‘云香’水仙NtNAC1基因的克隆及功能分析

Cloning and Functional Analysis of NtNAC1 Gene in Narcissus tazetta ‘Yunxiang’

本研究利用RT-PCR技术从‘云香’水仙的根中克隆得到了NtNAC1基因,并通过qRT-PCR技术分析了该基因的时空表达模式以及在逆境和逆境相关激素处理下的表达变化,结合分析过表达该基因的转基因烟草植株的表现等解析了该基因的功能。结果显示:NtNAC1基因全长1083 bp,其ORF为918 bp,编码306个氨基酸,N端含有NAM保守结构域,聚分在SNAC1亚组,与香蕉MusaSNAC1、小麦TaNAC47亲缘关系较近。实时荧光定量分析显示,NtNAC1的表达水平随花器官花瓣和副冠的发育呈先上升后下降趋势,始花期达到峰值;NtNAC1在根和叶中高表达,受ABA、MeJA、SA、H_2O_2、NaCl、PEG诱导上调,推测该基因可能参与‘云香’水仙花器官的形态建成及响应非生物胁迫。进一步构建表达载体转化烟草,获得9株转基因植株。在高盐和干旱胁迫处理下,转基因植株根系发达、长势更好、存活率高,表明水仙NtNAC1过表达能提高烟草对高盐、干旱的耐受性,在抗逆中起正向调控作用,可作为提高水仙抗性的优良遗传基因资源。

NtNAC1 was cloned by RT-PCR from the roots of Narcissus tazetta ‘Yunxiang’. The gene was further characterized by analyzing its tempo-spacial expression patterns in N. tazetta ‘Yunxiang’ plants, as well as changes in its expression in the leaves and roots under different stresses and stress-related hormones, and by analyzing the NtNAC1 over-expressing transgenic tobacco plants. The results showed that the cloned full-length cDNA was 1083 bp long, containing an ORF of 918 bp long which would putatively encode a peptide of 306 amino acid residues. The deduced protein possessed a highly conserved DNA binding domain, NAM, at its N-terminal, and was grouped, together with its most closely related proteins, MusaSNAC1 and TaNAC47, into the SNAC1 subgroup on the phylogenetic tree. Quantitative real-time PCR results showed that the expression level of NtNAC1 in petals and coronas first increased and then decreased as the floral organs developed, and reached a peak in the early flowering period. NtNAC1 was abundantly expressed in roots and leaves, and was induced by the treatments of ABA, MeJA, SA, H2O2, NaCl and PEG, indicating that it may be involved in the morphogenesis of floral organs in addition to its principal role in the responses of plants to abiotic stresses. Nine transgenic tobacco lines carrying NtNAC1 were obtained by agrobacterium-mediated transformation and three of them were over-expressing lines. The NtNAC1 over-expressing lines showed better developed root systems and higher survival rates under the treatments of high concentrations of salt and mannitol, as compared with the non-transgenic plants. Our results showed that expressing NtNAC1 increased stress tolerance of plants and its potential in the improvement of abiotic stress resistance of N. tazetta could be further exploited.