Cloning And Quantitative Expression Analyses Of The Flowering Regulation Transcription Factor CONSTANS Homologous Gene In Brassica Napus L.

Brassica, one of the important oil crops, is also a traditional oil crops in CHINA. Flowering is an important process of a shift from vegetative growth to reproductive growth, and it has a certain relationship between the rapeseed growth period, pollination and the best period of development of rape seed and rapeseed yield increase. Based on previous studies on flowering pathways in Arabidopsis, we chose a key transcriptional regulation factor gene CONSTANS (a key transcriptional regulation factor gene in the photoperiod pathway) homologous gene sequence(published in the NCBI) and cloned CO homologous gene from different Brassica napus L. by RT-PCR method with GAPDH(glyceraldehyde3-phosphate dehydrogenase) as the reference gene, the expression levels of CO gene, were analyzed in different tissues, time pionts, ripening between the different Brassica napus L. by qRT-PCR(quantiative Reverse transcript PCR). We concluded that the following results:(1) The cloning of CO homologous gene in Brassica napus L.:CO genes were cloned from the early line, and late line respectively. They have1101bp CDS sequences, designed as A-CO-1, B-CO-1, respectively. The result of sequencing indicated that they have a high homo logy with the known CO gene. A-CO-1have two base site difference with the known sequence(the Genebank number AF016010.1), the B-CO-1have one different base site, and then these nucleotide differences ultimately result in the changes in the amino acid sequence of the predicted protein. The protein amino acid sequence analysis was showed that the A-CO-1and B-CO-1amino acid sequence of the predicted protein contains two B-box domains and a CCT domain. So, we speculate that the A-CO-1and B-CO-1CO share the similar function with their homologous gene CO, but need further functional validation.(2) Comparision analysis of CO gene between different maturity materials: Nucleotide and amino acid sequencing analysis of A-CO-1and B-CO-1, cloned from early line and late line, respectively, was carried out. The result showed that there have three differences in nucleotide bases and one difference in amino acid site, whether these change of the amino acid site can affect the gene function,we need further studies.(3) Expression of CO gene in different tissues: The expression of CO gene was existed in all tissue tested, there were high expression levels in bolting period, so we inferred that accumulation of CO gene related to the flowering in Brassica napus L.. In early line D626-6, in the root and stem, the high relative expression appeared in AM of bolting period I, in the leaf and bud, appeared in AM of bolting period II, then in the flowering and silique, appeared in AM of full-blossom period. In late line D125-5, in the CO gene of the root, the high relative expression appeared in AM of squaring period; in the stem, appeared at NOON of squaring period; in the leaf, appeared in PM of initial flowering period, in the bud, appeared in PM of bolting period I, in the flower and siniquae, appeared in PM of initial flowering period and AM of full-blossom period, respectively. The highest relative expression levels of CO gene in different tissues showed that the early line have higher expression levels than the late line.(4) Expression of CO gene in different growth period: There was highest CO gene expression at leaf in each growth period. In squaring period, the highest expression was appeared in leaf in different materials; In bolting period I, for the late line D125-5, the highest expression was appears in leaf, for the early line D626-6, the highest expression was appeared in bud; In bolting period Ⅱ and initial flowering period, the highest expression was appeared in leaf, after were bud and stem; In full-blossom period, for the early line D626-6, the highest expression was appeared at leaf, for the late line D125-5, the highest expression was appeared at bud; In flowering end period, the highest expression was appeared in leaf. The highest relative expression of CO gene, in different growth period of both early line and late line, was appeared in AM or PM.(5) Expression of CO gene in different materials:At AM of bolting period II (before the flowering) and initial flowering period, the early line D626-6had significant highest CO gene expression level than the late line in leaf; At NOON, the early line D626-6had higher expression level than the late line D125-5; But, at PM, the early line D626-6had significant lowest expression level than the late line D125-5.