此外,该序列中还发现一些光响应元件如GA-box、GAG-motif、I-box等。
应用FootPrinter3.0软件对龙眼、拟南芥(NC_003076.8)、水稻(AF397034.1)、葡萄(EF222286)、芒果(GU338039.1)、山核桃(DQ989226)6个物种LFY同源基因启动子序列进行比较,找出保守区域,绘出系统发生树。结果显示6个物种LFY启动子区域共有的保守基序有4个(图3),保守区域数目不多,说明LFY同源基因间启动子序列差异较大。
表1应用PlantCARE预测LLFY启动子顺式作用元件
Table1Cis-actingregulatoryelementsanalysisofLLFYpromotersequencebyPlantCARE
位点名称
Site Name
|
位置
Position
|
信号序列
Signal sequence
|
位点功能
Function of site
|
CAAT-box
|
-802、-527、-447、-98
|
CCAAT
|
启动子区域顺式作用元件
Common cis-acting element in promoter and enhancer regions
|
-391、-190
|
gGCAAT
|
TATA-box
|
-689、-514、-211、-125
|
TATA
|
转录起始-30核心启动子元件
core promoter element around -30 of transcription start
|
circadian
|
-595
|
CAANNNNATC
|
参与生理周期调控的顺式作用元件
cis-acting regulatory element involved in circadian control
|
ARE
|
-182
|
AAACCA
|
厌氧诱导必要的顺式作用元件
cis-acting regulatory element essential for the anaerobic induction
|
MBS
|
-579、-346
|
CAACTG
|
干旱诱导的MYB结合位点MYB binding site involved in drought-inducibility
|
-169
|
CAGTTA
|
-238
|
CAACTG
|
MRE
|
-246
|
TTAGGTT
|
光响应MYB结合位点
MYB binding site involved in light responsiveness
|
ABRE
|
-476
|
GCCACGCACT
|
脱落酸响应顺式作用元件cis-acting element involved in the abscisic acid responsiveness
|
GATA-motif
|
-442
|
CCCTATC
|
部分光响应元件
part of a light responsive element
|
I -box
|
-775
|
AAGATAAGGCT
|
部分光响应元件
part of a light responsive element
|
Sp1
|
-378
|
GGGAG
|
光响应元件
light responsive element
|
MNF1
|
-376
|
AGGGCAC
|
light responsive element
光响应元件
|
GAG-motif
|
-112
|
CATCTCT
|
部分光响应元件
part of a light responsive element
|
GA-motif
|
-13
|
AAGGAAGA
|
部分光响应元件
part of a light responsive element
|
Skn-1_motif
|
-722
|
ATG AC
|
胚乳表达需要的顺式作用元件
cis-acting regulatory element required for endosperm expression
|
O2-site
|
-776
|
GATGATATGG
|
玉米醇蛋白代谢途径顺式作用元件
cis-acting regulatory element involved in zein metabolism regulation
|
-809 CATCATGCCAATGTAAATTAGCACTTGCAGATAGATGATAAGGCTAAAGCTGTAAACTTA
CAAT-box I -box
-749 TGTGTAAAGCGATTTCCATTTTTAGGAATGACTTGGCACTTGCAGACAGCCATAATTTTT
Skn-1_motif
-689 TATAAAACCTTGATATCAAAAATAAAAGGAAAAGTTTCATCCTTAGGGACAGAGGATGCA
TATA-box
-629 TTTAGCAAAGAAACTGCCCTAATAGCATCCACTTCAACGAAATCCACCTTCAACTGTAAG
circadian MBS
-569 GAATAGGCAAGCAACAAACCTTCTCTTAGAGCGACCGATTCACCAATTTTTGGGCTATAG
