Regulating JsTPS Promoters by JsMYB108 and JsMYB305 in Jasminum sambac
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摘要:
目的 探明茉莉香气合成相关转录因子JsMYB108和JsMYB305对3个萜烯合成酶基因JsTPS启动子的调控机制。 方法 以茉莉叶片DNA为模板，采用染色体步移法克隆3个JsTPS基因的启动子片段，预测启动子序列顺式作用元件，并在此基础上将3个JsTPS基因启动子片段分别构建到报告载体pGWB433，单独转化烟草叶片或与pK7FWG2.0-JsMYB108、pK7FWG2.0-JsMYB305效应载体共同转化烟草叶片以检测JsMYB108和JsMYB305对3个TPS基因启动子的激活作用；同时，通过酵母单杂交实验进一步检验JsMYB108和JsMYB305与3个TPS基因启动子的结合作用。 结果 克隆了3个JsTPS基因启动子片段，长度分别为 1 357、1 849、1 005 bp。这些序列中均含MYB识别位点，不同启动子序列包含不同顺式作用元件，如光反应元件、损伤响应元件、脱落酸诱导顺式作用元件等；叶片GUS染色和GUS活性检测结果显示3个JsTPS基因启动子片段均具有启动活性且JsMYB108和JsMYB305能不同程度地激活3个TPS基因启动子活性。JsMYB108使JsTPS1、JsTPS3、JsTPS4启动子活性增强至对照的1.96倍、6.47倍和4.15倍；JsMYB305使JsTPS1、JsTPS3、JsTPS4启动子活性增强至对照的1.57倍、15.18倍和3.12倍；酵母单杂交结果显示JsMYB108和JsMYB305能与JsTPS1、JsTPS3和JsTPS4启动子结合。 结论 JsMYB108和JsMYB305可以不同程度激活JsTPS基因启动子活性，推测这2个转录因子在茉莉花萜烯香气合成和代谢过程具有一定作用。 Abstract:Objective Regulation functions of JsMYB108 and JsMYB305 on the promoters of three terpene synthase genes (TPSs) relating to the aroma synthesis of jasmine were analyzed. Method The promoter fragments of JsTPSs were cloned by genome walking with the DNA of jasmine leaves as template to determine the sequences of the cis-acting elements in them. The fragments were constructed separately in the reporter vector pGWB433. Then, tobacco leaves were transformed with the reporter vector alone or with pK7FWG2.0-JsMYB108 and pK7FWG2.0-JsMYB305 effect vectors to detect the activation of JsMYB108 and JsMYB305 on the promoters. Binding of JsMYB108 and JsMYB305 to the promoters was verified by the yeast one-hybrid assay. Result The cloned promoter fragments of the three JsTPSs were 1 357 bp, 1 849 bp, and 1 005 bp with MYB recognition sites. The elements relating to light response, damage response, and abscisic acid induced cis-acting were predicted in different promoter sequences. The GUS staining and activity detection in tobacco leaves confirmed varying degrees of activity of the fragments by introducing JsMYB108 and JsMYB305. Comparing to control, JsMYB108 expanded the activities 1.96-fold on the promoter of JsTPS1, 6.47-fold on that of JsTPS3, and 4.15-fold on that of JsTPS4, while JsMYB305 did 1.57-fold, 15.18-fold, and 3.12-fold, respectively. The yeast one-hybrid assay further verified the bindings of JsMYB108 and JsMYB305 to JsTPS1, JsTPS3, and JsTPS4. Conclusion JsMYB108 and JsMYB305 could activate the promoters of JsTPS1, JsTPS3, andJsTPS4. These two transcription factors might play a key role in the synthesis and metabolism of aromatic terpenes in jasmine flowers. -
Key words:
- Jasminum sambac /
- transcription factors /
- TPS /
- promoter /
- regulation
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图 1 JsMYB108和JsMYB305转录因子与3个JsTPS基因启动子共同转化烟草后的GUS组织化学染色结果
A：阴性对照；B：阳性对照；C：单独注射proJsTPS1::GUS；D：proJsTPS1::GUS+35S::JsMYB108共同注射；E：proJsTPS1::GUS+35S::JsMYB305共同注射；F：单独注射proJsTPS3::GUS；G：proJsTPS3::GUS+35S::JsMYB108共同注射；H：proJsTPS3::GUS+35S::JsMYB305共同注射；I：单独注射proJsTPS4::GUS；J：proJsTPS4::GUS+35S::JsMYB108共同注射；K：proJsTPS4::GUS+35S::JsMYB305共同注射。图中标尺为1 mm。
Figure 1. GUS histochemical staining on tobacco transformed with two JsMYB transcription factors and three JsTPS promoters
A: negative control; B: positive control; C: single injection of proJsTPS1::GUS; D: co-injection of proJsTPS1::GUS+35S::JsMYB108; E: co-injection of proJsTPS1::GUS+35S::JsMYB305; F: single injection of proJsTPS3::GUS; G: co-injection of proJsTPS3::GUS+35S::JsMYB108; H: co-injection of proJsTPS3 ::GUS+35S::JsMYB305; I: single injection of proJsTPS4::GUS; J: co-injection of proJsTPS4::GUS+35S::JsMYB108; K: co-injection of proJsTPS4::GUS+35S::JsMYB305. Scale bar = 1 mm.
