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花生NAC转录因子的克隆和功能分析

The Isolation and Functional Analysis of NAC Transcription Factors in Peanut (Arachis Hypogaea L.)

【作者】 邵凤霞

【导师】 毕玉平; 柳展基;

【作者基本信息】 山东师范大学 , 发育生物学, 2009, 硕士

【摘要】 花生是重要的油料作物和经济作物。我国是世界上种植花生的主要国家之一。花生产区主要分布于干旱和半干旱地区,干旱严重影响花生的产量和品质,是花生生产的首要限制因子。我国有70%的花生面积受到不同程度的干旱危害,平均减产20%以上,造成巨大的经济损失。因此,解决干旱对花生生产的影响是当务之急。植物在其生长过程中经常会遭受到不同程度的生物和非生物逆境胁迫,这些胁迫严重影响植物的生长发育,并对农业生产造成极大的负面影响。研究发现,在长期的生物进化过程中,植物自身已经发展了多种抵抗干旱胁迫的机制,以通过调节自身生理和代谢变化来抵御干旱。其中的一个重要机制就是通过逆境胁迫信号转导来启动逆境胁迫响应基因的表达。在胁迫信号转导途径中,转录因子由于能够调节多个胁迫响应功能基因的表达而备受关注。NAC转录因子是近十年来发现的植物特有的转录调控因子,在植物生长发育、激素调节和防御抵抗多种生物和非生物胁迫、及作物产量和品质等方面发挥重要作用。NAC转录因子的N端高度保守,含有NAM保守结构域,为DNA结合区,C端则高度变异,为转录激活区。我们从出口型大花生鲁花14不同发育时期种子混合cDNA文库中克隆鉴定出了2个花生NAC类基因,分别命名为AhNAC1(GenBank登录号为EU669863)和AhNAC2。利用RT-PCR技术,从花生cDNA中扩增得到这两个基因的cDNA全长。序列分析表明,AhNAC1基因的cDNA序列长度为1453 bp,其开放阅读框(ORF)长度为912 bp,编码303个氨基酸,预测其分子量为34.1 kD,等电点为7.6。而AhNAC2基因的cDNA为1573 bp,ORF为1050 bp,编码349个氨基酸,预测其分子量为39.1 kD,等电点为7.66。通过与其他NAC蛋白序列比对, AhNAC1和AhNAC2均聚类到ATAF亚家族中,预测AhNAC1可能在花生种子发育中起作用,而AhNAC2可能参与了干旱等非生物胁迫反应。在本实验中,我们利用半定量RT-PCR的方法分析了AhNAC2在干旱、盐渍、低温和ABA处理下的表达变化。结果表明:该基因的表达受到干旱诱导最为明显,且其表达随处理的时间出现峰值变化,而ABA处理后表达变化无明显差异。酵母转录激活实验、区域缺失试验和显色反应证明了AhNAC2的激活域位于C端,而基因枪转化洋葱表皮细胞的瞬时表达表明了AhNAC2-GFP融合蛋白特异定位在细胞核中。这些结果表明AhNAC2具有转录因子的特征。此外,为了功能验证,我们构建了AhNAC2基因的过量表达载体,并通过农杆菌介导的叶盘转化法成功转到烟草中,筛选到转基因株系,并对后代进行干旱耐受性分析,结果表明转基因烟草抗旱能力较野生型显著提高。而不同浓度ABA处理后,转基因烟草与野生型无明显差异。在正常生长状态下,过量表达的转基因株系可以诱导含有DRE/CRT元件的某些下游基因大量表达,而对照中表达量较低,因此推测AhNAC2基因可能具有诱导非生物胁迫相关的COR/RD基因表达的能力。此外,离体叶片的失水实验也证明了转基因株系的耐旱能力提高。这些都暗示AhNAC2在干旱胁迫信号途径中起一定作用。本研究从花生中分离鉴定出两个NAC类转录因子AhNAC1和AhNAC2,并进行初步功能分析,发现AhNAC2在干旱胁迫响应中起到调控作用。这不仅有利于理解花生响应干旱胁迫的分子机理,而且为进行花生抗旱遗传改良储备了基因资源。

【Abstract】 Peanut is an important oil and cash crop. China is one of the vital peanut-cultivated countries in the world. Peanut distributes mainly in arid and semiarid region. Therefore, drought significantly affects the yield and quality of peanut. It is reported that 70% of peanut area in China are in various degree of drought and the average decrease in yield was more than 20%. Therefore, it is essential to resolve the negative effect of drought stress to peanut production.Plant is often exposed to biotic and abiotic stresses of varying degrees in growth process which affected plant growth and development badly and cause huge negative effect to agriculture. Plant has established some suitable and resistance mechanisms in the long evolution to reduce the damage by adjusting physiological and metabolic pathway. One of the important mechanisms is to induce the expression of stress response genes by regulating the stress signal transduction, and transcription factors have drawn more and more attention owing to their ability for regulating of many stress response factors.NAC transcription factors are the new type transcription regulatory factors which are unique to plants. They play an important role in regulation of plant growth and development, hormone level development, responses to stresses, and crops yield and quality. NAC transcription factors contain a NAC domain in the conserved N-terminal, which may function as DNA-binding region, while the various C-terminal serves as transcriptional activation region.We isolated two NAC-like genes from the cDNA library of different stages seeds of the export peanut cultivar‘luhua14’by RT-PCR and named as AhNAC1 (GenBank accession number EU669863)and AhNAC2. Sequence analysis shows that the AhNAC1 cDNA has a length of 1453 bp which contains a single open reading frame(ORF) of 912 bp encoding a protein of 303 amino acids with an estimated molecular mass of 34.1 kD and an isoelectric point of 7.6. While the AhNAC2 cDNA is 1573 bp and contains an ORF of 1050 bp. It encodes a protein of 349 amino acids with an estimated molecular mass of 39.1 kD and an isoelectric point of 7.66. Compared to other NAM members, AhNAC1 and AhNAC2 belong to ATAF subfamily. We supposed that AhNAC1 may play a role in the seed development and AhNAC2 may be involved in drought response pathways.In this study, we characterized the AhNAC2 expression in response to drought, salinity, low temperature and ABA by semiquantitative RT-PCR analysis. The results indicated that the AhNAC2 expression was strongly induced by dehydration, and varied along with the drought treatment hours. However, exogenous ABA treatment didn’t change obviously the expression of AhNAC2.Yeast transactivation analysis, domain deletion andβ-galactosidase filter assays showed that the transactivation activity of AhNAC2 was conferred by its C-terminal. Transient expression analysis in onion epidermal cells by genegun indicated AhNAC2-GFP fuse protein was specially localized in nuclear. All the results indicate that AhNAC2 is a transcription factor.Overexpression vector was constructed to examine the function of AhNAC2. By using Agrobacterium mediated leaf-disk tobacco transformation, the recombinant was transformed to Nicotiana tabacum L. The transgenetic strains have been derived after screening. Resistance analysis on these strains reveals that the strains overexpressing AhNAC2 gene have higher level of drought resistance than the wild strains. Besides, in normal condition, some downstream genes containing DRE/CRT element expressed more in the transgenetic strains than the wild strains. The above results indicate that AhNAC2 may induce the expression of abiotic stress related COR/RD genes. Besides, water loss analysis showed that the transgenetic strains were more resistant to drought. These results indicate a role of AhNAC2 in drought stress regulation. In conclusion, we have isolated two NAC transcription factors, AhNAC1 and AhNAC2, and verified their physiological functions. These results will not only help us to understand the mechanism of peanut resistant to drought, but also provide the gene for genetic improvement of peanut drought resistance.

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