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Seed Germination and Seedling Establishment Assays After surface sterilization of the seeds, stratification was conducted in the dark at 4°C for 3 d. Next, ;100 seeds of each genotype were sown on MS plates lacking or supplemented with 0.5 mM ABA, 150 mM NaCl, or 400 mM mannitol. In the analyses of iDDA1 lines, 1 mMABAwas used and b-estradiol was added to medium at 10 mM final concentration as stated. To score seed germination, radicle emergence was analyzed at 72 and 96 h after sowing. Seedling establishment was scored after 5 d as the percentage of seeds that developed green expanded cotyledons and the first pair of true leaves. Root Growth Assays Seedlings were grown on vertically oriented MS plates for 4 to 5 d. Afterward, 20 plants were transferred to new MS plates lacking or supplemented with 10 mM ABA. The plates were scanned on a flatbed scanner after 10 d to produce image files suitable for quantitative analysis of root growth using ImageJ version 1.37 software Phylogenetic Analysis DDA1 homologs were searched in different databases, Plaza (https:// bioinformatics.psb.ugent.be/plaza/), Phytozome (https://www.phytozome.net), Gramene (https://www.gramene.org), TAIR (https://www.arabidopsis.org), and GenBank (https://www.ncbi.nlm.nih.gov), using web-hosted BLAST applications (https://blast.ncbi.nlm.nih.gov/Blast.cgi). All truncated DDA1 sequences were excluded in subsequent analyses. Sequences were aligned using ClustalW2 (https://www.ebi.ac.uk/Tools/msa/clustalw2/) with the Gonnet protein comparison matrix and default parameters (gap opening penalty of 10 and gap extension penalty of 0.1 for initial pairwise alignment; gap opening penalty of 10 and gap extension penalty of 0.2 for the multiple alignment; with residue-specific and hydrophilic residue penalties, a gap separation distance of 4 and a 30% delay divergent cutoff) and then manually inspected to check for possible misalignments (Supplemental Data Set 1). The best model fitting the alignment was selected using ProtTest (Abascal et al., 2005), which corresponded to a Jones-Taylor- Thornton model for amino acid substitution with a discrete gamma distribution (four categories), a proportion of invariant, and an initial BIONJ tree. This model was used to conduct a maximum likelihood analysis with bootstrapping using 1000 replicates using the MEGA 5 (https://www. megasoftware.net/) software. The gaps/missing treatment parameter was set to complete deletion. The more distant homologs, from Selaginella moellendorffii and Picea glauca, were used as an outgroup to root the resulting phylogenetic tree. |
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玉砌的强: 金币+15, 翻译EPI+1, ★★★很有帮助 2014-12-09 21:54:10
玉砌的强: 金币+15, 翻译EPI+1, ★★★很有帮助 2014-12-09 21:54:10
| 种子萌发和幼苗建立测定种子表面消毒后，分层在黑暗中进行在4℃下3天。接着，100种子每种基因型的播种在MS平板缺乏或补充有0.5mM的ABA，150 mM氯化钠，或400毫甘露醇。在iDDA1行分析，使用与1 mMABAwas B-雌二醇加入到培养基中，在10mM的最终浓度如所述。得分种子发芽，胚根出现在播种后72和96小时进行分析。苗成立了进球后5天作为种子的百分比绿色发展子叶扩大和第一对真叶。根生长测定幼苗生长在垂直取向的MS平板进行4至5天。随后，20株植物被转移至新的MS平板缺乏或补充用10mM的ABA。将平板上扫描平板扫描仪10 d后产生图像文件适用于根的定量分析使用ImageJ1.37版本软件的增长系统发育分析DDA1同源进行检索在不同的数据库，广场上（http：//bioinformatics.psb.ugent.be/plaza/），Phytozome（https://www.phytozome.net）Gramene（https://www.gramene.org），TAIR（https://www.arabidopsis.org），和GenBank中的（https://www.ncbi.nlm.nih.gov），使用Web托管应用BLAST（https://blast.ncbi.nlm.nih.gov/Blast.cgi）。所有截断DDA1序列被排除在随后的分析。序列使用对齐ClustalW2（https://www.ebi.ac.uk/Tools/msa/clustalw2/）与贡内特蛋白质比较矩阵和默认参数（空位开放罚0.1初始配对比对10和缺口延伸罚分;差距所述多个开口的0.210和缺口延伸罚点球排列;与残基的具体和亲水残基的处罚，间隙4分离距离和30％的延迟发散截止），然后手动检查，以检查可能的偏差（补充数据集1）。使用被选为最佳模型拟合的对齐ProtTest（阿瓦斯卡尔等人，2005），其对应于一个琼斯Taylor-氨基酸替代桑顿模型离散伽玛分布（4种），的不变的比例，和一个初始BIONJ树。这种模型被用来进行最大似然分析引导使用1000次重复使用的MEGA5（HTTP：// WWW。megasoftware.net/）软件。差距/缺少治疗参数为设置完成删除。更遥远的同源物，从卷柏moellendorffii和云杉青冈，被用作外群根的导致进化树。 |
2楼2014-12-09 12:58:21