图 2 瞬时转化烟草叶片中GUS活性检测
图中“*”表示处理与对照相比有显著差异（P＜0.05）。
Figure 2. GUS quantitative detection of gene expression
* indicates significant differences with control at P＜0.05.
图 3 Y1H[proJsTPSs-pAbAi] 诱饵菌株最低AbA抑制浓度筛选
Figure 3. Screening for lowest AbA inhibitory concentration of Y1H [proJsTPSs-pAbAi] bait strain
图 4 酵母单杂验证JsMYB108和JsMYB305与JsTPS启动子的互作
Figure 4. Yeast one-hybrid verification on interaction between JsMYBs and JsTPS promoters
表 1 引物序列
Table 1. Sequences of primers applied
引物名称
Primer name引物序列（5′- 3′）
Primer sequences (5′- 3′)用途
UsageJsTPS1DNA-F ATGGGCAGCCAAGTTTATGCATC 扩增 DNA 序列
DNA amplificationJsTPS1DNA-R GAGATAGGGTTGTGGAACGCTA JsTPS1-GW-GSP1 ATTGTGCGAAGGTCCTCGTTTTGCTTGTAA 扩增启动子序列
Promoter sequence amplificationJsTPS1-GW-GSP2 GTTACGGAGCGTCGAGCAACTTCAATGC JsTPS1-PRO-F TTTCATAGGGAAATTGGTGC 验证启动子序列 JsTPS1-PRO-R TTATATCTAATCAAATGCTTCTAGC Confirming promoter sequence JsTPS3-PRO-F ATGACAAGGTATTAATGCTTGGCG JsTPS3-PRO-R TCTTGATATGTATTACTAGTAATTTAACAAC JsTPS4-PRO-F GTGATATTGATTCCAAATCTGGTC JsTPS4-PRO-R GATAACAAGTAAAACGGAAAATCAGG pAbAi-proJsTPS1-F AATTCGAGCTCGGTACCCGGGCACATTCAAGCTTGAAATACCGG 扩增酵母单杂启动子片段 pAbAi-proJsTPS1-R ATACAGAGCACATGCCTCGAGCAAGCGCATGTAAACGCTAGAC Amplification of promoter sequence
for yeast one hybridpAbAi-proJsTPS3-F AATTCGAGCTCGGTACCCGGGTGCATTTGGATGTAACATTAAAATCA pAbAi-proJsTPS3-R ATACAGAGCACAATGCCTCGAGGTCACATCATGGTTGTGACGAACA pAbAi-proJsTPS4-F AATTCGAGCTCGGTACCCGGGGCCGCCATAAGAAAATTCGG pAbAi-proJsTPS4-R ATACAGAGCACATGCCTCGAGGCTCGTGTACTACATATCGTTTATTAATTAC pAbAi-F GTTCCTTATATGTAGCTTTCGACA 验证上游片段-pAbAi pAbAi-R CCATCTCGAAAAAGGGTTTGCC Confirming cloning sequence in –pABAi vector pGADT7-JsMYB108-F gtaccagattacgctcatatgATGGAGCATCATGTTAAAGGAGATG 构建pGADT7-JsMYBs
Construction of pGADT7-JsMYBs vectorspGADT7-JsMYB108-R cagctcgagctcgatggatccCTACTGTTGTAGGAAATTCCACATGTC pGADT7-JsMYB305-F gtaccagattacgctcatatgATGGACAAGAAAATATGCAATAGCTC pGADT7-JsMYB305-R cagctcgagctcgatggatccTTAATCCCCATTAAGTAACTGGATGG pGADT7-F TAATACGACTCACTATAGGG 验证pGADT7-JsMYBs pGADT7-R AGATGGTGCACGATGCACAG Confirming the construction of
pGADT7-JsMYBs vectors
