AtNEK6在棉花旱盐胁迫响应中的功能分析

Functional Characterization of AtNEK6 Overexpression in Cotton Under Drought and Salt Stress

【目的】AtNEK6是在拟南芥中发现的一种NIMA相关激酶,在拟南芥中过表达AtNEK6能够促进植物的生长发育,提高植物的耐盐耐旱性。通过将AtNEK6转化棉花,研究其在抗逆中的分子机理,为培育耐旱耐盐碱棉花新品种提供理论基础和种质资源。【方法】采用农杆菌转化法,将来源于拟南芥的AtNEK6导入棉花,通过实时荧光定量PCR分析转基因株系中AtNEK6的表达量;通过观察转基因植株表型和扫描电镜观察细胞表皮细胞,分析转基因棉花的生长发育情况;利用甘露醇和NaCl模拟干旱处理和盐处理分析转基因棉花的耐盐耐旱能力,通过测定相关生理指标,鉴定AtNEK6对转基因棉花耐逆的贡献。【结果】利用卡那霉素筛选获得转基因幼苗,通过PCR鉴定获得10个不同的转基因株系;利用qRT-PCR分析筛选出表达量较高的L7、L17、L25株系。正常条件下,与野生型相比,转基因棉花的株高增高、叶面积增大,生长发育得到了促进,通过扫描电镜观察发现转基因棉花叶片的细胞表面积与野生型并无明显差异,细胞周期相关基因CYCB1;1和CYCA3;1及生长发育相关基因GhGRF5、GhEOD、GhAN3和GhEBP1的表达量均上调。通过耐盐耐旱性分析,正常条件下,与野生型相比,转基因株系的根长、鲜重和干重均无明显差异,仅侧根数增多;在250 mmol·L^(-1)甘露醇处理条件下,转基因株系的根长、侧根数、鲜重和干重均显著高于野生型,表现出更好的生长状态;在200 mmol·L^(-1)NaCl处理条件下,转基因株系的侧根数、鲜重和干重均显著高于野生型,相比于野生型生长状态更好。温室中正常生长1月龄的转基因棉花,在300 mmol·L^(-1)甘露醇处理条件下,转基因植株的SOD活性比野生型提高了0.65倍、0.42倍和1.45倍,CAT活性提高了0.65倍、0.64倍和0.42倍,MDA含量降低了0.51倍、0.41倍和0.22倍;250 mmol·L^(-1)NaCl处理结果与甘露醇类似,转基因棉花的耐盐生理指标均优于野生型。另外,相关胁迫响应基因GhAREB、GhDREB、GhNCED和GhLEA5在转基因棉花中的表达量均显著高于野生型棉花。【结论】AtNEK6通过参与细胞周期和生长发育的调控过程促进棉花的生长发育,同时提高棉花在逆境中的耐盐耐旱性。

【Objective】 AtNEK6 is a NIMA related kinase in Arabidopsis thaliana. Overexpression of At NEK6 in Arabidopsis can promote plant growth, and improve the salt tolerance and drought tolerance of plants. By transforming At NEK6 into cotton, the molecular mechanism of its resistance to stress was studied, so as to provide theoretical basis and germplasm resources for breeding new cotton varieties with drought tolerance and salinity tolerance.【Method】The AtNEK6 gene was introduced into cotton by the Agrobacterium transformation method, and the expression level of AtNEK6 in transgenic lines was analyzed by real-time PCR. The growth and development of transgenic cotton were observed by observing the phenotype of transgenic plants and observing epidermal cells by scanning electron microscope. Mannitol and NaCl were used to simulate drought tolerance and drought tolerance of transgenic cotton by simulated drought treatment and salt treatment. The contribution of At NEK6 to the stress tolerance of transgenic cotton was identified by measuring related physiological indexes.【Result】The transgenic seedlings were screened by Kanamycin, and 10 different transgenic lines were identified by PCR. qRT-PCR analysis was used to select L7, L17 and L25 with higher expression levels. Under normal conditions, the transgenic lines were exhibited higher height and larger leaf than wild-type plants. But the cell surface area of transgenic cotton leaves was not significantly different from that of wild type by scanning electron microscope. The expressions of cell cycle related genes CYCB1, 1 and CYCA3, 1 and growth related genes GhGRF5, Gh EOD, GhAN3 and GhEBP1 were upregulated in the transgenic lines. Salt and drought tolerance of transgenic cotton was analyzed. On the normal 1/2 MS medium, the root length, fresh weight and dry weight of transgenic lines were not significantly different from those of wild type, and the number of lateral roots increased. However, in medium containing 250 mmol·L-1 mannitol, the root length, the lateral root number, fresh weight and dry weight of transgenic lines were significantly higher than those of wild type, showing a better growth state. In medium containing 200 mmol·L-1 NaCl, the number of lateral roots, fresh weight and dry weight of the transgenic lines were significantly higher than those of the wild type. Cotton seedlings of 30 days normal growth in the greenhouse were treated with 300 mmol·L-1 mannitol, the SOD activity of the transgenic plants was increased by 0.65 times, 0.42 times, 1.45 times compared with the wild type, and the CAT activity was increased by 0.65 times, 0.64 times, 0.42 times, and the MDA content was decreased. 0.51 times, 0.41 times, 0.22 times. Similarly, the changes of physiological indexes in transgenic lines in 250 mmol·L-1 NaCl treatment were higher than the ones in WT. In addition, the expression levels of related stress responsive genes GhAREB, GhDREB, Gh NCED 和 GhLEA5 in transgenic cotton were significantly higher than those in wild type cotton, which further showed that overexpression of AtNEK6 in cotton could increase salt tolerance and drought tolerance of plants.【Conclusion】AtNEK6 promotes the growth of cotton by participating in the regulation of cell cycle and growth. At the same time, it improved the salt tolerance and drought tolerance of cotton in adversity.