Cloning And Expression Analysis Of Cold-regulated Genes In Citrus Plants

China has become the second largest country of citrus crop production, where most citrus species originated. The world citrus industry has been seriously influenced by periodically freezing temperatures. A series of cold waves in the last century had a major impact on citrus production in Floririda and California of America and in south China. Therefore, a degree of cold hardiness is a desirable trait for introduction into commercial citrus cultivars. Much effort has been made toward improvement of cold tolerance of citrus crop through various conventional breeding programs; however, to date little progress has been made mainly because the fruit of the hybrids tends to contain high levels of poncirin which gives it a disagreeably bitter taste. In addition, polyembryony, long juvenility periods and the quantitative inheritance of cold tolerance have also been limiting factors for producing cold-hardy citrus verieties. Use of molecular biology techniques such as gene cloning, gene manipulattion and genetic transformation can overcome problems associated with conventional breeding and provide new approaches for understanding and improving cold tolerance in Citrus. It requires the availablities of genes that can be used in genetic transformation studies. Poncirus trifoliata (L.) Raf., a citrus relative, is widely used as the main rootstock of commercial citrus cultivars in China because it can enhence cold tolerance and resistance to some pathogens and insects of the grafted citrus plants. It is very likely that compared with cold-stress sensitive citrus, trifoliate orange may possess and express special cold-responsive genes/proteins that funtion to enhence cold tolerance. However, very little is known about the identity of genes/proteins that render trifoliate orange cold tolerant. In present study, suppression subtractive hybridization, at transcriptional level, and proteomic analysis, at translation level, were employed to isolate cold-regulated genes differentially expressed in citrus. The main results were as follows:The lethal treatments for leaves of trifoliate orange and Miyamoto satsuma mandarin (Citrus reticulata Blanco.) were -6℃for 60 min and 40 min, respectively, which provides important reference for experiment design of subsequent studies.It was improved for Clontech PCR-Select^TM cDNA Subtraction Kit to conveniently apply in citrus plants by introducing a housekeeping gene actin (sene primer: 5'-CACACTGGAGTGATGGTTGG- 3', antisense primer: 5'-ATTGGCCTTGGGGTTAAG AG-3') to detect the efficiency of adopter ligation and subtractive hybridization. It was needed to prolong adopter ligation time from overnight to 20 h according to the requirement of the kit manual that the adoptor ligation efficiency must be higher than 25%.Ptcor8, a cold-regulated gene of 849 bp size, was isolated from the leaf of P. trifoliata naturally cold acclimated in field. Bioinformatic analysis reveals that Ptcor8 is a cDNA full-length gene which belongs to cold acclimation protein WCOR413 family and possesses a potential ORF of 621 bp sizes encoding a cold acclimation protein WCOR413 homolog of 206 amino acid residues. Predicted by bioinformatic analysis tools such as ProParam, PSORT, MotifScan, CCD, TMHMM and NetPhos, the protein coded by Ptcor8, with molecular weight 22.8kD and pI 9.4, possesses five hydropathicity regions, five transmembrane helix, six serine phosphorylation sites and one WCOR413 domain. Molecular evolutionary investigation demonstrates that all members of WCOR413 family derive from green plants, dividing into two groups-PM (plasma membrane) type and TM (thylakoid membrane) type, and that Ptcor8 protein belongs to PM type. Ptcor8 gene has been submitted to GenBank on NCBI and an accession number of EU077497 was given to it. Preliminary expression analysis of Ptcor8 showed that this gene unexpressed at 26℃, expressed from 17℃to -6℃and expressed not shorter than 1 d when induced by 4℃, suggesting that Ptcor8 gene may involve in cold acclimation of P. trifoliata induced by low temperature.Differentially expressed protein profiles from leaves of P. trifoliata and C. reticulata cv. Miyamoto were determined by two-dimensional electrophoresis technique. Of 22 Differentially expressed protein spots analyzed by MALDI-TOF-TOF MS, 4 proteins showed significant homology to the proteins in GenBank. Amino acid residue of resistance protein, the homolog of protein spot #2 from P. trifoliata, was selected as target sequence for degenerate primer design. PCR was performed using degenerate primers and P. trifoliata genomic DNA as template. The PCR product of about 700 bp size was cut down, purified, cloned and sequenced. A fragment of 676 bp size nucleic acid sequence (Ptcorp) was obtained. Blast retrieval showed that Ptcorp shares 88% homology with an EST of cold acclimated Bluecrop (Vaccinium corymbosum) library (Accession number: CF811080), indicating that Ptcorp have association with cold acclimation. Semiquantitative RT-PCR analysis demonstrated that Ptcorp gene was upregulated by cold stress which was in accordance with the former result of protein expression profile.