抱茎独行菜种皮粘液质相关基因TTG1的克隆、表达分析及功能鉴定

Cloning,Characterization,and Functional Analysis of Seed Coat Mucilage-related GeneTTG1 from Lepidium perfoliatum

抱茎独行菜(Lepidium perfoliatum L.)为十字花科具典型粘液质繁殖体植物,而TTG1基因(Transpa-rent testa glabra 1)所编码的蛋白是调控种皮细胞分化并影响粘液质释放的转录因子。目前关于TTG1基因在粘液质繁殖体植物中的研究报道较少,为探究TTG1基因在抱茎独行菜粘液质发育中的作用,本研究利用同源克隆技术获得抱茎独行菜TTG1基因cDNA开放阅读框(ORF)序列,命名为LpTTG1。序列分析表明,该基因ORF全长为1032 bp,编码343个氨基酸,含有WD40基序;qRT-PCR分析结果显示,该基因在抱茎独行菜各组织中均有表达,反映了该基因功能的多样性;免疫组织化学定位结果表明,LpTTG1在种子发育过程中内珠被和外珠被的表达水平变化与外珠被粘液质的合成过程相一致,推测该基因可能参与调控抱茎独行菜种皮的发育及粘液质的形成。将LpTTG1基因转化拟南芥,该基因的过量表达显著促进了粘液质合成途径下游基因AtMUM4在角果中的表达,表明该基因有可能参与粘液质合成途径调控,并促进下游产物MUM4的产生。然而,对LpTTG1转基因拟南芥与野生型植株表型的比较发现,两者种子形态及粘液质分泌与释放方式均无显著差异,这可能是因为抱茎独行菜种皮发育和粘液质形成是一个多基因调控的复杂过程,某一基因的过量表达也许不会引起明显的表型变化。

Lepidium perfoliatum, an annual herb plant species of Brassicaceae, has a typical myxospermy. The TTG1 （ Transparent testa glabra 1） gene encodes a putative transcription factor, which has been identified to play a role in epidermal cell differentiation and mucilage release. Till now, research on the TTG1 gene in myxospermy plants has been rarely reported. To identify TTG1 gene function, mucilage related gene LpTTG1 from L. perfoliatum was cloned in the present study. The full length ORF of the TTG1 gene from L. perfoliatum was isolated by homologous cloning, and was found to be 1032 bp long, encoded 343 putative amino acidsand contained WD40 motifs, named as LpTTG1. The qRT-PCR results showed that LpTTG1 was widely expressed in different tissues of L. perfoliatum, which may reflect diverse functions of LpTTG1. Moreover, immunolocalization analysis indicated that the expression of LpTTG1 changed in inner and outer integuments and corresponded to the synthesis of mucilage in outer integument cell layers, suggesting that LpTTG1 mainly regulates the development of the seed coat then applies the effect on mucilage production. Furthermore, overexpression of LpTTG1 in Arabidopsis could significantly enhance the expression of AtMUM4 （which developmentally regulates mucilage production downstream） in silique, which means LpTTG1 attends to the mucilage regulation pathway and generates more downstream product-MUM4 in promotion of mucilage synthesis. In our experiment, however, no significant difference in seed morphology and release pattern, or the secretion amount of mucilage between LpTTG1 overexpression transgenic line and WT, was observed. One possible explanation may be that mucilage synthesis and release is a complex process in Arabidopsis, and is regulated by many genes with functional redundancy, therefore increasing the transcription level with one of them during development may not result in significant phenotype change.