| Plant polyphenols is a general name of multi-hydroxy phenolic compounds. Polyphenols have high antioxidant capacity in vitro and many advantages, such as natural, efficient, low toxicity, etc. Accordingly, the biochemical and molecular biological study on the polyphenols attracted wide attention of scientists around the world, which will be the main direction of natural antioxidants development in the future. Potato contains a large amount of antioxidant active substances, including polyphenols, vitamin C and carotenoids. Especially, colored potato tubers are rich in antioxidant content, mainly because the red or purple color of tubers is attributed to the accumulation of anthocyanins. Therefore, study on anticancer effects of potato polyphenols in vitro and clarification of molecular basis of anthocyanins biosynthetic pathway in potatoes can provide theoretical and practical basis for further development of natural antioxidant from potato.In this thesis, different colored Solanum tuberosum were used as material, through phenolics determination, antioxidant activity determination, chlorogenic acid determination, anthocyanin determination and cancer cell proliferation assay, the anticancer activity and mechanism of potato polyphenols were analyzed. Meanwile, the anthocyanins biosynthetic gene CHI was isolated by homologous clone. Additionly, the genomic structure and expression of potato anthocyanins biosynthetic genes were analyzed, and then white potato varieties were transformed with anthocyanins synthesis gene3GT. The results are as follows:1. The determination of potato polyphenols compounds. Using two different methods to prepare polyphenols extract of four potato cultivars and one wild species. The total phenolic, chlorogenic acid, anthocyanin contents and antioxidant activity were determined, the results showed that:(1) S. pinnatisectum contained the highest total phenolics, chlorogenic acid content and antioxidant activity, followed by purple tubers ’Bora Valley’ and’Purple Majesty’, red tuber’Mountain Rose’and then white tuber ’Northstar’. The anthocyanin content of colored potatoes was higher than white potato tubers.(2) A positive correlation was found between total phenolics, chlorogenic acid content and antioxidant activity.(3) The results were consistent between the two extraction methods used.2. Analysis of inhibitory effect of potato polyphenols extract on the proliferation of human Caco-2colon-cancer cells and HepG2liver-cancer cells. The polyphenols extract of five varieties and three phytochemicals were investigated for their antiproliferative activity against cancer cells by morphological observation and XTT assay, the results indicated that:(1) The polyphenols extracts from all varieties tested had noticeable antiproliferative activity against Caco-2and HepG2cancer cells. S. pinnatisectum extract had the lowest proliferation and colored potato cultivars’Mountain Rose’,’Bora Valley’and’Purple Majesty’had higher anticancer effect than the white cultivar ’Northstar’. Moreover, the inhibition of colon-cancer and liver-cancer cell proliferation by potato extracts was attributed to the concentration of phenolic compounds and interrelated with the antioxidant activity significantly.(2) Chlorogenic acid and anthocyanins both can inhibite cancer cells growth. Chlorogenic acid had stronger inhibiting activity than pelargonidin chloride and malvidin chloride.3. Cloning and expression analysis of anthocyanins biosynthetic genes in potato species.(1) A gene named CHI (chalcone isomerase) was isolated from the wild species (S. pinnatisectum) by RT-PCR. The open reading frame of the gene was657bp long and encoded a putative protein of218amino acids with a molecular weight of24.46kDa and a theoretical pI of7.68. The multiple alignment of the amino acid sequence of CHI and that of other homologues showed that CHI had a high identity with homologous members from Solanum lycopersicum.(2) Analysis of CHI mRNA level by semi-quantity RT-PCR showed that it was expresed in roots, apical buds, flowers, stolons, swelling stolons and mature tubers, especially highly expressed in swelling stolons and roots, and lightly in the stolons and tubers. The level of CHI mRNA became weaker in the apical buds and flowers, and disapeared in stems and leaves.4. Analysis of the genomic structure and expression of anthocyanins biosynthetic genes.(1) Five gDNAs encoding anthocyanins biosynthetic genes CHS、CHI、F3H、DFR and3GT were isolated from red potato cultivar ’Chieftain’ and white potato ’Superior’ The sequence analysis indicated that the full length of the CHS DNA contained two exons and one intron, F3H DNA contained three exons and two introns, DFR DNA contained six exons and five introns, CHI and3GT DNAs did not contain introns.(2) The spatial expression analysis of CHS, CHI, F3H, DFR and3GT genes in red potato (’Chieftain’and’Desiree’) and white potato (’Superior’) indicated that no anthocyanins biosynthetic genes transcripts may cause no anthocyanins accumulated in the process of tuber formation and lead to the white flesh potato tubers consequently.5. Integrated3GT gene into the genome of white potato cultivar’Superior’by Agrobacterium-mediated transformation.18kanamycin-resistant plant lines were obtained, of which3plant lines were positive by PCR. |
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