Cloning And Function Analysis Of Chalcone Isomerase Gene From Purple-fleshed Sweet Potato [Ipomoea Batatas (L.) Lam]

Anthocyanins are the most important pigments of plants. They are harmless and of easy incorporation in aqueous media and can be used as natural water-soluble colorants. Moreover, anthocyanins have antioxidant activity, and play a vital role in the prevention of cancer and diabetes. Therefore, due to the enormous potential of natural anthocyanins as healthy pigments, there are numerous research reports about the biosynthesis pathway of anthocyanins. Purple-fleshed sweet potato is a special cultivar in sweet potato [Ipomoea batatas (L.) Lam.] due to the valuable hereditary feature and economic value. As a fine raw material, which contains a high content of anthocyanins, it attracted wide attentions in the past few years. As so far, there was no data to confirm which the biosynthetic pathway of anthocyanidins in purple-fleshed sweet potato was the same as that in the model plants. Chalcone isomerase (CHI, EC 5.5.1.6) is one of the key enzymes in the anthocyanin biosynthesis pathway catalyzing the stereospecific isomerization of chalcones into their corresponding (2S)-flavanones. But it had no report on what the role CHI plays in the biosynthesis of anthocyanins in purple-fleshed sweet potato. Therefore, in this paper, the gene structural traits, expression pattern and functions of chi gene from purple- fleshed sweet potato were investigated. The main results in the paper were presented as follows:1. Full-length cDNA sequences of chi gene were isolated from purple-fleshed sweet potato (Ipomoea batatas (L.) Lam) cultivar’Yamakawamurasaki’and was designated Ibchi. The full length of Ibchi genes was 844 bp, containing a 732 bp open reading frame (ORF) which encoded a 243 amino acid polypeptide. The estimated molecular weight of IbCHI was 25.5 kDa and the estimated isoelectric point was 5.09. IbCHI had the conserved amino acids residues, which were essential for activity of CHI, including T50, Y108, N115 and S192. SOPMA prediction results indicated that the secondary structure of IbCHI was composed of 42.24% alpha helices,17.05% extended strands, 8.14% Beta turns and 32.57% random coils. IbCHI shared high homology and common source in evolution with the CHI from other plants.2. The ORF of Ibchi gene did not contain intron in different sweet potato cultivars or strains ’A5’,’Yamakawamurasaki’and’Yubeibai’. Ibchi gene had more than three copies in the genome of sweet potato cultivars or strains’Yamakawamurasaki’,’A5’ and’Yubeibai’. Ibchi gene showed significant restriction fragment length polymorphism (RFLP) among different sweet potato cultivars or strains, implying that the flanking sequences of Ibchi gene in different sweet potato cultivars or strains presented significant genetic variation.3. The prokaryotic expression vector of Ibchi gene was constructed and the conditions for soluble recombinant protein IbCHI induction were studied in the paper. The optimum condition was 18℃,0.2 mM IPTG,180 rpm,10 h. Soluble recombinant protein IbCHI was purified using his-tag affinity chromatography, and was used for preparation of anti-IbCHI serum.4. The eukaryotic vectors of Ibchi gene for subcellar location research were constructed and transformed into onion epidermal cell.The fluorescence signal of fusional protein IbCHI-EGFP was detected, which showed that IbCHI was located in plasma membrane, cytoplasm and nucleus in onion epidermal cell.5. The eukaryotic overexpression vector of Ibchi gene was constructed and transformed into Arabidopsis thaliana chi gene deficiency mutant (tt5), which made the seeds of transformants recover the former phenotype of wide type. It showed that Ibchi gene encoded a functional CHI enzyme and had an important function in anthocyanin synthesis pathway.6. Expression patterns of Ibchi gene in leaves, stems, tuber skin and tuber flesh in different strains and cultivars of sweet potato (’A5’,’Yamakawamurasaki’,’A6’,’Zijing’ and’Yubeibai’) were investigated. The expression of Ibchi gene in different tissues was different significantly:the tissues with high anthocyanin contents got high expression level of Ibchi gene, including the purple tuber flesh and skin of’A5’ and’Yamakawamurasaki’, tuber flesh of ’A6’, purple leaves and stems of’Yamakawamurasaki’and’Zijing’; while the tissues with low anthocyanin contents got low expression level of Ibchi gene, including green leaves and stems of’A5’and’A6’, and white tuber flesh of’Yubeibai’and’Zijing’. Meanwhile the relative content of IbCHI protein in storage roots of different sweet potato cultivars or strains (’A5’,’Yamakawamurasaki’and’Yubeibai’) were also different significantly. Generally, the variation trends of expression of Ibchi gene and the relative content of IbCHI protein were consistent with that of anthocyanin content in sweet potato. According to the results above we concluded that IbCHI was a key enzyme in the biosynthetic pathway of anthocyanin in sweet potato.7. In different sweet potato cultivars or strains (’A5’,’Yamakawamurasaki’and’Yubeibai’), there were fifteen genetic variation sites in the nucleotide sequence of the three Ibchi genes, and in which there were five basic sites are different between white-fleshed cultivar’Yubeibai’and purple-fleshed cultivar (strain)’Yamakawamurasaki’and’A5’8. In different sweet potato cultivars or strains (’A5’,’Yamakawamurasaki’and’Yubeibai’), there were seven genetic variation sites in the amino acid sequence of the three IbCHI protein, and in which there were four amino acid residues are different between white-fleshed cultivar’Yubeibai’ and purple-fleshed cultivar (strain)’Yamakawamurasaki’and’A5’. We suggested that the catalytic activity of IbCHI from’Yubeibai’would be affected by the mutations in amino acid residues.9. Sucrose can induce the biosynthesis of anthocyanin in the cotyledon and epicotyl of transgenic type (tt5+Ibchi) and wild type (Ler) of Arabidopsis thaliana. Moreover, data showed that anthocyanin synthesis of transgenic type was more sensitive to sugrose than that of wild type.10. Transgenic type (tt5+Ibchi) and wild type of Arabidopsis thaliana showed more tolerant in high ligh stress than tt5 mutant. The chlorophy II fluorescence parameters (Fv/Fm and Yield) in the leaves of tt5 mutant declined more rapidly than that of transgenic type and wild type under high ligh stress.11. Transgenic type (tt5+Ibchi) and wild type of Araobidopsis thaliana showed more tolerant than tt5 mutant in the condition of drought stress. The leaves of tt5 were withered more seriously than that of transgenic type and wild type. Meanwhile, the chlorophy II fluorescence parameters (Fv/Fm and Yield) in leaves of tt5 mutant also declined more rapidly than that of transgenic type and wild type under drought stress. After rehydration treatment, the Fv/Fm and Yield value in leaves of transgenic type and wild type can return to the level of control, while tt5 mutant can not.