Molecular Cloning, Expression Analysis Of Defence Related Genes From Citrus Infected By Ca. Liberibacter Asiaticus

Huanglongbing is one of the most devastating diseases of citrus, the disease is caused by a phloem-limited fastidiousα-proteobacterium,which was named Candidatus Liberibacter spp. Because Candidatus Liberibacter spp and the mechanism of the interaction between Candidatus Liberibacter spp and citrus in the infection process of the pathogen remain unclear, the citrus breeding in resistance to huanglongbing failed to achieve a major breakthrough. Studying the host responses mechanism would help understand the disease mechanism of HLB and may contribute to developing new tools to mannage this devastaing disease.In the present study, according to results of Citrus sinensis SSH library analysis, primers of two up-regulation EST sequences were designed; the full-length cDNA of MYB and CAD type gene were cloned and their sequences were analysed. The expression characteristics of two genes were analysed under Ca. Liberibacter asiaticus treatment by Real-time quantitative PCR. According to the above, the mechanisms that Ca. Liberibacter asiaticus infected Citrus sinensis and Citrus sinensis defensed Ca. Liberibacter asiaticus were explored. All of these provided the basis of molecular biology for the research in resistance to Ca. Liberibacter asiaticus. At the same time, they provided theoretical and technological basis for controlling huanglongbing and the citrus breeding in resistance to Ca. Liberibacter asiaticus. So the prevention and control of HLB get further development.We cloned the full-length cDNA of MYB and CAD by rapid amplification of cDNA ends (RACE) from the leaves of Citrus sinensis. And then, the characteristics and functions of the two genes were analysed by different bioinformatics methods. The expression characteristics of two genes were analysed under Ca. Liberibacter asiaticus treatment by Real-time quantitative PCR.The results and conclutions of this research are as follows:1. A MYB type gene was cloned by rapid amplification of cDNA ends (RACE). This gene was termed as CsMYB, and submitted in GenBank (accession No. of HQ841074). The full-length cDNA sequence of CsMYB is 1306 bp, including an open reading frame (ORF) of 909 bp and the typical 26 bp poly-A. Bioinformatics analysis showed that the gene putatively encodes a protein which has 302 amino acids with molecular weight 32.97KD, and its theoretical pI is 8.5. The CsMYB gene contains two typical conserved motifs: R2 and R3.2. The CAD(cinnamyl alcohol dehydrogenase) gene was cloned by rapid amplification of cDNA ends (RACE). This gene was termed as CsCAD, and submitted in GenBank (accession No. of HQ841075). The full-length cDNA sequence of CsCAD is 1124 bp, including an open reading frame (ORF) of 897 bp. Bioinformatics analysis showed that the gene putatively encodes a protein which has 298 amino acids with molecular weight 32.45KD, and its theoretical pI is 6.4. The CsCAD gene contains two typical conserved motifs: ADH_N and ADH_zinc_N.3. The expression profiles of two genes under the treatment of Ca. Liberibacter asiaticus (Las) was investigated by Real-time qPCR. The results showed that CsMYB gene expression varied at different times infected by Las.The expression profile of CsMYB gene presents the trend of 'low-high-low'. Compared with control, the expression level is low and presents a weak upward trend in a month after inoculation. The expression raises to higher level as the pathogen proliferating, the level is twice and three times of the control after one month. The symptom starts to appear after three months. And later, with more severe disease, the expression starts to descend, even it is lower than control and keeps a low level.Therefore the CsMYB gene is speculated to be a involved in the procedure induced defense of Las.The expression model of CsCAD gene is also 'low-high-low'.Compared with control, its growth is higher than CsMYB. About 20 days, the level of CsCAD remains little changed. But after one month, the expression raises rapidly and the highest level is 10 times of the control. And 3 months later, the expression begins to fall and remains a lower level which still is higher than control.4. According to the results of the sequences and quantitative analysis of CsMYB and CsCAD , the CsMYB gene is speculated to be a transcription factor and the CsCAD gene to be a cinnamyl alcohol dehydrogenase, they involve in the induced defense procedure .