Analysis Of The Differentially Expressed Genes In Fingered Citron Under Cold Stress

Fingered citron (Citrus medica L. var. sarcodactylis Swingle) is one of the specific economic trees in China, which has a high ornamental and economic value while poor resistance of cold. The development of Fingered citron industry was seriously hampered by the cyclical frozen disaster of plants. People have been dedicated to solving the problems of poor tolerance of plants by the way of plant-cold-resistant breeding. The research of mechanism of plant cold tolerance is the theoretical basis of the cold resistance breeding. The progress of their research directly restricts the process of cultivation of varieties resistant to cold and improvement of existing varieties. In order to investigate the mechanisms of resistance to cold stress, we studied the differences in gene expression and compared the physiological and biochemical changes under 4℃cold acclimation in 2 years old potted plants of Fingered citron by the research methods of suppression subtractive hybridization, reverse northern blot and quantitative PCR.1. Differentially expressed fragments was screened by suppression subtractive hybridization (SSH) using the experimental test materials of 2 years old Fingered citron (C. medica cv.'Qingpi') growed at 4℃for 24 h. The plants without cold treatment was used as the "driver" and the plants after cold treatment was used as the "tester"; The total RNA was respectively extracted from "driver" and "tester" leaf tissue and mRNA was isolated and then reverse transcriptased into a double stranded tester cDNA and driver cDNA; After two rounds of subtractive hybridization and PCR amplification, the differentially expressed fragments in Fingered citron under cold stress were enriched. We obtained 200 clones, including 111 different fragments which the screened colony PCR products concentrated in 250 bp-1 kbp were sequenced. The sequence analysis revealed that 57 fragments were showed high similarities at the amino acid level to the genes with known functions.Reverse Northern dot-blotting indicated that 87% of cold-induced genes were positive clones and the expression of them appear to be up regulated.2.2 years old Fingered citron which growed at 4℃for 24 h was used as experimental test materials. Full-length cDNA sequence of GRAS and ERF gene were cloned in Fingered citron using the RACE and RT-PCR method. The results showed that the GRAS fragment in Fingered citron was highly associated with GRAS genes of Arabidopsis and the sequence alignment showed that it had a conserved domain of GRAS. Therefore, we named the GRAS gene in Fingered citron as CmsGRAS (Genbank accession number:JF440647), which had an ORF of 1236 bp and encoded 413 amino acids. The sequence alignment results showed that the full-length ERFcDNA in Fingered citron was associated with Arabidopsis AtERF6 and had a conservative AP2/EREBP domain. Therefore, it was named as CmsERF6 (Genbank accession number:HQ698835), which had an ORF of 999 bp and encoded 309 amino acids;3.2 years old Fingered citron and Poncirus (Poncirus trifoliata Raf.) under 4℃treatment for different hours were used as experimental test materials; In order to understand the differences of molecular mechanisms in cold tolerance between Fingered citron and Poncirus, mRNA expression levels of GRAS and ERF genes were detected and physiological indicators of cold stress were measured; Fluorescence quantitative PCR method was used to study the mRNA expression levels of GRAS and ERF genes in Fingered citron and Poncirus under low temperature stress, the results showed that the expression of GRAS genes in Fingered citron and Poncirus are both on the rise with the increase of stress time. Within 24 h the expression of GRAS gene in the Poncirus was reached 800 times of the control. The increase of its expression may be related to low temperature stress response; The influence of low temperature stress at 4℃for 12 to 24 hours was both strong in Fingered citron and Poncirus, but it is more intense in Fingered citron, indicating that Fingered citron was got more cold damage; The results of quantitative PCR analysis showed that ERF6 was induced both in Fingered citron and Poncirus during cold acclimation, but the trend of expression levels were obviously different. The expression of ERF6 rapidly increased in cold-sensitive Fingered citron after 12 hours of 4℃cold treatment and returned to control levels after 48 hours; While in cold-toleranted Poncirus, there was only a slight increase within 24 hours of 4℃cold treatment and significantly increased by about 4000 times compared with the control group after 48 hours; We speculated that ERF6 may be involved in cold stress response both in Fingered citron and Poncirus but it may have a different mechanism of resistance to cold in Fingered citron and Poncirus, and particularly may play an important role in the strong cold tolerance in Poncirus.