【目的】类钙调磷酸酶B互作蛋白激酶（CBL-interacting protein kinase，CIPK）是一类丝氨酸/苏氨酸激酶，在干旱、低温等非生物逆境的信号传递中起着重要的作用。以中国野生燕山葡萄（Vitis yeshanensis‘Yanshan’）为材料，筛选到1个CIPK转录因子，暂命名为VyCIPK9（登录号：MK942080），探究其特性与功能。【方法】利用实时荧光定量PCR （quantitative real-time PCR，qRT-PCR）技术分析VyCIPK9基因在葡萄不同组织器官及不同诱导下的表达模式，同时构建VyCIPK9基因植物表达载体，将VyCIPK9基因转化到烟草中，探究盐胁迫处理下VyCIPK9基因过表达对转基因烟草种子、幼苗和植株生长发育及其生理特性的影响。【结果】VyCIPK9基因在葡萄不同组织器官中特异表达，主要在老叶中高度表达；VyCIPK9基因对吲哚乙酸（indole acetic acid，IAA）、茉莉酸甲酯（methyl jasmonate，MeJA）诱导和盐胁迫响应强烈；在盐胁迫条件下，VyCIPK9转基因烟草种子发芽率均比野生型烟草高、幼苗生长状况均比野生型烟草效果好；植株生长4周后，测定其生理生化指标，发现VyCIPK9转基因烟草植株生理生化指标中相对电导率、脯氨酸含量和抗氧化酶活性均高于野生型烟草植株，这说明盐胁迫下VyCIPK9转基因植株受到的伤害程度小于野生型烟草植株。【结论】过表达VyCIPK9基因可以增强转基因烟草植株的耐盐性，为进一步了解VyCIPK9基因在葡萄抗非生物逆境胁迫中的作用提供了一定的理论依据。
Cloning and functional analysis of VyCIPK9 gene in Chinese wild grape (Vitis yeshanensis‘Yanshan’)
【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. Yan-shan 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-1 of 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 36μmol · 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 treat-ments 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 dif-ferent tissues and organs and different plant growth regulators were analyzed;the plant expression vec-tor 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 tobac-co were sterilized and cleaned, they were first cultured in MS medium. After 5 days, they were trans-planted 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 ger-mination 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 tobac-co (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 con-ductivity meter (DDS-308A, Shanghai Leici). The content of chlorophyll, proline, malondialdehyde, su-peroxide dismutase (SOD), peroxidase (POD), catalase (CAT) and hydrogen peroxide (H2O2) were mea-sured by spectrophotometer (Beijing Solabao Technology Co., Ltd.).【Results】The expression of the Vy-CIPK9 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 concen-trations of the salt stress. The VyCIPK9 gene was transferred into tobacco. It was found that seed germina-tion 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 physiologi-cal 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 chloro-phyll 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.
万方数据
维普
