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title: 蒺藜苜蓿花发育基因TCP 和WFL1 的功能分析
Other Title: Functional Analysis of Floral Symmetry Development Genes TCP and WFL1 in Medicago truncatula
author: 阳天泉
Advisor: 陈江华
Issued Date: 2016-11
Major: 植物学
Abstract: 花对称性(Floral Symmetry)是被子植物花结构的典型特性之一,根据其花
冠对称性可分为辐射对称(actinomorphy),两侧对称(zygomorphy)以及不对
称(asymmetry)三种形式。已有研究表明,CYC 同源基因在不同物种都调控两
侧花型发育。在豆科植物百脉根(Lotus japonicus)和豌豆(Pisum sativum)中,
CYC 类基因通过复制产生3 个拷贝,它们共同决定豆科植物两侧对称花型背腹
极性的发育。
在蒺藜苜蓿(Medicago truncatula)Tnt1 插入突变体库中,我们筛选到MtCYC1
和MtCYC2 基因突变体。其中MtCYC2 突变体(lob standard 1,ls1)为单位点隐
性突变,表现为背部花瓣上部出现对称凹陷。MtCYC1 突变体(normal petals,
npe)花发育与野生型无明显差异。通过遗传杂交获得的纯合双突变体npe/ls1,
背部花瓣内部对称属性消失,其形状和大小类似于内部不对称的侧部花瓣,导致
整个花器官两侧对称形态丢失。基因表达分析显示,MtCYC1 和MtCYC2 具有相
似的表达模式,都在发育的背部花瓣具有最高水平的表达。这些结果表明
MtCYC1 和MtCYC2 是花瓣背部属性建立的关键因子,对背部花瓣属性建立有部
分功能冗余。MtCYC3 在npe/ls1 双突变体中表达较野生型上调,说明MtCYC3
在背部花瓣的表达可能受MtCYC2 和MtCYC1 抑制。为了调查MtCYC1 和MtCYC2
如何维持其在背部花瓣表达,以及它们发挥作用的机制,我们做了进一步研究。
首先在MtCYC2 基因的启动子区域预测到TCP 蛋白的结合位点GGGCCCT,酵
母单杂交结果显示:MtCYC1 和MtCYC2 都能与该靶序列结合。为了进一步证
实MtCYC1 和MtCYC2 调控MtCYC2 基因,我们设计了一个effector-reporter 瞬
时表达系统。将MtCYC2 基因的启动子与荧光素酶基因融合作为reporter,将
MtCYC1 和MtCYC2 分别与CaMV35S 启动子融合作为effector。瞬时共表达实验
显示,MtCYC1 和MtCYC2 都负调控MtCYC2 的表达。酵母双杂交和双分子荧
光互补实验显示,MtCYC1 和MtCYC2 存在相互作用,并且它们自身能够形成
同源二聚体。这些结果表明:MtCYC1 和MtCYC2 以形成同源或者异源二聚体
形式发挥作用。
通过正向遗传学筛选突变体库发现一个新的突变体:5 枚花瓣形态和大小类
似旗瓣(dorsalized petals,dsp1),柱头和雄蕊不能形成野生型弯曲的形态,雄蕊直立散生,不能正常包围柱头。遗传分析表明该突变体是由单位点显性基因控
制。dsp1 突变体表型为所有花瓣背部化,我们猜测DSP1 基因与NPE 和LS1 可
能有调控关系。通过遗传杂交获得dsp1/ls1 双突变体,表型与dsp1 单突变体相
似,说明DSP1 可能是位于LS1 的上游调控因子。qRT-PCR 检测MtCYC 基因在
野生型和dsp1 突变体背景中的表达差异,结果发现:在dsp1 突变体花芽中,
MtCYC1 和MtCYC2 表达量都显着上调。MtCYC1 在dsp1/ls1 双突变体中表达上
调。原位杂交也显示:MtCYC1 和MtCYC2 只在野生型背部花瓣表达;而在dsp1
突变体所有的花瓣都有表达。结果说明:DSP1 在花发育早期抑制MtCYC1 和
