Cloning, Expression And Molecular Evolution Of BnBOR1s In Brassica Napus

Boron (B) is an essential mineral nutrient for the growth and development in higher plants. Oilseed rape (Brassica napus), which is the fifth-largest crops after rice, wheat, corn and soybean, is one of the major oil crops in China. Oilseed rape is sensitive to B deficiency. B deficiency causes the symptom of "flowering without seed setting", or even no seed harvest. In China, the Yangtze River region is the major oilseed rape production zone and accounts for more than 80% production of rapeseed. The main production area of oilseed rape is deficient in soil available B. Considerable genotypic variations in response to B deficiency exist among different cultivar. Boron transporters (BOR) are ubiquitous in higher plant kingdom and mediate B translocation, which maintain B homeostasis for higher plants and have an important role both in tolerance to B deficiency and toxicity. Based on the identification of B efficiency for some oilseed rape cultivars, there were some works about physiological mechanism of high B efficiency and B-related QTLs mapping had been done in our lab. In this study, B-related traits were investigated at seedling stage under various B treatments. The full-length cDNAs and genomic sequences of the BOR1 genes were cloned in B. napus. Through bioinformatic analysis and RT-PCR, the phylogenetic trees and expression profile were basically clarified. The relationship between BORs and B-related QTLs were discussed. The main results are as follows:1.The difference of physiology between high and low B efficiency cultivars under various B treatmentsUnder different B levels with solution culture, the dry weight, root length, boron accumulation and boron transfer coefficient of Qingyou10 (high boron efficiency cultivar) and Westar (low boron efficiency cultivar) were investigated. The results showed that significant difference, which QingyoulO had normal growth while Westar showed typical signs of B deficiency, between QingyoulO and Westar was found at the B level of 0.25μM. Boron accumulation coefficient indicated that the high B efficiency of Qingyou10 was partly attributed to the higher B transport from root to shoot than Westar.2. Cloning and structure features of BOR1 genes from B. napusUsing homology-based Rapid Amplification of cDNA Ends (RACE) technology, six boron transporter genes were cloned from B. napus, and thus named as follow: BnBOR1;1a, BnBOR1;1c, BnBOR1;2a, BnBOR1;2c, BnBOR1;3a and BnBOR1;3c. Southern blot and gene cloning showed that there are likely only six BnBOR1 genes in whole genome of B. napus. Altenative splicing in the form of intron retention and competed intron splicing exist in BnBOR1 genes family. Altenative poly(A) tailing sites were detected by 3'-RACE. There is a thirty-three bp inverted repeated fragment in upstream sequence of BnBOR1;1a that located the region between TATA box and 5'-UTR. The inverted repeated fragment is most likely a new Brassia-specific MITE-like structure. Four BnBORl, which are BnBOR1;1a, BnBOR1;1c, BnBOR1;2a and BnBOR1;2c, had lossed intron one and intron six.3. Molecular evolution of BnBOR1 gene familyAll of the BnBOR1 members formed a small branch with a 100% bootstrap support, which were further clustered with AtBOR1 to form a distinct Brassicaceae BOR1 branch. It was clear that BnBOR1 were the orthologous genes to AtBOR1 in B. napus. Brassica BOR1 genes were separated into three distinct groups:BnBOR1;1a and BnBOR1;1c in GroupⅠ, BnBOR1;2a and BnBOR1;2c in GroupⅡ, and BnBOR1;3a and BnBOR1;3c in GroupⅢ. The group I is closely related to the groupⅡ. The result indicated that the BnBOR1;1a, BnBOR1;2a and BnBOR1;3a were originated from the AA genome and the others from the CC genome. The reciprocal blast suggested that each group was comprised of two members, one of which is from B. rapa and the other from B. oleracea. The result implied that the six genes were formed companying with the triplication and allotetra-ploidization of the B. napus. The detail about triplication process of Brassiceae tribe is still unclear. In this study, it was suggested that the triplication of Brassiceae tribe comprised two distinct processes, one is heterologous diploidiztion and the other is autotetra-ploidization.There is codon bias in BnBOR1 gene family, while no significant differences exist among gene family. Ka/Ks of coding region sequences of BnBOR1 are from 0.0249 to 0.0978, more lower than one, suggesting that there is no positive selection and only intensive purifying selection in BnBOR1 genes family. Even though, six possible positive selection anino acid sites in BnBOR1 gene family were also detected by using the Bayes empirical bayes (BEB) calculation of M8 (beta&ω) model in PAML 4.4 software. Only the probability of L700 reaches significance level. LRTs (M0 vs. M2) with group III as foreground provided significant rejection of null MO model, after Bonferroni's correction (P<0.025, df=1), indicating that the groupⅢ(BnBOR1;3a and BnBOR1;3c) evolve under divergent selective pressure comparing with the others.4. Expression profile of BnBORl genes familyNorthern blot reveal that BnBORl are major expressing in roots at seedling stage. Different from AtBOR1 which was not affected in transcribe level by B supply, the expression of BnBOR1 were induced obviously in the case of B deficiency. Under B normal and deficiency treatments, the expression profile of BnBORl investigated in field experiment. Though with high sequence similarities, BnBOR1 members show expression differentiation of tissue specificity. The expression of Group III (BnBOR1;3a and BnBOR1;3c) genes could be detected in all tested tissues, while the other four showed similar tissue-specific expression profile, which expressed strongly in roots and stems, moderately in flowers, weakly in buds, and undetectable in leaves and siliques. The accumulation of BnBOR1;1c, BnBOR1;2a and BnBOR1;2c were up-regulated distinctly in the case of B deficiency. There were distinct differences between the QY10 and Westar. The BnBOR1;1c was expressing higher in QY10 than Westar in all tested tissues regardless of B supply. Moreover, QY10 have more accumulation of BnBOR1 than Westar in flower organ.5. Boron transporters were mapped onto TN genetic mapTwo molecular markers from BOR genes, BnBOR2-A and BnBOR7-A, were located on linkage group of TN population. BnBOR2-A was mapping in the A1 and BnBOR1-A was mapping in the A3. Moreover, BnBOR7-A was mapping in two previous reported B-related QTLs region (Jinpeng Yang,2007), which one was BCRLN (Ratio of B concentration in root under low B to under normal) and the other was BARSL (Ratio of root to shoot on B accumulation under low B). It is suggested that the BnBOR1-A was probably the candidate gene for these QTLs.