Screening And Studying On The Genes Related To Anthocyanin Biosynthesis In Turnip

A cDNA library was constructed from each of 'Tsuda' turnip, which is light-sensitive in pigmentation of root peel, and 'Yurugi Akamaru' turnip, which is light-insensitive. Using these two turnips grown in light or dark conditions, four subtraction libraries were constructed as follows; library 1 from red root-peel of 'Tsuda' treated in light subtracted and white peel treated in dark condition, library 2 from white root-peel of 'Tsuda' treated in the dark and red peel of 'Yurugi Akamaru' treated in light, library 3 from white root-peel of 'Tsuda' treated in the dark and red peel treated in light, and library 4 from red root-peel of 'Yurugi Akamaru' treated in light as well as in the dark and white peel of 'Tsuda' treated in the dark. From these libraries, unique gene fragments were selected and were fundamentally analyzed by bioinformatics methods. cDNA microarrays were manufactured with these fragments after PCR amplification. Total RNA was isolated from 'Tsuda' and 'Yurugi Akamaru' treated in different conditions and labeled with cy3 or cy5 during the synthesis of cDNA, and then the cDNAs were hybridized with the cDNA microarray to test the expression of the genes and gene colony related to anthocyanin biosynthesis. The structure genes and regulator genes could be screened according to the hybridization consequence. Anthocyanins in root peel were identified and quantified with a UV-visual spectrophotometer. 'Tsuda' and 'Yurugi Akamaru' turnips were treated in four light conditions, i.e., dark, sun light, constant light and UV-A light. Expression of PAL, F3H, DFR, ANS and MYB genes in root peel involved in the anthocyanins biosynthetic pathway was investigated by Northern blotting analysis respectively. The main results are as follows.1. From cDNA library, 618 unique gene fragments were selected as well as 1425 fragments from subtraction library. Structure genes such as PAL, CHS, F3H, DFR and ANS, and regulator genes fragments such as bHLH, WD40, MYB and bZIP were screened from subtraction libraries involved in anthocyanidin biosynthesis. PCR products of these fragments were purified and dropped on glass microslides to perform cDNA microarray analysis.2. From subtraction library, 1424 fragments were mapped into metabolism approaches of KEGG database. Some gene fragments such as PAL, CHS, F3H, DFR, ANS related to anthocyanidin biosynthesis were found in library 1 and library 4. They were thought involved in anthocyanidin biosynthesis in light-sensitive 'Tsuda' and light-insensitive 'Yurugi Akamaru'. Reverse subtractions, i.e., library 1 and library 3, were compared. In library 1, 54 metabolism approaches including flavonoid biosynthesis were found. In library 4, 48 metabolism approaches including flavonoid and alkaloid biosynthesis approach were found. CHS, bHLH and bZIP gene fragments were found in library 2.3. Microarray prepared from unique gene fragments of cDNA library was used to hybridize to samples, revealing that genes expressed in 'Tsuda' treated with light were moreabundant than those in the dark. At the same time, the genes expressed in 'Yurugi Akamaru' were more abundant than those in 'Tsuda' in dark condition. The functions of some unknown genes require further study.4. The hybridization results of microarray made from unique gene fragments of subtraction library showed that the expression of genes in turnips treated in sunlight and UV-A radiation condition were more than those in darkness. At the same time, expression of some genes, which were related to anthocyanidin biosynthesis, was more enhanced. These results showed that root-peel of'Tsuda' could be colored by irradiation of sunlight and UV-A.5. The accumulation of anthocyanins in peel of'Tsuda' turnip roots was increased as light-exposure time was longer; however, in light-insensitive 'Yurugi Akamaru' the accumulation of anthocyanins had no relationship with light-exposure time.6. The Northern blotting results showed that in 'Tsuda', the expression of F3H, DFR, PAL and ANS could be induced by irradiation of constant light and UV-A. Expression of these genes was correlated with light-exposure time under constant light and UV-A. The expression of MYB had no relationship with light-exposure time. UV-A was thought to be a key factor, which induced the expression of structure genes involved in anthocyanidin biosynthesis in 'Tsuda'. In 'Yurugi Akamaru', the expression of F3H and PAL could be induced by irradiation of constant light and UV-A. Under constant light and UV-A, these two genes and MYB expressed similarly to those in 'Tsuda'. The expression of DFR could not be found in 'Yurugi Akamaru'. Instead a special expression of ANS was found.