Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (22): 4230-4240.doi: 10.3864/j.issn.0578-1752.2018.22.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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

FAN Xin(),ZHAO LeiLin,ZHAI HongHong,WANG Yuan,MENG ZhiGang,LIANG ChengZhen,ZHANG Rui,GUO SanDui,SUN GuoQing()   

  1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2018-06-08 Accepted:2018-07-23 Online:2018-11-16 Published:2018-11-16

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Abstract:

【Objective】 AtNEK6 is a NIMA related kinase in Arabidopsis thaliana. Overexpression of AtNEK6 in Arabidopsis can promote plant growth, and improve the salt tolerance and drought tolerance of plants. By transforming AtNEK6 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 AtNEK6 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, GhEOD, GhAN3 and GhEBP1 were upregulated in the transgenic lines. Salt and drought tolerance of transgenic cotton was analyzed. On the normal 1/2MS 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, GhNCED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.

Key words: AtNEK6, transgenic cotton, salt and drought tolerance, functional analysis

Fig. 1

Acquisition and identification of transgenic cotton A: The schematic structure of pBI121-AtNEK6 vector; B: Callus of tobacco; C: Seeding of transgenic plants; D: Identification of positive transgenic cotton plants; WT: CK; 1-15: AtNEK6 transgenic line; E: Expression level of AtNEK6. ** indicate significant difference at P<0.01 level. The same as below"

Fig. 2

AtNEK6 promotes the growth of transgenic cotton A: Growth phenotype of plant from WT and transgenic lines, Bar=10 cm; B: Morphology of leaves from WT and transgenic lines, Bar=5 cm; C: Height of plant from WT and transgenic lines; D: The difference of leaf area between WT and transgenic lines, 1-8: The first true leaf to the eighth true leaf; E: Leaf epidermal cells of WT and transgenic lines under SEM, Bar=50 μm; F: Leaf epidermal cell area of WT and transgenic lines; G: Transcript levels of cell cycle-related/ growth-related genes in WT and transgenic lines revealed by qRT-PCR. * indicate significant difference at P<0.05 level, respectively. The same as below"

Fig. 3

Characteristics of transgenic plants under drought and salt stress A: Performances of WT and transgenic cotton after treatment of 250 mmol·L-1 Mannitol and 200 mmol·L-1 NaCl for 7 days; B: Root length; C: Lateral roots number; D: Fresh weight; E: Dry weight"

Fig. 4

Identification of drought and salt tolerance of AtNEK6-overexpressing cotton in greenhouse and enzyme activity detection A: Performances of WT and transgenic cotton under drought and salt stress; B: CAT activity; C: SOD activity; D: MDA content"

Fig. 5

Expression analyses of stress response genes in WT and AtNEK6-overexpressing cotton under drought and salt conditions"

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