Cloning And Expression Of A Novel Alliinase Gene From Garlic Roots And Selencing Of Alliinase Genes By RNAi

Garlic(Allium sativum L.) was a very important medicinal plant. Alliicin and other sulfur compounds, did much benefit to human’s health. During tissue damage, they are generated by enzymatic catalysis of alliinase. So, alliinase was a very important enzyme in the genus Allium. It was found alliinase from garlic bulbs and leaves was different from roots. There were many researches about alliinase from garlic bulbs and leaves, but very few about alliinase from garlic roots.In according to part cDNA sequence of alliinase from garlic roots in GeneBank, the full cDNA sequece and genome sequence were cloned by RACE and RT-PCR techniques. The deduced amino acid sequence was analysised through bioinformatics softwares. Transcriptional levels of different alliinase genes in different tisues were analyed by semi-quantitative RT-PCR and real-time fluorescence quantitative PCR. According to codon bias of Pichia pastoris, the gene code for alliinase from garlic bulbs was changed, and then successfully expressed in Pichia pastoris system pIC9K/GSl15.Also, a particle bombardment-mediated transformation system had been established in garlic. RNAi plant expression vectors for silencing alliinase genes have been constructed and transformed into garlic. Five trangenic garlic plants were regenerated and analysed by PCR, Southern blot, semi-quantitative RT-PCR, real-time fluorescence quantitative PCR and activity assay of alliinase.The results were summarized as bellow:1. The full cDNA sequence of alliinase from garlic roots was cloned with length of2007bp and the biggest open reading frame of1342bp. The deduced amino acid sequence contained463amino acids, with a molecular mass of52.3kD and predicted isoelectric point of6.95. The genome sequence of alliinase from garlic roots contained3extrons and2introns with length of2217bp. Identities of deduced amino acid sequence with I and II root alliinase of onion is70%and61%respectively, but with a bulb alliinase of garlic was only54%.2. The deduced anino acid sequence was analysised using bioinformatics softwares. A signal peptide was identified in N terminus with a possible cleavage site between residues29and30. Conserved domains were found as follow:EFG-like domain in N terminus; PyrP binding domain in the middle with a Lys residue at position287from the N terminus as a possible PyrP binding residue; Conserved domains belongs to aspartate aminotransferase family; Alliinase catalysis domain near C terminus. Besides, there were four potential glycosylationsites and9cysteine residues, eight of which were predicted to form4disulfide bridges.3. Southern blot analysis of genomic DNA from garlic bulbs indicated root alliinase was likely encoded by a family of related genes. Semi-quantitative RT-PCR analysis showed that a bulb alliinase gene of garlic was high expression in bulbs and shoots, but no expression in roots. On the other hand, a root alliinase gene of garlic was high expression in roots, but very low expression in bulbs and shoots. Real-time fluorescence quantitative PCR analysis suggested that the expression level of root alliinase gene in roots was27times and9times high as expression level in bulbs and shoots, respectively. 4. According to codon bias of Pichia pastoris, the gene code for alliinase mature protein from garlic bulbs was changed, and then successfully expressed in Pichia pastoris system pIC9K/GSl15. The specific activity of alliinase from the recombinant pPIC9K-Alliich/Pichia-54was highest after induced expression72h by methyl alcohol. It was68.95U/mg, much lower than that of the extracted alliinase from garlic blubs.5. A particle bombardment-mediated transformation system in garlic had been established. Using the calli induced from the root segments of in vitro garlic (Allium sativum. L) plantlets as recipients, six combinations of particle bombardment parameters were designed for the development of transformation procedure of this plant. Transgenic garlic plantlets were successfully recovered from four bombardment combinations. The highest transformation frequency of2.4%was obtained when the calli were bombarded once at shoot distance9cm with pressure1100psi and at shoot distance12.5cm with pressure1300psi helium, respectively. The resistant plantlets to antibiotics were analyzed by PCR and Southern blot. The results showed that hygromycin phosphotransferase (HPT) selectable marker gene and P-glucuronidase (gus) reporter gene had been integrated in the genome of galic. GUS expression assay of bombarded calli, somatic embryos, embryogenic roots, regenerated shoots and leafs showed that resistant tissues distinctly appeared blue.6. Six RNAi plant expression vectors for silencing alliinase genes had been constructed. Three of them targeted for the bulb alliinase gene and three for root alliinase gene. These vectors were transformed into garlic recipient plants. Five trangenic garlic plants transformed with RNAi vector targeted for bulb alliinase gene were generated. The gene insertion of these trangenic garlic plants was confirmed by PCR and Southern blot assay. By semi-quantitative RT-PCR and real-time fluorescence quantitative PCR analysis, transcriptional levels of bulb-alliinase gene in fresh bulbs and leaves of transgenetic garli plants decreased significantly. The highest silencing efficiency was more than90%. Activity assay of alliinase in transgenic garlic lines exhibited that total activity of alliinase also decreased.The new gene cloned in the research would be a good choice for recombinant expression of alliinase. The results of this study would be useful for analyzing contribution of different alliinase to edible quality of garlic. The newly established transformation procedure could be used in the improvement of galic by genetic approach.