MtCYC2 的表达;dsp1/ls1 双突变体中可能是MtCYC1 发挥着建立背部花瓣属性
的功能。
植物的表皮蜡质覆盖于植物各器官和组织表面,是植物自我防护的一道屏
障,在植物适应干旱环境及各种非生物和生物逆境中发挥重要的作用。蜡质是特
长脂肪酸链的衍生物,C16 或者C18 脂肪酸在内质网上延伸成特长链脂肪酸。在
模式植物拟南芥中,长链脂肪酸的合成途径以及蜡质合成已有大量研究,而豆科
模式植物蒺藜苜蓿研究较少。本研究在Tnt1 插入突变体库中正向筛选到一个新
的单位点隐性突变体wfl1(winkled flower and leaf 1),花瓣不能正常展开并包裹住雄蕊,只外露出不可育的雌蕊,叶片融合。根据候选基因与突变体的连锁分
析,以及反向筛选该候选基因的突变体,确认突变表型,成功定位wfl1 目的基
因。WFL1 基因编码一类β-酮脂酰-CoA 合酶(KCS),催化长链脂肪酸延长的起
始步骤。该论文对其功能进行了初步研究:扫描电镜显示野生型和wfl1 突变体
叶片表皮蜡质差异很明显,突变体表皮蜡质晶体密度降低,蜡质立体形态变得更
短更薄。TB 测试检测叶片表皮渗透性,结果显示wfl1 突变体叶片比野生型更容
易被染色,说明突变体叶片表皮通透性增加。杂合子自交后正常植物与突变体比
例符合3:1,说明wfl1 是单位点隐性基因控制。以拟南芥21 个KCS 蛋白序列
为探针,在蒺藜苜蓿基因组数据库中得到28 个同源性较高的序列。系统进化树
显示,蒺藜苜蓿的28 个KCS 蛋白在KCS 家族的4 亚类(FAE1-like,FDH-like,
CER6,KCS1-like)都有分布,但是分布的数目变化较大。WFL1 与拟南芥FDH
基因(AT2G26250)关系最近,并且wfl1 突变体与fdh 突变表型相似。结果表明,尽管KCS 基因在FDH-like 亚类分化较大,但是基因功能保守。qRT-PCR 检测
WFL1 的表达模式,结果显示WFL1 在植物营养期和生殖期顶端、花瓣、柱头和
雄蕊中有很高的表达,其次是茎、叶和萼片。进一步用原位杂交实验调查WFL1
不同组织和不同发育时期的表达,结果显示在叶原基发育过程中的外层细胞表达
较强;花原基早期发育阶段各轮器官,如萼片,花瓣,柱头,雄蕊都有表达,在
花药发育成熟后期表达下调,而在花瓣,柱头及胚珠发育过程中持续高表达。通
过气相色谱-质谱联用(GC-MS)分析叶片脂肪酸成分和含量,结果显示wfl1 突
变体叶片脂肪酸含量显着低于野生型;脂肪酸成分发生剧烈变化,超长链脂肪酸
比例显着增加,亚麻酸比例显着减少,不饱和度降低。结果说明WFL1 基因功能
缺失引起蒺藜苜蓿超长链脂肪酸代谢紊乱。
关键词:花发育,背腹极性,TCP 基因,KCS 基因,蒺藜苜蓿
English Abstract: 

Floral symmetry is a characteristic feature of floral structure and diversity among
angiosperm. There are three main types of floral symmetry including actinomorphy,
zygomorphy and asymmetry. Among these, zygomorphy is considered the more
specialized form. Advances in molecular evolution of zygomorphy have shown the
key regulatory role of CYC-like gene, TCP gene family members, in determining the
development of floral zygomorphy. Studies found that CYC genes from Lotus
japonicus and Pisum sativum are essential for the establishment of dorsoventral (DV)
development in legume plants.
