Isolation And Functional Analysis Of ICE1Gene From Brassica Campestris

The abiotic stresses serve as important environment factors that influence normal growthof plants. Among these factors, low temperature, drought stress and salt are the mostimportant factors. The low temperature can damage of different degree or even death of plants,and caused the huge loss of agricultural and forestry production. Plants develop a variety ofsophisticated mechanisms to rapidly sense a changing environment and protect themselvesfrom environmental stress. Plants develop a sophisticated mechanism, which can distinguishadversity information to adjust growth of plant through the signal express, and finally protectthem from environmental stress.Brassica napus (Brassica campestris.L) is one of the most important oil crops in China.As a new Brassica napus breed, Longyou6can be planted in the arid and cold regions ofNorthern China, where the extreme low temperature can reach-20℃to-30℃. It is the onlyone winter Brassica napus breed that lives through the winter can in the central and northernregion and He Xi region of Gansu province.This research isolated a cold-resistant gene ICE1from Longyou6.The expression in different tissues and low temperature stress of Brassica napus ICE1were analyzed by real time RT-PCR method. We studied the sequence characteristic andresistance physiological function of transgenic tobacco of Brassica napus ICE1. The mainresults are showed as follow:1. The new cold tolerance gene ICE1(the register number of GenBank is JF268687) wascloned from Longyou6by using RACE (Rapid Amplification of cDNAEnds) technique. Thisnew gene had a full-length of1737bp with an open reading frame (ORF) of1500bp, andcomprised499amino acid residues with the predicted molecular mass of53.2KD andisoelectric point of5.0. The result of phylogenetic tree showed that Brassica napus ICE1wascloser to BcICE1and BchICE1. Bioinformatics showed that Brassica napus ICE1protein wasa hydrophilic protein and not includes signal peptide sequence. The protein had a total of2-4transmembrane regions and two large polarity connection ring.2. The analysis results of real time RT-PCR indicated that Brassica napus ICE1wasexpressed in different tissues (stem, leaf, hypocotyls). The expression was the highest inhypocotyls, secondly was leaf and the lowest was stem. The expression of ICE1in Longyou6and Tianyou2Brassica napus were induced by low temperature stress after48days low temperature stress. However, the former had a higher level of gene transcription of ICE1thanthe later.3. The fusion protein pBI121-ICE1-GFP was expressed transiently in onion epidermis.The green fluorescence stimulated by GFP in cell nucleus was observed under the confocalfluorescence microscopy. The results indicate that Brassica napus ICE1is localized in the cellnucleus.4. We conducted an expression carrier pBI121-ICE1-GFP, and then transformed it intotobacco by the CaMV35S promoter. With the plasmid DNA as positive control and wildtobacco as negative control, the PCR result showed that regenerated pants had amplifiedobvious DNA band, which had the same position with amplification band of the positiveplasmid. Compared with non-transgenic plants，transgenic lines exhibited high resistance tokanamycin and followed with Mendelian single-copy inheritance theory. The above resultsindicated that Brassica napus ICE1gene have been integrated into tobacco.5. When transgenic and non-transgenic plant stressed by2℃with seven day, Thenon-transgenic tobacco wilting serious but transgenic tobacco changed little. All transgeniclines of Brassica napus ICE1had higher chlorophyll content, greater photosynthetic rate,relative water content, soluble proteoglycan, the activities of SOD, less electrolyte leakageunder the same stress conditions。These results suggested the cold tolerance of transgenictobacco plants with Brassica napus ICE1gene was improved.In conclusion, Brassica napus ICE1gene can be induced to express by low temperaturecondition, and improved significantly the cold resistance of transgenic tobacco plants.Brassica napus ICE1gene can provide powerful genetic resources and molecular tool forimproving cold resistance of plants.