摘要
为研究百脉根(Lotus corniculatus L.)C2钙依赖蛋白激酶基因LcC2DP1在不定根形成过程中的功能,通过RACE法从百脉根中克隆LcC2DP1基因,利用qRT-PCR检测其时空表达模式,并通过农杆菌介导的瞬时表达系统在百脉根中过量表达LcC2DP1并鉴定其功能。结果显示:LcC2DP1基因全长705 bp,编码235个氨基酸,分子质量为25.95 ku,与蒺藜苜蓿同源性最高(82%);在百脉根不定根分化过程中持续表达,表达部位为根、茎和叶片;与野生型亲本(WT)相比,转LcC2DP1基因百脉根(TP)的不定根分化提前1~2 d;在不定根分化的9~15 d ,其总根长分别是WT的168%、155%,根体积分别是WT的249%、161%,根尖数分别是WT的156%、137%。TP百脉根的总根长(P<0.01)、根体积(P<0.01)和根尖数(P<0.05)表现出一定的发育优势,表明LcC2DP1基因可能与百脉根不定根发育调控相关。
植物根系具有固持地上组织和吸收水分、养分的双重功
百脉根(Lotus corniculatus L.)是一种优质的多年生豆科牧草,也是一种良好的蜜源和护坡固土植物,其由匍匐茎发生的不定根所构成的根系系统十分发达。本研究通过RACE扩增法克隆Leo百脉根C2CDPK编码基因LcC2DP1,利用qRT-PCR技术检测该基因的组织表达特异性,通过农杆菌介导的瞬时表达系统对其不定根分化调控功能进行初步分析,以期为揭示百脉根根系发育调控机制提供依据。
所用植物材料为百脉根(Lotus corniculatus ‘Leo’),种植于贵州大学贵州省农业生物工程重点实验室试验田。农杆菌(Agrobacterium tumefaciens)菌株GV3101和pSH737植物表达载体均保存于笔者所在实验室。
笔者所在课题组前期分析百脉根不定根发育转录组数
引物 Primer | 序列 Sequence | 用途 Function |
---|---|---|
A041-1(GSP1) | CGTAGGGATCTTGGCG | 5' RACE扩增 |
A041-2(GSP2) | ATCCATTCTGTGTCCTTC | 5' RACE amplification |
A041-3(GSP3) | TTGGAGCAGGAAACAACAG | |
S042-1 | CATCGTTTGGAACAGCAATACACT | 3' RACE扩增 |
S042-3 LcC2DP1-F LcC2DP1-R |
TTCAATCCAAAACTGGCAGACACGC TGTTGAATTGAAAGATCTGC GAGGGTGTAGAGGATCCA |
3' RACE amplification 全长扩增 Full length amplification |
使用相关的生物信息分析在线工具进行LcC2DP1基因的分子特征分析,其中,理化性质(蛋白质分子质量、等电点、分子式)采用ExPASy ProtParam tool(https://web.expasy.org/protparam/)、编码蛋白的亲疏水性采用Protscale(https:∥web.expasy.org/protscale/)、蛋白结构分析采用SOPMA(https:∥npsa-prabi.ibcp.fr/cgi-bin/)和SWISS-MODEL(https://swissmodel.expasy.org/)进行。利用Plant-mPLoc(http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/)对LcC2DP1基因进行亚细胞定位预测。利用BEDTool
为探究LcC2DP1在百脉根不同组织和不同时期中的表达情况,分别收集Leo百脉根的根、茎、叶以及不定根形成过程中0、3、6、9、12 d根部组织为样本。以百脉根UBI(DQ249171.1)作为内参基因,通过IDT(https://sg.idtdna.com/site/home/home/sessiontimeout)在线软件设计LcC2DP1和UBI的扩增引物(
引物 Primer | 序列(5'-3') Sequence | 用途 Function |
---|---|---|
LcUBI-F | CTTCACCTTGTCCTTCGTCTG | 内参基因 |
LcUBI-R | TGGATCTTAGCCTTCACGTTG | House-keeping gene |
QLcC2DP1-F | TTTCACGCCAAGATCCCTAC | 目的基因 |
QLcC2DP1-R | GAGTGTATTGCTGTTCCAAACG | Gene of interest |
利用限制性内切酶EcoRⅠ和XbaⅠ(TaKaRa, 大连)分别酶切pSH737和pMD18T- LcC2DP1质粒,酶切片段回收后用T4 DNA连接酶(TaKaRa,大连)4 ℃连接过夜,构建LcC2DP1基因的植物表达载体,该基因由35S启动子驱动表达,以GUS::NPTII作为报告基因和筛选基因。采用YEP液体培养基培养所获得的阳性工程菌至对数生长期。参照杨少彤
基于百脉根不定根分化转录组筛选的Lc1g3v0026680序列,对LcC2DP1基因进行RACE扩增,获得的LcC2DP1全长为705 bp,编码235个氨基酸,该序列上游有起始密码子ATG,下游有终止密码子TGA。为了验证拼接序列的正确性,设计引物对LcC2DP1编码区进行扩增,得到1条约700 bp的特异性条带,序列测定结果表明LcC2DP1编码区与拼接结果序列完全一致(

