Analysis Of CRF Family Genes, Cloning And Preliminary Function Research Of BnaCRF8s In Brassica Napus

Phosphorus(P) is an essential macro-element for plant growth and development. Oilseed rape(Brassica napus) is an important oil-crop in China, which has a high demand for P and is hypersensitive to P deficiency. In this study, we analyzed the basic characteristics of the cytokinin response factor(CRF) gene family in Brassica napus. Four Brassica napus CRF family genes named BnaCRF8 s which were significantly induced under low P conditions were identified from our previous RNA-seq database. Subsequently, the cDNA and genomic full sequences of BnaCRF8 s were isolated from “Eyou Changjia” which is a P-efficient cultivar. The function of BnaCRF8 s was revealed based on bioinformatic analysis, gene temporal-spatial expression analysis, subcellular localization and transgenic analysis. The molecular mechanism of BnaCRF8 s involved in P homeostasis was preliminarily discussed in this research. Our main results are as follows.1. The basic characteristics of the CRF gene family in Brassica napusBased on the genomic database of Brassica napus published previously, we identified 36 homologous CRF family genes in B. napus of 12 AtCRF family genes in Arabidopsis. The results of bioinformatic analysis show that 36 genes are scattered on 16 chromosomes except A4, A10 and C9, the total length of the genes varies from 495 bp to 1117 bp, the length of the amino acids of the genes varies from 164 to 362, most of the genes have only one exon except four of them. Phylogenetic analysis shows that BnaCRFs family genes can be divided into three groups. Gene structure analysis indicates all the genes contain AP2/ERF and CRF conserved domains. Further protein analysis shows that all the proteins are stable, hydrophilic and partially disordered. They are not secreted and transport proteins, and are localized in nucleus functioning as a regulator. 2. Cloning and phylogenetic analysis of BnaCRF8 s in B. napusThe expression profile of the B. napus family genes were analyzed based on our previous RNA-seq data generated from B. napus seedlings under high and low P conditions. The results showed that four BnaCRF8s(BnaC2.CRF8, BnaA2.CRF8, BnaCn.CRF8 and BnaA7.CRF8) were the most highly induced genes under low P stress in B. napus. Thus, we cloned the full-length genomic and cDNA sequences of the genes from P-efficient cultivar “Eyou Changjia”. Bioinformatics analysis shows that there is only one exon for four genes, the sequence similarity among genes is up to 90.33%, the similarity of amino acid among genes is 89.64%. Phylogenetic analysis indicates that two genes(BraA2.CRF8 and BraA7.CRF8) from B. napus A genome and two genes(BnaC2.CRF8 and BnaCn.CRF8) from B. napus C genome are clustered together with the homologous genes from B. rapa A genome and B. oleracea C genome, respectively. 3. Preliminary function analysis of BnaCRF8 s in B. napusWe analyzed the expression levels of four BnaCRF8 s in shoot and roots of the Pefficient B.napus cultivar “Eyou Changjia” under high and low P conditions. The results showed that four BnaCRF8 s were strongly induced by low P condition in both shoot and roots. The real time quantitative PCR results showed that the expression levels of the genes increased gradually when suffered from a no P environment, and recovered to a very low level with P re-supply. The expression levels of the genes were investigated in bud, pod handle, pod shell and seeds of B. napus under low P and normal P levels. The results showed that the expression levels of the genes in pod handle were significantly higher under low P condition than that under normal P condition, but the opposite results were detected in pod shell and bud. No significant differences were observed between the expression levels of the genes under both P levels in seeds except BnaA2.CRF8 which had a higher expression level under normal P condition compared to low P condition. The subcellular localization result showed that BnaC2.CRF8 was localized in nucleus. Over expression of BnaCRF8 s in Arabidopsis led to reduced growth of shoot and root and decrease of biomass in transgenic lines, indicating BnaCRF8 s negatively regulated P-starvation response.