In this study, we isolated and identified MtCYC1 and MtCYC2 genes mutants with
Tnt1 insertion in M. truncatula. The MtCYC2 mutant plants exhibited the deficiency in
dorsal petals development (Lob Standard 1, ls1). The genetic analysis showed the
defect phenotype of ls1 was controlled by one recessive allele. In contrast, the mutant
of MtCYC1 gene did not give rise to a detectable phenotype in flower, similar to wild
plants (Normal Petals, npe). Intriguingly, the double mutant npe/ls1 results in strong
phenotypic changes such as more strong developmental deficiency in dorsal petals
and loss of internal asymmetry that was similar to internal asymmetrical lateral petals
in floral shape and size, ultimately resulting in the loss of whole floral symmetry.
Gene expression analysis showed that MtCYC1 and MtCYC2 have a similar
expression pattern, highly transcripted in dorsal petals. These results suggested that
MtCYC1 and MtCYC2 play a critical role in the control of dorsal petals development
in M. truncatula, particularly MtCYC2, and have partially functional redundancy.
Besides, we also isolated an additional CYC gene, MtCYC3, which might play a role
in lateral petals development. The MtCYC3 gene showed the increased expression
level in the dorsal petals of double mutant npe/ls1 compared with the wild type. This
implied that MtCYC3 may be repressed by MtCYC2 and MtCYC1 in the dorsal petals.
Subsequently, we further dissected the regulatory mechanism underlying the
molecular basis of MtCYC1 and MtCYC2 in dorsal petals in M. truncatula. We
initially investigated the known TCP gene family binding sites in the promoter region

of MtCYC2 and found the presence of GGGCCCT sequence for TCP protein binding,
implying that the MtCYC2 gene may be regulated by itself or MtCYC1 protein. Yeast
one-hybrid were carried out and the result showed that either MtCYC1 or MtCYC2
can recognize the GGGCCCT sequence from MtCYC2 promoter region. In order to
confirm this fact in vivo, we constructed a effector-reporter transient expression assay
in tobacco. The promoter sequence of MtCYC2 gene fused with luciferase gene were
considered as reporter, and coding sequences of MtCYC1 or MtCYC2 with CaMV35S
promoter were designed as effector. Co-injection of the reporter and effector in
tobacco mature leaves found that MtCYC1 and MtCYC2 protein may be negative
regulators. In addition, as previously reported in other plants, our finding showed that
MtCYC1 and MtCYC2 can form homodimer or heterodimer by protein interaction
that was confirmed by yeast two-hybrid and bimolecular fluorescence
complementation (BiFC) experiment. Besides, the MtCYC1 and MtCYC2 protein
were found to locate in cell nucleus. These results suggested MtCYC1 and MtCYC2
protein exert their function in cell nucleus by homo- or hetero-dimer protein complex.
Additionally, we identified a novel mutant in M. truncatula mutant library, in which
all five petals of the mutant were changed into dorsal petals, called dsp1 (Dorsalized
Petal 1). Compared with wild type, this mutant displayed some distinct features such
as stigma and stamens erection, scattered anthers unable to enclose the stigma. The
genetic analysis showed that the phenotype of dsp1 was controlled by one dominant
allele. Combined with the phenotype characterization of npe and ls1, we proposed a
hypothesis that DSP1 might be a key negative regulator for dorsal petals development
via repressing the MtCYC1 and MtCYC2. To test it, we crossed the dsp1 and ls1, and
obtained the double mutant dsp1/ls1. The dsp1/ls1 mutant had a quite similar floral
phenotype to dsp1, strongly implying that DSP1 may be a upstream regulator of
MtCYC2. The expression levels of MtCYC genes were performed in different mutants
and wild type by quantitative RT-PCR. The results showed that the expression levels
of both MtCYC1 and MtCYC2 in flower bud were significantly up-regulated in dsp1
mutant, compared with that in wild plant. Consistent with in situ hybird results,
MtCYC1 and MtCYC2 just expressed in dorsal petal of wild type, whereas they
ABSTRACT
VI
ectopic expressed in all five petals in dsp1. The expression of MtCYC3 gene in dsp1
mutant reduced to the half of that in wild plant. In dsp1/ls1, MtCYC1 was
up-regulated compared with wild plant, while MtCYC3 was similar to its expression
in dsp1. These observations suggested that DSP1 also play essential role in dorsal
petals determination by repressing the MtCYC1 and MtCYC2 in early flower
development except for dorsal petals.
