Subcellular Localizations, Expressions And Functional Analysis Of Csice And Cscbf Transcription Factors Related To Cold Stress In Tea Plant[Camellia Sinensis(L.)O.Kuntze]

Low temperature is one of the major environmental factors, limiting geographical distribution, which also effects on crops yields and qualities. A great loss occurred on the world because of the low temperature each year. In recent years, the mechanism of chilling tolerance in plant had been a hot spot in the plant biology researches. Now the transgenic technology was used widely. Engineering method can be used to improve plant chilling tolerance. The genetic transformation system had not successfully established in tea plant. It was difficult to know gene biological function by transgenic tea plant. I transformed target genes from tea plant to Arabidopsis in this study to know their biological functions.Cultivar’yingshuang’was used as the material in this study. The ORF of CsICE and CsCBF were cloned. Following their subcellular localizations, gene expressions and biological functions were investigated.The main results were as follows:1. The ORF of CsICE and CsCBF were cloned. The subcellular localization results revealed that CsICE localized in cell nucleus, while CsCBF was mainly localized in cell nucleus, and also expressed in cell membrane and cell cytoplasm.2. The expression levels under different stresses were analyzed, including cold stress (4℃), ABA treatment(500μM), and osmotic stress (10%PEG6000). After ABA treatment, the expression peaks of CsICE and CsCBF were the highest in all the stresses,40and28.5times higher than control respectively. Under cold stress, the expression peaks of CsICE and CsCBF were lower than ABA treatment,22times and7times higher than control respectively. As for dehydration stress, the expression levels of CsICE and CsCBF were the lowest, only5times and3times higher than control respectively. CsICE expression trend was similar with low temperature treatment, while change rate was faster. The expression of CsCBF was in a steady state, except at a time of2h, which the expression level was significantly upregulated.3. The gene transcript levels of CsICE and CsCBF in different tissues, including roots, stems, leaves, buds, flowers and fruits were analyzed. CsICE and CsCBF both expressed in all tissues, but at different levels in different tissues. The highest expression level of CsICE was in the buds, significantly higher than other tissues, followed by in leaves and roots, and stems and fruits were lowest. The highest expression level of CsCBF was in the roots and shoots, followed in the leaves and stems, lowest levels in flowers and fruits.4. CsICE and CsCBF genes were transformed into Arabidopsis respectively by inflorescences disseminated method, using pBI121-GFP and pCAMBIA1301-GUS expression vectors. Transgenic plants were obtained after antibiotics screening.5. There were no phenotypic differences between CsICE transgenic plants and wild-type Arabidopsis. However, CsCBF overexpression plants displayed strongly phenotypic alterations, including stunted plants, smaller leaves, greener leaves, thicker blades, and lathy leaf shape. Under chilling stress, wild-type Arabidopsis had more purple leaves, which might be due to the anthocyanin generation. And wild-type Arabidopsis suffered more harmer under low temperature.6. The expression levels of CsCBF transgenic plants were analyzed by real time fluorescent quantitative PCR under cold stress. The expression level significantly increased after4h treatment, which value was15times higher than control. The results showed that cold treatment could induce the expression of CsCBF gene in Arabidopsis and was up-regulated.7. The cold resistance ability of CsCBF transgenic plants was analyzed by measuring physiological parameters under cold stress. Proline contents in CsCBF transgenic plants were higher than wild-type Arabidopsis and rose higher compared with wild-type Arabidopsis; MDA contents in wild-type Arabidopsis were higher than CsCBF transgenic plants and rose higher compared with transgenic plants; the maximal photochemical efficiency Fv/Fm and the potential photochemical efficiency Fv/Fo declined to a greater extent in wild-type Arabidopsis compared with transgenic plants; the qN of transgenic plants tended to increase and the qN of wild-type Arabidopsis was a downward trend. As a result, transgenic plants had better cold resistance ability than wild-type Arabidopsis. CsCBF gene in Arabidopsis might play a role in enhancing plants to resist chilling treatment.