Cloning And Analysis Of WRKY Gene In Tomato

Plants take advantage of a variety of mechanisms to adapt biotic and abiotic stress reaction. About the study between plants and the environment, people are more and more focusing on the plant gene regulation at the transcriptional level, the research on the transcription factor has become a focus of the current plant gene research.When the plants were stimulated by the external environment, it would pass through a series of signals, eventually induced the expression of defense response gene, the transcription factor played an important role in this process. Recently, MYB, bHLH proteins, MADS domain proteins, WRKY zinc finger proteins, AP2 domain proteins, bZIP proteins and other transcription factors had been found in plants. Among the numerous transcription factors, WRKY transcription factors were a new type of transcription factor which has been discovered in recent years, they were named for the highly conserved amino acids WRKYGQK in the N-terminal. The specific zinc finger motifs located downstream of the conserved regions, which only in the transcription factors in plants. It had been identified that WRKY protein could specifically combine the (T) (T) TGAC (C / T) sequence, WRKY domain played an important part in this process, which interacted with other proteins to activate or inhibit the defense response gene transcription.WRKY genes which had rapid, transient and tissue-specific expression features could be induced by the external environment, and could participate in biotic and abiotic stress responses. When the higher plants were injured by a variety of pathogen, WRKY gene would express quickly and largely. WRKY transcription factors perform regulative functions in plant defense responses, plant growth and development, plant metabolism and signal transduction. In addition, some WRKY proteins also had self-control ability.Rapid amplification of cDNA ends (RACE) was built based on the mRNA reverse transcription and PCR, making use of this technique the complete sequence of gene could be known from the limited fragments, or even from unknown gene fragments. RACE had many advantages in obtaining the gene coding sequence that the cDNA library did not have.Real-time fluorescence quantitative PCR ( FQ-PCR) technology was a highly sensitive nucleic acid quantification technique, which was developed on the basis of the qualitative PCR, Adding fluorescent group in the PCR reaction, the whole PCR process could be detected by the cumulative amount of fluorescence signal, finally the unknown template also could be quantitativly analyzed by using of the standard curve. Taking advantage of quantitative PCR, the researcher not only could quantify DNA, but also RNA, including genomic DNA copy number, viral DNA copy number and transgene copy number. Although the WRKY genes in Arabidopsis, tobacco and rice had been studied more, the tomato WRKY transcription factor was rarely reported so far. This study is to obtain WRKY resistance gene in tomato, which would lay the foundation for breeding disease-resistant tomato.In this study, tomato seedlings were used as experimental materials, the specific primer was designed according to the cloned WRKY transcription factor gene fragment and tomato EST sequences; The WRKY gene partial cDNA sequence has been cloned by RT-PCR technology from tomato seedlings which were treatment by 100μM JA, We obtained 3'cDNA sequence using the RACE technology, it was 1028 bp, and encoded a polypeptide of 187 amino acids residues; The 5'cDNA was 823 bp. The fragment was inserted into the pMD19-T vector, and transformed into E. coli DH5α.The recombinant plasmidwas digested with Pst I and EcoRΙ, and sequenced. Through splicing and further carried out operation on related bioinformatics analysis. The result indicated that the full long cDNA was about 1740bp, and the longest open reading frame was 1083bp. The full long cDNA which encoded a protein containing one conserved domain WRKYGQK and the C2H2 motif about 360 amino acids residues. Therefore, we suggest that the WRKY we had cloned belonged to WRKY family groupΙΙ. The Blastn result showed that it had 91%,88%,85%,86%,80% and 69% similarity with FJ654265.1 (E: 0.0), AK329773.1 (E: 0.0), FJ654264.1 (E: 0.0), EU755370.2 (E: 0.0), AY789641.1 (E: 0.0) and AB028022.1 (E: 0.0) respectively, which proved that the cloned genes belonged to WRKY gene family.Making use of relevant software to analyze the structure of the cloned WRKY genes, nd further to preliminarily predict the function. The result suggested that WRKY protein molecular weight was 39.77KD, and theoretical pI value was 8.57.Secondary structure analysis showed that theα-helix,β-folding and random coil in WRKY protein accounted for 28.55%, 21.80% and 49.65% respectively. Analysis indicated that the protein had no transmembrane helix zone, it was not a transmembrane protein. In addition, it was not found its signal peptide, thence we concluded that it was not a secreted protein.To determine whether elicitors could induce expression of tomato WRKY gene, we analyzed the transcript levels of WRKY gene induced in various stress. Real-time PCR indicated that tomato WRKY gene can be induced in biotic stress such as NaCl(100mM), 4℃and signal molecular JA (100μM), SA (1mM). In salt stress, the trend was initially increasing, with the highest expression after NaCl(100mM) treated 12 hours; In cold stress, it changed little; However, after JA (100μM) treated 12 hours, the expression significantly increased. In addition, in order to study the genetic effects on tomato growth, WRKY gene expression in the roots, stems, leaves had been explored. The result showed that WRKY gene expressed in roots, stems and leaves, with the highest expression in young leaves, while not observable in stems. The data further indicated that tomato WRKY genes may be involved in the regulation of the defense response as well as the growth and development.