Identification And Functional Analysis Of Stress-related Genes In Brassica Napus

Brassica napus is an important oilseed plant, which contains oil content, accounting for 35%-45% of its dry weight. Rapeseed oil is also good edible oil that is rich in fatty acids. Rapeseed oil is also an important industrial raw material and is the optimal sourceoffeed for developing biodiesel. Therefore, developing rape and increasing oil content can not only make an important contribution to energy security strategy but also ensure food security and improve farmer income in china. However, Brassica napus often encounters abiotic stresses, such as high salinity, drought, cold and nutrient deficiency (such as P and k limitation), which result in plant growth retardation and reduce agricultural productivity. Transference of stress tolerance genes into plant genome by gene engineering strategies to produce new varieties of stress resistance will bring new chance for reform of conventional breeding.Compared with model species such as Arabidopsis and rice, little is known about identification of abiotic stress-related genes and the molecular mechanism in response to abiotic stresses of Brassica napus. Hence, screening and identification of stress-related genes and study the molecular mechanism of stress tolerance could provide us the basis of effective genetic engineering strategies for improving stress tolerance of B. napus. In our study, over 500 genes related to high-salinity and drought stresses were identified from B. napus cDNA libraries using macroarray analysis, and some interested genes were chosen for further expression and functional study. The main results are as follows:1. Screening and identification of high-salinity/drought-related genes from B. napus cDNA libraries by macroarray analysisIn total,313 high-salinity-responsive genes (including 172 up-regulated and 141 down-regulated genes), and 477 drought-responsive genes (including 288 up-regulated and 189 down-regulated genes) were identified by cDNA microarray.154 genes were induced by both high salinity and drought, whereas 104 genes were suppressed by both high salinity and drought, suggesting the existence of a substantial common regulatory system or a cross-talk between high-salinity and drought stresses.2. Expression patterns of the 13 stress-induced genes13 candidate genes were chosen for further study. These proteins of the 13 genes encoding belong to 6 protein families, including protein kinase (MAPKKK and CIPK), metallothionein proteins, auxin-responsive or-repressed proteins, lipid transfer protein, stress-related proteins and metabolic enzyme. The expression profiles of the 13 genes under salinity or drought stress were analyzed by real-time quantitative RT-PCR.6 genes were selected for analyzing expressions under ABA treatment. The results are as follows:among 13 genes, the expressions of 7 genes were induced by salt stress; 12 of the selected 13 genes were induced to up-express by treatment with 200 mM mannitol, of which 6 genes were induced by high-salinity stress simultaneously; further study revealed that 5 manitol-inducible genes were also induced by ABA, suggesting that a significant cross-talk may occur between drought and high-salinity stress signaling and between drought and ABA responses. Expression patterns of the 13 genes in different tissues/organs of B. napus suggest that these genes may play important roles in tissues/organs development.3. Expression analysis and functional identification of cold regulated gene BnCOR25Among 288 clones identified to be putative drought-responsive genes, one gene (cDNA) encodes putative novel cold-regulated protein with a calculated molecular mass of 25 kDa, and consequently designated as BnCOR25 (accession number in GenBank:HM 187577). The results of Real time RT-PCR displayed that BnCOR25 was mainly expressed in hypocotyls, cotyledons, stems, and flowers, but its mRNA was found at low levels in roots and leaves. Northern blotting indicated that BnCOR25 expression is significantly induced in roots by osmotic and drought stresses. The data also revealed that BnCOR25 gene expression may be mediated by ABA-dependent pathway. BnCOR25 protein may be chiefly localized on cell plasma membrane and cytoplasm. Overexpression of BnCOR25 in yeast enhanced the cell survival probability under cold stress, and overexpression of BnCOR25 in Arabidopsis enhances plant tolerance to cold stress. These results suggested that the BnCOR25 gene may play an important role in conferring freezing/cold tolerance in plants.4. Expression analysis of BnCIPK6 gene and histochemical assay of BnCIPK6 promoter activitiesAmong 154 genes identified to be putative induced by both high salinity and drought stress, one gene (cDNA) with deduced amino acid sequence homology to CBL-interacting protein kinases 6, and consequently designated as BnCIPK6, was