CAAT-box TATA-box
-509 TTACCTTTCCAAGGATTCGTTGTAGCAACTATGGCCACGCACTGATGATTATGGAAGCAA
ABRE
-449 CACCAATCCCTATCATGAGAGAGCTAGAGCAAATGGCTACATCTGAGTTTGAGTTTTAGG
CAAT-box GATA-motif
-389 CAATCATAAAGGGGAGGGCACCAACTAGGAACAGACGAAGCAGCAACTGATTTAGGCAAA
CAAT-box Sp1 MNF1 MBS
-329 ATAAACATGGCTACTTTAGCGTTTTGGAATTCTCGTTAGGAGAATGTAAGGTAAGTCACC
-269 AAGGCGAGACTCAGTTTGAGTGGTTAGGTTCCAACTGTCAAGTTCCAAACTAACAGAATA
MRE MBS
-209 TATGAACCACATTTTCCTTGGCAATAAAAACCATAAAAATCAGTTATTTGAGGGCAGTAT
TATA-box CAAT-box ARE MBS
-149 CGTGAACACAGAAACAGCAGCCTTTATAGCAAACCTACATCTCTCTGCTGCCCAATTCCA
TATA-box GAG-motif CAAT-box
-89 GAACATAAAAGATATTCACTGCTCACGAGTGCTAAGAGGGCAAAAACAAAACAAAAAATA
-29 AAAAATAAAGACGAAAAAGGAAGACAGACATG
GA-motif
|
|
图2龙眼LLFY基因启动子部分序列
启动子基本元件用方框表示;其它调控元件用阴影表示;起始密码子用粗体表示;引物用下划线表示
Fig.2PartialsequencesofLLFYpromoter
Potentialbasicelementsofpromoterareboxed.OtherTFBSareinshadow.Thestartcodonisshowninboldletters.Thereverseprimerisunderlined.
3讨论
启动子序列分析表明LLFY启动子区域含有丰富的可能是顺式作用元件的序列,LLFY表达调控受内在因素和多种外界条件的影响。如LLFY表达受生理周期的影响,可能是导致花芽分化具有周期性原因;启动子序列中含有多个光响应作用元件,说明LLFY的表达受光调控;生产中KCIO能够诱导龙眼成花,可能与LLFY启动子含有干旱诱导顺式作用元件有关,是由于施用KCIO产生了干旱胁迫。LLFY表达调控的复杂性也说明了LLFY可能具有多种功能。如LLFY启动子中还发现有厌氧诱导等顺式作用元、胚乳表达需要的顺式作用元件以及玉米醇蛋白代谢途径顺式作用元件等,这些元件是否与LLFY的其它功能有关有待进一步研究。龙眼LLFY启动子序列的克隆也为进一步采用酵母单杂交等方法获得与LLFY互作的调控蛋白创造条件。
对一些物种的研究表明在进化过程中,LFY同源基因的功能出现分化,不仅控制着花的发育,在被子植物的发育过程中还起着其它的调控功能。例如,单子叶植物水稻中LFY的同源基因RFL表达明显区别于双子叶植物,RFL基因在幼穗中表达,而在成熟的小花和叶子中没有表达。葡萄VFL在即将发育成花序的分生组织中、花序芽中都有表达,在花芽中的表达达到最高,在叶芽、叶片和卷须原基中也有微量表达,作者认为VFL除了在开花过程的重要功能外,还参与未分化的分生组织的维持。而龙眼LLFY参与花序的分化和维持,在“冲梢”过程LLFY表达量下降。葡萄等LFY同源基因启动子序列中都有MYB、ABRE和光响应等元件,龙眼中也发现了这些元件,但LLFY启动子中没有发现拟南芥中的CArG盒和GA的顺式作用元件,也没有找到葡萄中的茉莉酮酸酯诱导顺式作用元件、细胞分裂素调节基因ARR1结合位点等序列。将水稻、拟南芥与包括龙眼在内的4种果树LFY同源基因启动子进行保守序列分析,结果表明它们共同的保守序列只有4个,这为解释不同物种LFY同源基因存在表达和功能差异提供了证据。如果仅将4种果树的LFY同源基因启动子序列进行比较,保守序列达到24个(数据未列出),说明木本植物LFY同源基因启动子序列相似性更高。以上试验的分析结果也说明了龙眼LLFY基因作为花分生组织特性基因的保守性,但功能及表达调控上与其它物种存在差异。
图3FootPrinter软件对6个LFY同源基因启动子的分析结果
Fig.3AnalysisofsixLFYhomologgenes’promotersbyFootPrinter
设定元件长度为8,最多两个突变位点,子区域设定为100bp,不同颜色显示不同的转录元件。
Motifssizewas8withatmosttwomutations.Thesubregionsizewassetto100bp.Differentcoloursmeaneddifferentmotifs.
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