Cloning And Expression Analysis Of Zeaxanthin Epoxidase Gene (BnZEP) In Brassica Napus L.

Carotenoids have important functions in plants. Some carotenoids provide the main source of the pigment in colored flowers and fruits, some carotenoids are precursors of abscisic acid synthesis, and some participate in xanthophyll cycle, drain the excess light energy, play a role of anti-photo-oxidation in plants. ZEP gene play a predominant role in xanthophyll cycle, not only participate in carotenoid biosynthesis, but also participate in abscisic acid biosynthesis pathway. It is the key enzyme gene of the two pathways.We have experimented by using pure white flower HW243 and yellow flower Zhongyou 821 as materials, and cloned the cDNA sequence and DNA sequence of BnZEP based on molecular biology technologies. Also, we analyzed the relevant biological information of BnZEP and speculated ZEP protein. The main results were summarized as follows:(1) We designed degenerate primers according to the conserved sequence of the ZEP gene published in NCBI database, and isolated the 582 bp products (conserved sequence) of the white flower Brassica napus using PCR amplification. Comparied with ZEP genes of other plants, the nucleotide sequence of this fragment shares, respectively,89%,89% and 96% identity with that of Arabidopsis thaliana, Thellungiella halophila, and Chinese cabbage. The results showed that the amplified fragment is a part of the BnZEP. This fragment showed a high degree of homology with the Chinese cabbage.(2) Using rapid amplification of cDNA ends (RACE) technology, we got the full-length cDNA of BnZEP. The cDNA has the sequence length of 2228 bp (Genbank accession number GU361616). It contains an 2010 bp open reading frame (ORF) encoding 669 amino acid residues, a 57 bp 5'untranslated region and a 161 bp 3'untranslated region. Comparied with ZEP proteins, the deduced amino acid sequence of this protein shares 89%,97%,92%,68% and 65% identity with that of Arabidopsis thaliana, Chinese cabbage,Thellungiella halophila, lutea and corn respectively.(3) In addition, we analyzed the relevant information of ZEP protein by some bioinformatics softwares. The molecular weight of ZEP protein was 73841.01 D, and the theoretical isoelectic point was 6.5292. The protein included twenty kinds of basic amino acids. As the proportion of hydrophobic amino acids was much greater than polar amino acids, we suggested that ZEP protein was a hydrophobic polypetide. From the integral structure of ZEP protein, we found that random coils,α-helixes and extended chains were the maximum amount of ZEP protein structural elements, while theβ-turns were scattered throughout the whole protein.(4) According to the full length cDNA, we designed the specific primers and amplified the complete sequence of DNA using PCR.. The DNA has the sequence length of 3268 bp (Genbank accession number GU561839), contain 16 exons and 15 introns. The number of exons and introns are same with that of published Arabidopsis thaliana.(5) Using semi-quantitative PCR, we found that the BnZEP expression products wereas significantly different between white flower and yellow flower. The BnZEP expression products in white flower were more than that of in yellow flower. After analysis, we speculated that the BnZEP may has some relativity with the white flower character.