| The molecular mechanisms of adaptive evolution is the most essential part of the adaptive evolution, and is the key to understand adaptive evolution of species. At the molecular level, adaptive evolution can be divided into gene sequence level and gene expression regulation level. Undergone positive selection genes and gene expression level variations are typically existed among natural populations over the geographic range of a species, which are essential for the local adaptation to the heterogeneous environments. With the recent advancement of next generation sequencing, it is now possible to combine gene discovery with characterization of transcript expression, in addition, transcriptome analysis has also been performed to study the evolutionary patterns of genes, which is helpful for the study of adaptive evolution. Numerous studies have demonstrated that drought affects the normal growth and development of plants, agroforestry production and the stability of ecological environment. Studies based on physiological, biochemical, and genetic levels show that drought tolerance mechanism and strategy are complex and vary depending on species-specific drought-coping mechanisms and strategies. In recent years, drought resistance mechanism on desert plants is a hot research topic, and it mainly focuses on some typical desert plants such as Reaumuria songarica, Ammopiptanthus mongolicus and Nitraria tangutorum, and but litter on low herbaceous plants with strong drought adaptability, especially on Braya humilis. B. humilis mainly distributes in the Qinghai-Tibet Plateau and adjacent arid regions, and the phenotypes of B. humilis are various in the two regions, which make B. humilis are promising candidates to discover adaptation mechanism for plateau and arid region species. In addition, B. humilis have strong drought adaptation, so they are also good material to study plant drought resistance mechanisms.In present study, to study the adaptive differentiation and evolutionary molecular mechanisms on the two B. humilis ecotypes, we compared phenotypic performance of B. humilis ecotypes under controlled condition and their chromosome number, besides, basing on transcriptome sequencing data, we analyzed differential expression gene between two B. humilis ecotypes, and study the evolutionary selection patterns(positive selection genes) in two B. humilis ecotypes. In addition, aimed to characterize the physiological and molecular traits and expression patterns of a osmosis-responsive gene in B. humilis of HZ responding to different levels of osmotic stress induced by osmotic stress. Several important physiological parameters were examined, including the levels of RWC, soluble protein, MDA, and antioxidant enzyme activity. A osmotic-responsive gene, Bh CIPK12, was identified, cloned, and characterized. Finally, a series of genes associated with antioxidant activity were characterized. Main conclusions are as follows:1. Phenotypic performance of B. humilis ecotypes under controlled condition:(1) HZ ecotype shows stronger seed vigor;(2) The growth and development of BHS are faster than HZ B. humilis, and HZ B. humilis can‘t bolt under controlled condition;(3) BHS is diploid(n=14), HZ is tetraploid(n=28).2. RNA-seq, de novo assembly and function annotation:(1) 22.4 Mb and 21.6 Mb clean reads were obtained respectively from HZ and BSH samples. A total of 40,762 and 46,485 unigenes with a mean length of 833 and 764 bp were generated for HZ and BHS ecotypes, respectively. A total of 72,833 non-redundant unigenes with a mean length of 812 bp were obtained from two ecotypes;(2) B. humilis showed very high homology with three Brassicaceae species: Arabidopsis thaliana, Arabidopsis lyrata, and Capsella rubella;(3) Most of unigenes had a significant hit in the public databases, the largest biological process terms were response to salt stress, response to cadmium ion and regulation of transcription.3. Differential unigene abundance between B. humilis ecotypes:(1) A total of 7,499 unigenes were differentially expressed in BHS ecotype compared with HZ ecotype. The largest biological process terms were response to environmental stress,(2) 33 TF families showed differential expression between two B. humilis ecotypes. The AP2/ERF, bHLH, MYB, and WRKY families were the four largest differential expression families;(3) 126, 119, and 77 DEUs were assigned into vegetative to reproductive phase transition of meristem, regulation of flower development, and flower development biological process terms.4. Positive selection genes:(1) 145 unigenes were identified as under positive selection. GO analysis showed that the largest terms were response to salt stress and response to cold;(2) 25 unigenes were shared between DEUs and positive selection genes. Two well-known stress and hormone related gene EIN2 and KEG, and a flowering-related gene ELF6, were also among the positively selected unigenes and DEUs.5. Physiological adaptations to osmotic stress in HZ B.humilis:(1) A tolerance threshold between 20 and 30% PEG-6000 was identified for HZ B. humilis. The water status and oxidative damage below this threshold were maintained at a relatively constant level during the 12 h of treatment. However, once the threshold was exceeded, the water status and oxidative damage were obviously affected after treatment for 4 h;(2)The soluble protein results suggest that B. humilis maintains a vigorous resistance to osmotic stress and that it may play a greater role in osmotic regulation at late stages of stress;(3) Moreover, SOD and CAT may be important at preventing oxidative damage in plants at early stages of stress, while POD may be more involved in some biological processes that resist osmotic stress at the late stage, especially in severely damaged plants.6. Cloning, characterization of a osmotic stress-responsive gene in HZ B. humilis:(1) A differential display cDNA fragment was amplified from B. humilis leaves treated with 30% PEG-6000 for 4h. The fragment identified was approximately 1600 bp in length. The coding sequence and ORF were 1060 bp in length and 490 amino acids, respectively. The predicted molecular mass was approximately 54.99 kDa, and the pI was 7.66;(2) We hypothesized that the expression levels of BhCIPK12 would first increase and then decrease with a reduction in water status and that the marked increase in expression would be stimulated by a sharp water loss in the leaves.7. Transcript variation in antioxidant related genes of osmotic-stressed HZ B. humilis plants:(1) Among 59 target genes, 35 genes were identified as up-regulated genes, including 12 antioxidants genes, 12 signal genes,11 photosynthesis genes. 23 genes were identified as down-regulated genes, including 4 antioxidants genes, 1 signal genes,18 photosynthesis genes. HEMC is common to up-regulated and down-regulated categories;(2) Most analyzed antioxidants genes effectively play a important role at early and middle stage of stress. Except for CIPK3, analyzed signal genes were responsible for signal transduction at early and middle stage of stress;(3) Most photosynthesis genes were repressed during the whole process of stress, some up-regulated photosynthesis genes, like GHLH, PHYB, all were up-regulated at early and middle stage of stress, and down-regulated at late stage. |
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