Gao Lei, Guo Su-Min, Cui Yong-Lan, Zhuge Qiang, Huang Min-Ren
The Key Laboratory of Forest Genetics and Gene Engineering, Nanjing Forestry University, Nanjing 210037, China.
Yi Chuan. 2007 Apr;29(4):490-8. doi: 10.1360/yc-007-0490.
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 microarray 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.
忽地笑具有平行脉序，主脉有许多侧脉呈纵向平行排列。每条纵向叶脉之间有二级侧脉（SLV）。一般来说，二级侧脉不明显。在本文中，材料是一种忽地笑突变体，称为凸起二级侧脉突变体（RSLV），因为其叶片背面有许多凸起的二级侧脉。其生长势比野生型弱，叶片非常薄。此外，RSLV的雄蕊完全退化。从RSLV突变体和野生型（WT）叶片构建了两个cDNA文库。从文库中，通过从5'端单通道测序获得了3122个EST，去除载体序列后每个EST长度均超过100 bp。经过多步筛选，基于这些EST设计了512个70聚体寡核苷酸DNA探针用于附着在微阵列载玻片上。通过微阵列在转录水平比较了RSLV突变体和WT叶片的基因表达谱。微阵列实验结果通过定量实时PCR（QRT-PCR）进一步证实。我们鉴定出5个基因，其在RSLV突变体和WT叶片之间的表达变化超过2倍。它们分别编码韧皮部蛋白2（PP2）、铁蛋白、果胶甲酯酶（PME）、叶绿素a/b结合蛋白（CAB蛋白）和丙酮酸脱羧酶（PDC）。此外，通过RACE分别从RSLV和WT中获得了这5个基因的全长cDNA序列。讨论了这些基因的差异表达与RSLV突变体表型形成之间的关系。
