Analysis of the function of the AS2 gene family in development of leaves

拟南芥的不对称叶片1 （ASYMMETRIC LEAVES1, AS1）和不对称叶片2 （ASYMMETRIC LEAVES2, AS2）基因参与了叶脉系统（包括突出的中脉）的建立以及对称叶片的发育。这些基因也可能通过抑制1类knox同源盒基因的表达，维持叶片细胞处于发育决定状态。AS2基因编码一种具有新结构域（AS2结构域）的蛋白质，该结构域包含半胱氨酸重复序列（C-motif）、保守的甘氨酸和亮氨酸拉链样序列。拟南芥基因组包含42个可能编码AS2结构域的基因（AS2和ASL1-41）。AS2、ASL1、ASL2、ASL3和ASL4蛋白包括AS2结构域的大多数氨基酸残基，这些氨基酸残基在这些蛋白中是保守的。ASI或AS2的功能缺失突变导致叶片向下卷曲，而AS2的过表达导致AS1存在时叶片向上卷曲。过表达ASL1的转基因植株叶片也呈上卷曲状。(1)为了了解AS2和ASL1-4中AS2结构域在拟南芥叶片发育中的功能，我们分离了ASL1、ASL2和ASL3基因的功能缺失突变。由于ASL1和ASL3的单一突变至少在营养阶段没有导致表型，因此使用as1， as2， by和asls突变体的遗传分析正在进行中。(2)含有gfp融合AS2的稳定转化在细胞核中显示荧光信号。AS2中的c基序是核定位所必需的。将ASL1::GFP和ASL3::GFP融合构建体导入洋葱表皮细胞后，荧光信号主要在细胞核内检测到。这些蛋白可能是调节某些基因（如1类knox基因）表达的核因子。

The ASYMMETRIC LEAVES1 (AS1) and the ASYMMETRIC LEAVES2 (AS2) genes of Arabidopsis thaliana are involved in the establishment of the leaf venation system, which includes the prominent midvein, as well as in the development of a symmetric flat lamina. The genes might also function in maintaining leaf cells in a developmentally determinate state, probably by repressing expression of class 1 knox homeobox genes. The AS2 gene encodes a protein with a novel domain (AS2 domain) that contains cysteine repeats (C-motif), conserved glycine, and a leucine-zipper-like sequence. Arabidopsis genome contains 42 putative genes (AS2 and ASL1-41) that potentially encode th e AS2 domain. The AS2, ASL1, ASL2, ASL3 and ASL4 proteins include most amino acid residues in t he AS2 domain that are conserved among these proteins. Loss-of-function mutations in ASI or AS2 generated downwardly curled leaves, although overexpression of AS2 resulted in upwardly curled leaves in the presence of the AS1. The transgenic plants that overexpressed ASL1 also showed upwardly curled leaves. (1) To understand the functions of the AS2 domain in the AS2 and ASL1-4 in the leaf development in Arabidopsis, we have isolated loss-of-function mutations in ASL1, ASL2 and ASL3 genes. As a single mutation in ASL1 and ASL3 resulted in no phenotype at least in vegetative stage, genetic analysis using the as1, as2, by and asls mutants is in progress. (2) The stable transformant contained GFP-fused AS2 showed the fluorescence signals in nuclei. The C-motif in AS2 was required for the nuclear localization. When the fusion constructs of ASL1::GFP and ASL3::GFP were introduced into onion epidermal cells, fluorescence signals were detected mainly in nuclei. These proteins may be nuclear factors that regulate expression of some genes such as class 1 knox genes.