respectively. Furthermore, the full-length cDNA sequences of the 5 genes were separately obtained from RSLV and WT by RACE. The relationship between differential expressions of the genes and the formation of the RSLV mutant phenotype were discussed.