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为了丰富棉花中NAC转录因子的功能研究,挖掘胁迫衰老相关NAC基因,本研究基于陆地棉(Gossypium hirsutum)基因组数据,克隆并分析了JUNGBRUNNEN1的同源基因GhNAC63及其启动子,并通过qRT-PCR技术分析了GhNAC63在不同衰老特性棉花品种子叶发育过程中的表达模式,以及GhNAC63在不同生长条件下,对不同胁迫处理的响应。构建了35S::GhNAC63过表达载体,转入拟南芥(Arabidopsis thaliana),同时利用pYL156-pYL192病毒诱导的基因沉默体系,抑制棉花中GhNAC63的表达,并观察棉花表型。基因序列分析结果表明GhNAC63定位于陆地棉染色体骨架scaffold246.1上,其启动子序列包含多种胁迫响应相关的顺式元件;基因表达模式结果表明,GhNAC63在纤维发育初期、花、真叶、茎及子叶中优势表达,在子叶发育中期表达量达到最高,并能够响应多种逆境胁迫,其中GhNAC63对乙烯胁迫的响应,在各种环境下均表现敏感;转基因结果表明GhNAC63过表达导致拟南芥对乙烯和干旱更敏感,而抑制棉花中GhNAC63的表达可以使植株生长更加健壮。本研究初步证明GhNAC63在拟南芥中负调控乙烯胁迫,同时负调控棉花生长发育,丰富了棉花中NAC转录因子的研究,为培育抗胁迫衰老转基因棉花材料奠定了基础。
In order to enrich the function of NAC transcription factors in cotton and to explore the NAC genes related to stress and senescence, GhNAC63 and its promoter of JUNGBRUNNEN1 were cloned and analyzed based on Gossypium hirsutum genomic data and analyzed by qRT-PCR The expression patterns of GhNAC63 during cotyledon development of cotton cultivars with different senescence characteristics were analyzed. The response of GhNAC63 to different stress treatments was also studied under different growth conditions. The 35S :: GhNAC63 overexpression vector was constructed and transferred into Arabidopsis thaliana. At the same time, the pYL156-pYL192 virus-induced gene silencing system was used to inhibit the expression of GhNAC63 in cotton and to observe the cotton phenotype. The results of gene sequence analysis showed that GhNAC63 was localized on the scaffold246.1 of upland cotton, and its promoter sequence contained multiple cis-components related to stress response. The results of gene expression showed that GhNAC63 was involved in the early development of fiber, And cotyledons, and reached the highest level in the mid-cotyledon development stage, and was able to respond to various stresses. Among them, the response of GhNAC63 to ethylene stress was sensitive in all environments. Transgenic results showed that GhNAC63 overexpression resulted in Arabidopsis thaliana It is more sensitive to ethylene and drought, while inhibiting the expression of GhNAC63 in cotton can make the plant more robust. This study proves that GhNAC63 negatively regulates ethylene stress in Arabidopsis, negatively regulates cotton growth and development, enriches the NAC transcription factor in cotton, and lays the foundation for the development of anti-aging transgenic cotton material.