chosen for further analysis. Transcripts of BnCIPK6 gene were induced by high salinity, osmotic stress, low phosphate, ABA and BL treatment. The results of the RT-PCR showed that BnCIPK6 was mainly expressed in flowers, at moderate level in cotyledons, but its mRNA was found at low levels in other tissues. We isolated the 850bp promoter of BnCIPK6, Histochemical assay revealed that the GUS staining was detected at relatively high level in hypocotyls and cotyledons in 12-day-old seedlings, but weak or no GUS signals in other tissues. The results of stress and hormone treatments revealed that the BnCIPK6 promoter is salt-/osmotic-/ABA-/BL-inducible.5. Screening and identification of BnCIPK6-interacting proteinsYeast two-hybrid analysis showed that BnCIPK6 interacts strongly and specifically with AtCBL1, AtCBL2, AtCBL3 and AtCBL9. Yeast two-hybrid analysis was performed using the BnCIPK6 as bait to screen the two-hybrid library of Brassica napus cDNAs constructed on the prey vector.27 unique proteins were identified as positive clones. The BnCIPK6-interacting proteins were related to various aspects of plant development, metabolism and signal transduction. The identified proteins included two calcineurin B-like proteins that are homologous to AtCBL1 and AtCBL3, respectively. The protein homologous to AtCBL1, designated as BnCBL1, was chosen for further study. BiFC assays also confirmed BnCIPK6 protein interaction with BnCBLl protein in vivo.6. Subcellular localization of BnCIPK6 and BnCBL1 protein and phosphorylation activities of BnCIPK6 and BnCIPK6T182D proteins in vitroThe results of subcellular localization showed that BnCIPK6:eGFP fusion protein can be observed at the plasma membrane, in the nucleus and cytosol, whereas BnCBL1 is plasma membrane-localized protein. The results of phosphorylation activities in vitro revealed that the BnCIPK6(M) mutant protein exhibited higher autophosphorylation activity, compared with BnCIPK6 protein, suggesting that the 182nd threonine residue in BnCIPK6 could be a critical target site for activation.7. Overexpression of the BnCIPK6, BnCIPK6(M) and BnCBL1 in Arabidopsis enhance plant tolerance to salt and low phosphate stress To study the function of BnCIPK6 and BnCBL1, the coding region of the BnCIPK6, BnCIPK6(M) and BnCBL1 were fused to the CaMV 35S promoter and used to transform Arabidopsis plants. Several transgenic plants were obtained by screening and identification. Transgenic lines with higher gene expression were selected for analyzing their phenotypes under various treatment conditions. The results showed that BnCIPK6, BnCIPK6(M) and BnCBL1 transgenic plants all enhance plant tolerance to salt and low phosphate stress. These results suggest that BnCBL1-BnCIPK6 may functionally interact with each other and that are involved in salt and low Pi (LP) stresses response in plant.8. BnCIPK6 and BnCIPK6(M) transgenic Arabidopsis were sensitive to abscisic acid (ABA)We tested the transgenic lines under ABA treatment. The results showed that overexpression of BnCIPK6 and BnCIPK6(M) increased plant sensitivity to ABA, especially the latter that show hypersensitivity to ABA. The expression of ABA-responsive Marker genes, such asABF3, ABF4 and RD29A, in the BnCIPK6(M) transgenic plants was remarkably higher than that in wild type under ABA treatment although there was no significant difference in expression levels of those genes between the transgenic plants and wild type in absence of ABA. We did not observe the obvious phenotype changes of BnCBL1 transgenic plants in response to ABA, compared to wild type plants. The results showed that BnCIPK6, but not BnCBL1, is involved in ABA response, suggesting that there may be another BnCIPK6 interaction CBL(s) involved in ABA signaling.9. BnCIPK6(M) overexpression transgenic plants show altered BR response phenotypes and increase plant sensitivity to BRZThe results revealed that overexpression of the constitutively active BnCIPK6 in Arabidopsis alters plants response to BR and increases plant sensitivity to BRZ. However, we did not observe the obvious phenotype changes of BnCBLl and BnCIPK6 transgenic plants in response to BR. The results revealed that the constitutively active BnCIPK6, which has higher kinase activity, may be involved in BR signaling pathway, suggesting that there may be another BnCIPK6 interaction CBL(s) involved inBR signaling.10. Arabidopsis cipk6 loss of function mutant shows less ABA sensitivity than the WT, but is more sensitive to salt and low phosphate stressesThe studies on Arabidopsis cipk6 loss of function mutant revealed that silencing of AtCIPK6 confers ABA insensitive growth phenotypes and enhances sensitivity to low phosphate stress, suggesting the AtCIPK6 gene is involved in response to salt, low phosphate and ABA. However, we did not observe obvious phenotypic changes under exogenous BL treatment.