Cloning and functional analysis of VyCIPK9 gene in Chinese wild grape (Vitis yeshanensis‘Yanshan’)

Abstract:【Objective】CIPK (CBL-interacting protein kinase) is a class of serine / threonine kinases that play an extremely important role in signaling abiotic adversities such as drought and high salt. Yanshan grape (Vitis yeshanensis), a Chinese wild grape originating in Shanhaiguan, Hebei Province, has strong resistance to cold, salt and drought. It was used as the material to study the characteristics and functions of the CIPK gene of V. yeshanensis‘Yanshan’in order to provide a clue for excavating grape CIPK genes and breeding new grape cultivars with abiotic stress resistance in the future.【Methods】 The 1-year old Yanshan nursery plants propagated bu cutting were transplanted in 20 cm × 30 cm pots. The plants were treated with plant growth regulator and abiotic stress. The treatments of plant growth regulators were: 0.5 mmol·L^-1 of indole-3-acetic acid (IAA), 0.1 mmol·L-1 of 6-benzylaminopurine (6- BA), 0.1 mmol·L^-1 of gibberellin acid (GA3), 3 mmol·L^-1of ethephon (Eth), 0.1 mmol·L-1 of salicylic acid (SA), 0.05 mmol·L^-1 of methyl jasmonate (MeJa), respectively, the samples were taken 0, 3, 6, 9, 12, 24, 48, and 72 hours after spraying on the leaves of the plants. The treatments of abiotic stress were:  (1) low temperature treatment: the whole plant of Yanshan grape was subjected to 4 ℃ low temperature (kept in light incubator with 16 h in light / 8 h in dark, light intensity 36 μmol·ms-1 ); (2) Drought stress treatment: when the soil water content of Yanshan grape plants in pots was 50% , drought treatment start; (3) Salt stress treatment: 200 mmol·L^-1 NaCl was used to irrigate the Yanshan grape plant, and the degree of solution outflow from the basin bottom was taken as the degree. All three abiotic stress treatments were carried out for 0, 3, 5, 7, 9, 11, 13 and 15 days. The leaves of mature Yanshan grapes in the mid of wines were collected, and then quickly frozen in liquid nitrogen and stored in -80 ℃ ultra-low temperature refrigerator for standby. The tissue specificity was analyzed by real-time quantitative PCR. The expression patterns of grape under abiotic stress (low temperature, salt and drought) induced by different tissues and organs and different plant growth regulators were analyzed; the plant expression vector of the VyCIPK9 gene was constructed. The VyCIPK9 gene was transformed into tobacco to verify function. After the seeds of the VyCIPK9 transgenic tobacco (OE#1, OE#2, OE#3) and wild-type tobacco were sterilized and cleaned, they were first cultured in MS medium. After 5 days, they were transplanted into MS medium containing 150 mmol·L^-1 and 250 mmol·L^-1 NaCl for salt stress treatment. The seeds germinated and the plants grew at 25 ℃, with 16 hours in light and 8 hours in dark. The germination rate was measured 7 days after the treatment. The root length (mm), plant height (mm) and fresh weight (g) of seedlings were measured 12 days after the treatment. The VyCIPK9 transgenic tobacco (OE#1, OE#2, OE#3) and wild-type tobacco continued to grow under the treatment of 150 mmol·L^-1 NaCl at 25 ℃, 16 h in light and 8 h in dark. Those plant physiological and biochemical indexes were measured 4 weeks after the treatments. The relative conductivity was measured and calculated by conductivity meter (DDS-308A, Shanghai Leici). The content of chlorophyll, proline, malondialdehyde, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and hydrogen peroxide (H2O2) were measured by spectrophotometer (Beijing Solabao Technology Co., Ltd.).【Results】The expression of the VyCIPK9 gene was the highest in the old leaves. The expression of the VyCIPK9 gene increased first and then decreased at 0-48 h induced by the seven plant growth regulators, and was most sensitive to ethylene and methyl jasmonate. The VyCIPK9 gene responded greatly to the salt stress under 200 mmol·L^-1 NaCl treatments, which laid a foundation for the verification of VyCIPK9 gene function under different concentrations of the salt stress. The VyCIPK9 gene was transferred into tobacco. It was found that seed germination rate and seedling growth of transgenic tobacco were better under salt stress. The physiological and biochemical indexes of transgenic tobacco were measured 4 weeks after the treatments. The physiological and biochemical characteristics of transgenic tobacco were stronger than those of the wild tobacco. With the extension of the salt stress treatment time, the chlorophyll and proline contents of the VyCIPK9 transgenic tobacco were significantly higher than those of the wild tobacco (p＜0.05), and the relative conductivity and malondialdehyde contents were significantly lower than those of the wild tobacco (p＜0.05), indicating that the photosynthetic characteristics and stress resistance of transgenic tobacco plants were stronger than those of wild tobacco, and the damage caused by the salt stress was lower than that of the wild tobacco. The oxidase activity of the VyCIPK9 transgenic tobacco was significantly higher than that of the wild tobacco (p＜0.05), and the content of H2O2 was significantly lower than that of chlorophyll in the wild tobacco (p＜0.05). It showed that the higher oxidase of the VyCIPK9 transgenic tobacco could help to reduce the content of H2O2, so as to reduce the damage of the salt stress.【Conclusion】The overexpression of the VyCIPK9 gene would enhance the salt tolerance of the transgenic tobacco plants.