The surface wax covers the various tissues and organs of terrestrial plant primary
aerial, which serves as an important defense barrier and plays important roles in a
variety of abiotic and biotic stresses. Plant cuticular waxes are complex mixture and
primarily comprised of long-chain fatty acids that were derived from 16- or 18-
carbon fatty acids in endoplasmic reticulum. The key genes involved into biosynthesis
pathway of long-chain fatty acids and wax formation were well-characterized in
Arabidopsis, but the key factors and molecular mechanism in M. truncatula remain
unknown. In our work, we screened the M. truncatula Tnt1-insertion mutant library
and identified a novel mutant with one recessive allele, called wfl1 (winkled flower
and leaf 1). The mutant had a strongly phenotypic defect, such as extremely fused and
wrinkled leaves, wrinkled petals that are unable to open, exposed pistil and infertility.
Combined linkage analysis and reverse genetic screen, we finally identified that the
target gene can encode a 3-ketoacyl-CoA synthase (KCS), which catalyzed initiation
step in long-chain fatty acids extension.
In this study, we primarily characterized the phenotype of wfl1 mutant, identified
the candidate gene and analyzed its function. The morphology of leaf cuticular waxes
of wfl1 mutant and wild plants was characterized by using scanning electron
microscope (SEM). The result showed that wfl1 leaves exhibited a more short and thin
crystal wax structure. Toluidine blue test performed on plants showed that wfl1 was
more easily to be dyed, indicating the reduction of cuticular waxes in wfl1 leaves that
would resulted in the increase of permeability of leaf epidermis. Genetic analysis for
selfing offspring of wfl1 heterozygote showed the numbers of the mutant and wild
type plants was followed 3:1 segregation ratio, suggesting that the wfl1 was controlled
by one recessive allele. Based on the protein sequence analysis, WFL1 gene encodes a
3-ketoacyl-CoA synthase, belonging to the KCS gene family.Based on the extensive
screen, we found 28 KCS genes in Medicago truncatula genome. Phylogenetic
analysis showed that these genes can be distinctly divided into four subfamilies:
FAE1-like, FDH-like, CER6 and KCS1-like. The WFL1 gene was homologous to the
FDH gene (AT2G26250) in Arabidopsis. The wfl1 mutant exhibited a similarly
phenotypic deficiency to fdh mutant. The result of quantitative RT-PCR showed the
WFL1 gene displayed a constitutive expression. High expression levels were found in
vegetative and reproductive shoots, stigma and pistil, followed by stem, leaf and
sepals. The temporal and spatial expression patterns were further confirmed by RNA
in situ hybridization. Transcripts of wfl1 gene were detected in outer cells of early leaf
primordium. At early developmental stage of floral primordium, the WFL1 can be
detected in the floral organs including sepals, petals, stigma and stamens. WFL1 was
highly transcripted in the stigma and ovule through the whole developmental stages,
but sharply reduced in the mature stamen. Analysis of methyl esters prepared from
total fatty acids of leaves showed that the content of fatty acid (FA) was significantly
decreased in the wfl1 plants. Compared to WT, the FA profiles were altered by the
decrease of linolenic acid (18C:3) proportions and dramatic increase of
very-long-chain FAs (>18C) proportions. The unsaturated FAs to saturated FAs ratio
(US/S) showed a significant reduction in wfl1.
Keywords: Flower development, Dorsoventral, TCP gene, KCS gene, Medicago
truncatula 

Degree Level: 博士
Place of Degree's Grantor: 北京
Grantor: 中国科学院研究生院
Content Type: 学位论文
URI: http://ir.xtbg.org.cn/handle/353005/10293
Appears in Collections:西双版纳热带植物园毕业生学位论文_学位论文

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Recommended Citation:
阳天泉. 蒺藜苜蓿花发育基因tcp 和wfl1 的功能分析[D]. 北京. 中国科学院研究生院. 2016.
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