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表 2 茉莉JsTPS1、JsTPS3和JsTPS4启动子序列中顺式作用元件预测
Table 2. Predicted cis-acting elements in JsTPS1, JsTPS3, and JsTPS4 promoters
顺式元件
Cis-element序列
Sequence位置 Position JsTPS1 JsTPS3 JsTPS4 G-Box
参与光响应的顺式作用调节CACGTT + 1157 − 1555 −846, −725 AuxRR-core
参与生长素的顺式作用调节元件GGTCCAT +345 ACE
参与光响应的顺式作用元件GACACGTATG −616 +503 TGACG-motif
茉莉酮酸甲酯反应性中涉及的顺式作用调节元件TGACG −261 −561,+ 1676 ,+863,
−1660 ,+1254 MBSI
MYB结合位点参与类黄酮生物合成基因的调控元件aaaAaaC(G/C)GTTA − 1318 I-box
光响应元件的一部分cCATATCCAAT −173 TGA-element
生长素反应元件AACGAC −153 − 1148 Box 4
涉及光响应性的保守 DNA 模板的一部分ATTAAT +136,− 1146 ,+449,
+413,+488+11,− 1246 ,−1083 ,+1711 ,
+869,+885,−1038 ,−1707 +188,+163 GC-motif
参与缺氧特异性诱导的调控CCCCCG + 1797 , −145−854,+172,−63 ARE
厌氧诱导必不可少的顺式作用元件AAACCA +374, −768 CCAAT-box
MYBHv1结合位点CAACGG −402 +894 CGTCA-motif
茉莉酮酸甲酯反应性中涉及的顺式作用调节元件CGTCA +261 − 1676 , +561, −863
+1660 , −1254 GATA-motif
光响应元件的一部分GATAGGA −582 −566 TATA-box
转录启动子周围−30个核心启动子元件TATA +40,− 1555 ,
+82,+1556 +726,+847,−434 ABRE
脱落酸反应性涉及的顺式作用元件ACGTG/TACGTGTC +213, − 1157 +619,
+618,+1130 A-box
顺式作用调节元件CCGTCC −46，−984 TATC-box
参与赤霉素反应的顺式作用元件TATCCCA −618 GT1-motif
光响应元件GGTTAA +58,−149,+100 TCA-element
水杨酸反应性涉及的顺式作用元件CCATCTTTTT − 1148 Sp1
光响应元件GGGCGG −350 GARE-motif
赤霉素反应元件TCTGTTG +1813 TGA-box
生长素反应元件的一部分TGACGTAA + 1254 3-AF1binding site
光响应元件TAAGAGAGGAA + 1092 AT1-motif
光响应模板的一部分AATTATTTTTTATT −252 TCT-motif
光响应元件的一部分TCTTAC −926 AE-box
光响应模块的一部分AGAAACTT −150 AT-rich element
富含AT的DNA结合蛋白的结合位点ATAGAAATCAA +282 O2-site
玉米醇溶蛋白代谢调控中涉及的顺式调控元件GATGA(C/T)(A/G)TG(A/G) −657 ABRE3a
参与脱落酸反应的顺式作用元件TACGTG +212,+618 +39 G-box
参与光响应的顺式作用调节TACGTG +212,+618,+ 1129 ,+1157 +39,−81,− 1555 MYB
MYB结构域CAACCA + 1295 −99,−1814,+ 1665 −447 MYB recognition site
MYB识别位点CCGTTG +402 −894 MYC
干旱诱导相关的顺式作用元件CATTTG −228,− 1170 ,+663−207, −482,
+457,+1334 +986 WUN-motif
损伤响应元件AAATTTCTT + 1253 as−1
水杨酸诱导型调控元件TGACG −261 +1822,−561,
+1676 ,+1254 Myb-binding site
MYB结合位点CAACAG +863,− 1660 W box
诱导子、受伤及病原体应答，结合 WRKY类转录因子TTGACC −1814 +858,+130 WRE3
顺式作用元件CCACCT +379 WUN-motif
损伤响应元件CAATTACAT −192
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