Cold Stress Response Of ERF6and Gras Gene In Some Citrus Plants And Their Promoter Sequence Analysis

The low temperature is one of the major environmental factors affect the yield and quality of economic crops. Citrus plants (Citrus) is one of the important economic crops in China, which has a far-ranging uses as fresh fruits or medicinal plants. Citurs has a very large acreage in our country. However, as a subtropical plants with poor resistance of cold, the cultivation range of Citurs has been seriously hampered by the cyclical frozen disaster of plants. People have been dedicated to use transgenic breeding to improve the cold resistance of those plants. The transcription factor proteins play an important role in the plant abiotic stress responses. ERF and GRAS, two transcription factors specific in plant, are reported to be invoved in above process. The citrus plant Fingered citron (C. medica cv. Qingpi), BenDiZao(C.reticulata cv. Succosa), OuGan(C reticulata cv. Suavissima) and ChuHongYou(C. maxima cv. chuhongyou) are various at cold resistance. Poncirus (Poncirus trifoliate Raf.) is close related to Citrus while with a high cold resistance. We studied the relationship between the different expressions of transcription factor ERF6and GRAS and the cold resistance strength in2years old Fingered citron, BenDiZao, OuGan, ChuHongYou and Poncirus, and then we cloned the promoter sequence of ERF6and GRAS gene in Fingered citron and preliminary analyzed their transcriptional regulation mechanism.1. We selected2years old Fingered citron, BenDiZao, OuGan, ChuHongYou and Poncirus as experimental materials to teste the relative electric conductivity (REC) in leaves of these five species, and then analyzed the cold resistance to calculate their semilethal temperatures (LT50) after treating above plants under a series of low temperatures for24h. The content of the malondialdehyde (MDA) of the five species were mesured and the cold resistance strengthes were compared after the plants under4℃treatmented for various of hours. The results indicated that the REC of Fingered citron and other four plants increased at various ratio with the temperature drop, all of them showing an S-curve shape; The LT50of Fingered citron, BenDiZao, OuGan, ChuHongYou and Poncirus were-5.2℃,-8.6℃,-8.3℃,-6.6℃and-10.0℃), respectively. The leaves’ MDA content of the five species increased at different extent with the time extention under-4℃treatment. The results of the cold resistance comparement among five plants showed in consistent with the results of MDA and REC comparement,i.e, the strength order of the cold resistance was Poncirus> BenDiZao> OuGan> ChuHongYou> Fingered citron; The differences of their cold resistance were also in consistent with the differences of semilethal temperatures among those five plants.2. We treated2years old Fingered citron, BenDiZao, OuGan, ChuHongYou and Poncirus under4℃for different hours, then detected the different mRNA expression levels of the GRAS and ERF genes among the five plants treatment by fluorescence quantitative PCR. After analyzing the relationship of the expression differences and the cold resistance strength between these two transcription factors, we made the responses of the ERF6and GRAS to cold stress clear in citrus plant. The results showed that the mRNA expression of the GRAS and ERF genes in Fingered citron, BenDiZao, OuGan, ChuHongYou and Poncirus under low temperature stress were all induced increase gradually. The mRNA expression of ERF gene in Fingered citron under low temperature stress showed an upward trend within12hours,4.3times increase of the control(0hours), then decreased gradually after12hours and finally recovered to the same level of the controls’ after48hours. Similarily, the mRNA expression of ERF gene in BenDiZao, OuGan, ChuHongYou and Poncirus also upregulated within36hours, respectively showing61,62,6and316increase times, and then decreased gradually after36hours. Under4℃, the mRNA expression of GRAS gene in Fingered citron, BenDiZao and Poncirus increased within36hours, showing the increase rate were9.6,408and1506times of control. The expression of GRAS gene in ChuHongYou and OuGan increased within12and24hours and the increase rate were16and1200times. These results indicated that upregulation the mRNA expression of the GRAS and ERF genes was related to the cold stress responses in citrus plants and the increase rate of the mRNA expression had a positive correlation with the cold resistance strength.3.2years old Fingered citron was used as experimental materials to extract the genomic DNA of Fingered citron and then to clone and analyze the promoter sequences of the ERF6and GRAS by Tail-PCR. Based on the full-length cDNA sequence of the GRAS and ERF gene, we designed the downstream specific primers to accquire the promoter sequences of the ERF6and GRAS genes. Nucleotide sequence analysis showed that the promoter sequence of ERF6gene consisted of847bp and the promoter sequence of GRAS gene consisted of999bp. By PLACE program, the functional elements analyse show the promoter sequences of the ERF6and GRAS genes contained a variety of conservative functional elements of the gene promoters in higher plants. The ICE1binding site, CBFs binding site and LTR of ERF6and GRAS genes are belong to the cis-acting elements involved in low-temperature responsiveness of plants, instructing that both the ERF6and the GRAS genes participate in the cold stress response of Fingered citron.The above results summarized that the mRNA expression of the GRAS and ERF genes in Fingered citron induced by low temperature, those two transcription fanctors involved in the cold stress response of citrus plants and improved the cold resistance of plant as the transcription factors of cold-responsive gene or cold-responsive gene itself.