The Functional Identification Of AMR1、GGP And GPP Genes Of Ascorbic Acid Synthesis Pathway In Tomato

Ascorbic acid (AsA) is an important antioxidant, playing important roles in various plant physiological processes. Ascorbic acid is an important vitamin widely distributed in fresh fruits and vegetables and many other plant species. As a kind of highly active substance, AsA is involved in many metabolic pathways. Ascorbic acid can scavenge the reactive oxygen species generated during ageing process and stress response, and thus help plants delay ageing and combat stress. AsA also acts as an important enzymatic cofactor and growth regulator, and thus regulating AsA snyhtehsis and accumulation is vital to plant growth devleppment and quality formation. Genetically engineering ascorbic acid biosynthesis has also made breakthroughs. However, in tomato, only a few of genes involved in ascorbate metabolism were cloned and identified. Regulation of ascorbic acid synthesis and metabolism by cloning the key genes in tomato awaits further investigationIn this study, some genes related to ascorbate biosynthesis were cloned from tomato. We preliminarily identify the function of AMR1, GGP and GPPs which are candidate genes in regulating tomato ascorbate biosynthesis. Then these genes were over-expressed or suppressed in tomato by Agrobacterium-mediated transformation. The main results are presented as following:1. AMR1 gene was cloned from tomato. It contains a complete open reading frame of 1283 bp, encoding 406 amino acids. GGP gene involved in ascorbate Smirnoff biosynthesis pathway was cloned from tomato. It contains a complete open reading frame of 1314 bp, encoding 437 amino acids. Two GPP genes (GPP1 and GPP2) involved in ascorbate Smirnoff biosynthesis pathway were also cloned from tomato. Their complete open reading frames are 822 bp and 807 bp respectively, encoding 273 and 268 amino acids respectively. The nucleotide sequences of the two GPP share 81% identity, whereas the two putative amino acid sequences have 80% similarity.2. The expression levels of AMR1, GGP, GPP1 and GPP2 were analysed in roots, stems, leaves, flowers and different development stages of fruits of tomato. Results showed that AMR1, GGP, GPP1 and GPP2 were constitutively expressed in various tissues, whereas different expression levels were found in different tissues. The results showed that the highest expression level of AMR1 is in roots, the highest expression level of GGP and GPPl is in leaves, whereas the highest expression level of GPP2 expression is in flowers. Ascorbic acid in roots, stems, leaves, flowers and different development stages of fruits of tomato cultivar Ailsa Craig (AC) were measured. The results showed that the total content of ascorbic acid were quite different in different tissues, with the lowest AsA in the roots and the highest AsA in the leaves. With the growth and development of fruits, the total content of ascorbic acid showed an increasing trend. Analysis of the correlation between gene expression and AsA accmualtion, AMR1 correlation was -0.64583, GGP correlation was 0.505171, GPP1 correlation was 0.731284, GPP2 correlation was 0.690334.3. The sense exrepssion and RNAi vector of AMR] and GGP were constructed and transformed into genome of tomato variety AC using the Agrobacterium-mediated transformation. The sense and co-suppression vector of GPP were also transformed to Ailsa Craig. The putative transgenic plants were identified by PCR, and results showed that the T-DNA was integrated into the tomato genome.4. QPCR were used to analyse the expression levels of the target genes in some transgenic tomato plants, and results showed that the transcript levels of target genes were elevated significantly in over-expressing transgenic lines and suppressed significantly in RNAi transgenic lines.5. Microplate reader was used to measure the total ascorbic acid content of leaves of transgenic lines with AMR1 over-expression or suppression. The results showed that the total ascorbate content in leaves decreased significantly in AMR1 over-expression lines, whereas the total asorbate content in leaves increased slightly in AMR1 suppressed transgenic lines. HPLC were used to measure the total ascorbic acid content of breaker fruits of AMR1 over-expressed and suppressed lines. The results indicated that the total ascorbate content in breaker fruits showed no significant differences between transgenic and wild-type plants.6. Microplate reader was used to measure the total ascorbic acid content of leaves of GGP over-expressed and suppressed lines. HPLC were used to measure the total ascorbic acid content of breaker fruits of GGP over-expressed and suppressed lines. The results indicated that the total ascorbate content in leaves and fruits showed no significant differences between GGP over-expression transgenic lines and wild-type plants, whereas the total asorbate content in leaves and fruits decreased significantly in GGP-suppressed transgenic lines.7. Microplate reader was used to measure the total ascorbic acid content of leaves of GPP 1 and GPP2 over-expressed transgenic lines. The results showed that the total ascorbate content in leaves increased slightly in over-expression lines. HPLC was used to measure the total ascorbic acid content of breaker fruits of GPPs co-suppressed transgenic lines. The results showed that the total ascorbate content in breaker fruits had no significant differences between transgenic and wild-type plants.