Cloning And Expression Of The Genes Related To Anthocyanin Biosynthesis In Turnip

There are three major plant coloring matters:betaine,carotenoid and anthocyanin,among which anthocyanin is a water-soluble pigment which presents in plants and decides the flower color of approximately 88%of the angiosperms.Mainly accumulating in vacuoles,Procyanidin has a variety of physiological functions,such as attracting insect pollinators,resisting hurt from low temperature and ultraviolet,controlling pests and diseases,as well as preventing oxidation and aging. As a branch of flavonoids biosynthetic pathway,anthocyanin biosynthetic pathway has become one of the life science research focuses currently in respect of its regulatory mechanism.Many important enzymes are involved in anthocyanin biosynthetic pathway,such as phenylalanine ammonia-lyase (PAL),which catalyzes the first reaction in phenylalanine metabolic pathway,chalcone-flavanone isomerase(CHI),which catalyzes intramolecular reaction of chalcone,and flavanone -3-hydroxylase(F3H),which can catalyzes synthesis of dihydrokaempferol from 4,5,7-trihydroxyflavanone.By using light dependent'Tsuda' turnip and light independent 'Yurugi Akamaru' turnip as additives,this study adopts RT-PCR methods to clone main enzyme genes of anthocyanin synthesis-PAL and CHI,as well as full-length cDNA sequence of F3H.Primer and Probe design is produced according to gene analogs between PAL,CHI and F3H of the two varieties.Unexposed roots 0 h,6 h, 12 h,18 h,24 h of Tsuda' turnip and 'Yurugi Akamaru' turnip are treated with UV-A.Expression quantity alteration of PAL,CHI and F3H genes are detected by using the method of northern-blotting. The main findings are as follows.(1) Open Reading Frame(ORF) of PAL entire gene of Tsuda' turnip and 'Yurugi Akamaru' turnip are both 2169 bp,with 722 amino acids encoded,and accession No.respectively as EU402423.1 and EU402424.1 in the GenBank.The homology between the PAL nucleotide sequence of the two turnips and PAL-1 of Brassica napus reaches 99%.The homology between the PAL amino acid sequence and PAL of Brassica napus is up to 99%.PAL domains exist in the amino acid sequences from No.67 to No.532.Nine positions are different between the nucleotide sequence of 'Tsuda' turnip and of 'Yurugi Akamaru' turnip.Deduced position differences of amino acid sequence are three.Open Reading Frame(ORF) of CHI entire gene of 'Tsuda' turnip and 'Yurugi Akamaru' turnip are both 756 bp,with 251 amino acids encoded,and accession No.respectively as EU402416.1 and EU402417.1 in the GenBank.The homology between the nucleotide sequence of the two turnips and CHI of Raphanus sativus reaches 99%.The homology between the CHI amino acid sequence and CHI of Raphanus sativus is up to 99%.CHI domains exist in the amino acid sequences from No.11 to No.222.Three positions are different between the nucleotide sequence of CHI gene of 'Tsuda' turnip and of 'Yurugi Akamaru' turnip.Deduced position differences of amino acid sequence are three.Nucleotide sequence of the F3H entire gene of 'Tsuda'yama Turnip and of 'Yurugi Akamaru' turnip is identical.ORF is1077 bp,with 358 amino acids encoded,and accession No. as EU402422.1 in the GenBank.The homology between the nucleotide sequence and F3H-1 of Brassica napus reaches 98%.Deduced homology between the amino acid sequence and F3H-1 of Brassica napus is up to 99%.F3H domains exist in the amino acid sequences from No.35 to No.342.(2) The results of northern-blotting indicated that,PAL,CHI and F3H gene expression of 'Tsuda' turnip and 'Yurugi Akamaru' turnip can be induced by UV-A;and that there is correlation between PAL,CHI and F3H gene expression and UV-A processing time.