1 材料与方法
1.1 材料
1.2 引物
表1 狗尾草肌动蛋白基因及目的基因荧光定量PCR引物Tab. 1 RT-PCR primers of actin genes and target genes of S. viridis |
| 基因登录号 Gene accession number | 引物序列（5'-3'） Primer sequence (5'-3') | 扩增长度 Amplification length/bp |
|---|---|---|
| Sevir.3G070600 | AGGGATGGGACAAAAAGAT ACTGGCATAGAGGGAGAGC | 301 |
| Sevir.3G232400 | TTCAGCCTATTGTGTGCGA GTTGTTGACGATGCCGTGC | 221 |
| Sevir.5G398400 | CTCTCAACCCAAAGGCAAAC GCTCATAGTCAAGGGCAACG | 348 |
| Sevir.5G470300 | TGGTAGGAATGGGGCAGAAG TTGCAACATACATGGCAGGG | 276 |
| Sevir.7G305900 | GCGGGAAATTGTAAGGGACA GAATGAAGGCTGGAAAAGGA | 184 |
| Sevir.8G042300 | GCTGGTTTTGCCGGTGACGA AGCCTTGGGGTTGAGTGGGG | 288 |
| Sevir.9G114100 | CTTCCAGCCATCTTTCATT CCAGACTCGTCGTACTCAG | 315 |
| Sevir.9G194900 | GGATGGGACAGAAGGACGC ATGGCTGGAACACTGAAGG | 259 |
| Sevir.1G249200 | AGCCGCAGGACGAGGACA CGGGCACCAGCGAGAAGA | 252 |
| Sevir.J003400 | ATGTTGCTGGTTTGTATGGTA GAGGCTCGTGAGTATGCG | 198 |
1.3 方法
2 结果与分析
2.1 狗尾草幼苗在干旱和盐胁迫处理下的生长情况
图1 狗尾草A10品系种子在含不同浓度梯度甘露醇（A）和氯化钠（B）的高渗培养基上第7天的萌发情况a1~e1 的甘露醇浓度梯度分别为0、100、200、300、400 mmol/L;a2~e2 的氯化钠浓度梯度分别为0、40、80、120、160 mmol/L。 Fig. 1 Germination of Setaria A10 lines on the 7th day on different concentrations of mannitol (A) and NaCl (B) hypertonic medium =Mannitol concentration gradients of a1-e1 were 0, 100, 200, 300 and 400 mmol/L, respectively. NaCl concentration gradients of a2-e2 were 0, 40, 80, 120 and 160 mmol/L, respectively. |
2.2 肌动蛋白基因actin的PCR产物电泳检测与产物特异性、通用性验证
图2 狗尾草8个肌动蛋白基因实时荧光定量PCR扩增片段电泳分析Fig. 2 RT-PCR amplification fragment electrophoresis analysis of 8 actin genes in S. viridis M:DL2000 Marker 1: Sevir.3G070600; 2: Sevir.3G232400; 3: Sevir.5G398400; 4: Sevir.5G470300; 5: Sevir.7G305900; 6: Sevir.8G042300; 7: Sevir.9G114100; 8: Sevir.9G194900; 9: Negative control. |
图5 狗尾草响应不同干旱及盐胁迫时肌动蛋白基因实时荧光定量PCR扩增片段电泳分析和转录组数据验证M：DNA分子标准DL2000;泳道1~9依次为：未作任何处理、100 mmol/L甘露醇处理、200 mmol/L甘露醇处理、300 mmol/L甘露醇处理、400 mmol/L甘露醇处理、40 mmol/L氯化钠处理、80 mmol/L 氯化钠处理、120 mmol/L 氯化钠处理、160 mmol/L 氯化钠处理。图B中标有相同小写字母者表示组间差异不显著（P>0.05）。 Fig. 5 RT-PCR amplification fragment electrophoresis analysis and transcriptome data validation of actin gene in response to different drought and salt stress of S. viridis M: DL2000 marker; The corresponding cDNA template for 1-9 was followed by untreated Setaria viridis, 100 mmol/L mannitol-treated, 200 mmol/L mannitol-treated, 300 mmol/L mannitol-treated, 400 mmol/L mannitol-treated, 40 mmol/L sodium chloride-treated, 80 mmol/L sodium chloride-treated, 120 mmol/L sodium chloride-treated and 160 mmol/L sodium chloride-treated Setaria viridis. The data in the figure B with the same lowercase letters indicate no significant difference between groups (P>0.05). |
2.3 狗尾草响应不同干旱及盐胁迫时RNA- seq筛选基因结果验证
图7 狗尾草响应不同干旱及盐胁迫时RNA-Seq筛选基因实时荧光定量PCR验证和转录组数据验证1~9对应的cDNA模板依次为未作任何处理、100 mmol/L甘露醇处理、200 mmol/L甘露醇处理、300 mmol/L甘露醇处理、400 mmol/L 甘露醇处理、40 mmol/L氯化钠处理、80 mmol/L氯化钠处理、120 mmol/L氯化钠处理、160 mmol/L氯化钠处理的狗尾草;A和B分别代表基因Sevir.1G249200和Sevir.J003400在9种不同处理条件下的实时荧光定量PCR验证;C和D分别代表基因Sevir.1G249200和Sevir.J003400在9种不同处理条件下的转录组数据验证。 Fig. 7 RT-PCR verification and transcriptome data validation of RNA-Seq genes in response to different drought and salt stress of S. viridis The corresponding cDNA template for 1-9 was followed by untreated Setaria viridis, 100 mmol/L mannitol-treated, 200 mmol/L mannitol-treated, 300 mmol/L mannitol-treated, 400 mmol/L mannitol-treated, 40 mmol/L sodium chloride-treated, 80 mmol/L sodium chloride-treated, 120 mmol/L sodium chloride-treated, 160 mmol/L sodium chloride-treated Setaria viridis; A and B represent the real-time PCR of Sevir.1G249200 and Sevir.J003400 under 9 different treatment conditions; C and D represent the transcriptome data validation of Sevir.1G249200 and Sevir.J003400, respectively, in 9 different treatment conditions. |