图1 LcC2DP1基因克隆及拼接翻译
Fig.1 LcC2DP1 gene amplification and mosaic translation
A:5' RACE克隆结果; B:3' RACE克隆结果; C:全长克隆结果 (1:阳性单菌落提取的质粒; M:DL2000 Marker);D:拼接翻译。A:5' RACE cloning results; B:3' RACE cloning results; C:Full length cloning results (1:Plasmids extracted from positive single colonies; M:DL2000 Marker);D:Mosaic translation.
对LcC2DP1基因结构进行分析,结果显示,其编码蛋白含235个氨基酸,相对分子质量为25 952.03,理论等电点为7.17,分子式为C1185H1737N305O344S6,脂肪指数为53.91,不稳定性指数为85.47,是一种不稳定蛋白。对LcC2DP1蛋白质进一步分析,发现其亲水性氨基酸较疏水性氨基酸多(

图2 LcC2DP1 基因分子特性
Fig.2 Molecular characteristics of LcC2DP1 gene
A:LcC2DP1蛋白疏水性; B:LcC2DP1蛋白二级结构预测; C:LcC2DP1的亚细胞定位预测; D:LcC2DP1蛋白三级结构预测; E:百脉根LcC2DP1与其他C2结构域蛋白的系统进化树。A:LcC2DP1 protein hydrophobicity analysis; B:LcC2DP1 protein secondary structure prediction; C:Subcellular localization prediction of LcC2DP1; D:Tertiary structure of LcC2DP1; E:Phylogenetic tree analysis of Lotus corniculatus and other C2 domain protein.
提取百脉根LcC2DP1基因上游2 000 bp序列进行顺式作用元件分析,结果(
元件 Element | 位点 Site | 功能 Function | 数量 Number |
---|---|---|---|
CAAT-box | 1,233,1 725,1 778,1 799 |
启动子和增强子区域常见的元件 Common elements in promoter and enhancer regions | 5 |
Box-4 | 16,1 495 |
参与光响应顺式作用调节元件 Participate in lighting response clocking components | 2 |
TATA-box | 10,11,12,13,1 976,1 977,1 978 |
转录起始核心启动子元件 Transcription initiation core promoter componentst | 7 |
MBS | 1 740 |
参与干旱诱导的顺式作用元件 Participate in drought -induced custody components | 1 |
AT1-motif | 1 370 |
参与光响应顺式作用调节元件 Participate in lighting response clocking components | 1 |
GT1-motif | 175,176 |
参与光响应顺式作用调节元件 Participate in lighting response clocking components | 2 |
扩增LcC2DP1基因片段,与pSH737植物表达载体连接,双酶切鉴定结果显示,重组质粒酶切后出现705 bp的目的基因条带(

图3 植物表达载体双酶切及农杆菌PCR电泳检测
Fig.3 The electrophoretic detection on double enzyme digestion of plant expression vector and PCR of Agrobacterium tumefaciens liquid
A:植物表达载体双酶切; B:农杆菌菌落PCR(M:DL 2000 Marker;1-2:不同阳性单菌落提取的质粒)。A:Verification of vector double enzyme digestion; B:PCR identification of Agrobacterium tumefactiens colony (M:DL 2000 Marker;1-2:Plasmids extracted from different positive single colonies).
通过qRT-PCR分析LcC2DP1基因在百脉根不同组织及不定根分化期间的差异表达情况,发现相较于内参基因UBI,LcC2DP1基因在根、茎、叶中均有表达,但主要表达部位为根(1.00±0.00)和茎(7.57±0.36),在叶中表达量(0.15±0.03)最低(

图4 LcC2DP1 基因表达特异性
Fig.4 LcC2DP1 gene expression specificity
*和**分别表示在0.05和0.01水平上显著性相关;WT:野生型植株;TP:转LcC2DP1植株;下同。A:LcC2DP1 基因在不同组织的表达; B:LcC2DP1基因在不定根分化过程中的差异表达。* and ** indicate significant correlation at the 0.05 and 0.01 levels, respectively;The same as follows.WT:Wild type;TP:Trans LcC2DP1 gene plants;A:The relative expression level of LcC2DP1 gene in plant different tissues; B:The expression of LcC2DP1 gene in the process of adventitious root development.
采用农杆菌介导的真空渗透法将植物表达载体pSHLcC2DP1转化到百脉根中。GUS组织化学染色显示,外源基因转化后3~15 d均可观察到百脉根叶片显示蓝色,外源基因瞬时转化百脉根后可在叶片和根部组织有效表达,随着时间的延长蓝色逐渐加深,到第9天时蓝色最深(

图5 不定根分化9天时的转基因植株观察与鉴定
Fig. 5 Observation and identification of transgenic plants after adventitious root differentiation for 9 days
A:GUS组织化学染色叶片和根部; B:不定根发育情况; C:RT-PCR检测(M:DL2000 marker; 1:水; 2:pSHLcC2DP1质粒; 3:WT; 4~7:瞬时转化LcC2DP1第9天的百脉根。A:GUS organizational chemical staining blade and root; B:Adventitious root development; C:Expression in the 9th day RT-PCR detection of transient transformation of Lotus corniculatus(M:DL2000 marker; 1:Water; 2:pSHLcC2DP1 plasmid; 3:WT; 4-7:Instantaneous transformation of LcC2DP1 Lotus corniculatus on the 9th day).
由

图6 百脉根不定根分化9~15 d期间的根系发育指标比较
Fig.6 Comparison of root development indicators of the Lotus corniculatus during 9-15 days
A:根系总根长; B:根体积; C:根尖数; D:根系总表面积; E:根平均直径。A:Total root length; B:Root volume; C:Root number; D:Root total surface area; E:Root average diameter.
C2结构域蛋白作为与钙离子结合的一类功能型蛋白,在调控植物生长发育、抗逆性以及信号转导方面起着重要的作用。本研究从Leo百脉根中克隆了LcC2DP1基因,序列分析发现该基因具有典型的C2结构域,属于C2域蛋白家族基
系统进化分析发现LcC2DP1基因与狭叶羽扇豆、蒺藜苜蓿、刺毛黧豆等豆科植物具有较高的同源性,其中与蒺藜苜蓿的同源性最高为82%,说明LcC2DP1在豆科植物中具有较高的保守性。对该基因进行的组织特异性表达分析表明,LcC2DP1基因在百脉根牧草的根、茎和叶片组织中均有表达,但这种组织表达存在着明显的差异,以根和茎组织中的表达量较高,而叶片中的表达量较低。马佛明
张兰军
本研究通过对LcC2DP1基因的克隆、生物信息学分析、组织特异性表达、瞬时表达分析,发现LcC2DP1基因属于C2蛋白基因,可能参与百脉根不定根发育调控过程,在后续的研究中,将进一步验证LcC2DP1是否具有钙依赖蛋白激酶相应的生理功能,进行百脉根稳定遗传转化和创制突变体植株,明确LcC2DP1基因在不定根发育过程中的生物学功能。
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