胡杨油菜素内酯合成酶基因CPD （PeCPD）的克隆及功能分析
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摘要
油菜素类固醇(brassinosteroids, BRs)是一类新发现的植物生长调节物质,被称为第六大类植物激素,在植物整个生长和发育中起重要的作用,其中包括细胞伸长与分裂、维管束分化、光形态建成、叶和根的发育、衰老、育性以及对逆境胁迫的响应等。BR生物合成途径及其遗传调节是当今植物生物学研究的热点问题。目前已经取得了重要进展,但主要集中在拟南芥、水稻、番茄和豌豆等这些一年生草本植物,而在多年生木本植物,尤其是林木中鲜有研究。CPD (constitutive photomorphogenesis and dwarf)是一个与BR合成相关的基因,最早鉴定于拟南芥,编码一种细胞色素单加氧化酶CYP90A1,在BR生物合成过程中催化22-OH-CR向22-OH-4-en-3-one转变。为探讨木本植物CPD基因的功能,本研究以极端耐旱、耐高温和耐盐碱的珍稀树种胡杨(Populus euphratica)和耐逆性相对较差的箭杆杨(P. nigra var. thevesitina)为对象,从中克隆到了与拟南芥CPD同源的cDNA全序列,构建了相应的植物表达载体,并将这些载体分别导入拟南芥cpd突变体(Atcpd)和野生型(Atwt),获得了多种转基因系。在此基础上,对这些转基因系的形态学、解剖学、遗传学、细胞学、生理学和分子生物学特征进行了详细的分析与比较。主要结果如下：
一、采用电子克隆和3'RACE策略,从胡杨和箭杆杨中克隆出了与拟南芥CPD同源的cDNA全序列(命名为PeCPD和PnCPD);对PeCPD和PnPCD的生物信息学分析显示：
1) PeCPD和PnCPD cDNA分别长1746bp和1553bp,二者总长不同,但ORF大小完全相同,都编码一个由476个氨基酸组成的蛋白质,氨基酸序列一致性为97%。
2) PeCPD和PnCPD编码的蛋白与CYP90A蛋白家族具有67-92%的同源性,因此确定该蛋白属于CYP90A家族(分别命名为PeCYP90A和PnCYP90A)。
3) PeCYP90A与PnCYP90A间的同源性显著高于PeCYP90A/PnCYP90A与毛果杨CYP90A之间的同源性。
4) PeCYP90A/PnCYP90A与木本植物甜橙、番木瓜和山茶CYP90A的进化关系相反地小于PeCYP90A/PnCYP90A与草本植物豌豆和豇豆CYP90A的进化关系。
5)不同CYP90A之间,根据信号肽序列预测的亚细胞定位也有所不同。
二、采用Real-Time qRT-PCR法分析了不同派(section)杨树及胡杨不同组织(器官)之间PeCPD基因表达水平,结果显示：
1)在青杨(P. cathayana,青杨派)、胡杨(P. euphratice,胡杨派)、箭杆杨(P. nigra var. thevesitina,黑杨派)、大叶杨(P. tomentosa,大叶杨派)和新疆杨(P. euphratice,白杨派)中的相对表达量分别为1.0,1.4,1.1,0.8和1.1,胡杨中的表达量最高,大叶杨中的表达量最低。
2)在胡杨中,成熟叶中的表达量最高,中间叶次之,幼叶最低。PeCPD在根中完全不表达,在叶脉和枝形成层中低表达。
三、将PeCPD cDNA插入植物表达载体pMDC43,构建了由花椰菜花叶病毒启动子(CaM35S)驱动的PeCPD表达载体35S::PeCPD,由CaM35S驱动的PeCPD-GFP融合表达载体35S::PeCPD-GFP,以及由拟南芥CPD基因启动子(AP)驱动的PeCPD表达载体AP::PeCPD.
四、将35S::PeCPD-GFP、35S::PeCPD和AP::PeCPD分别导入拟南芥cpd突变体(Atcpd),分别得到3(C35CG1/2/3)、2(C35C1/2)和3个(CAPC1/2/3)Atcpd-PeCPD转基因系。对以上各转基因系的形态学、遗传学、生理学和解剖学研究显示：
1)T1代C35CG1/2/3、C35C1/2和CAPC1/2/3系与Atcpd(?)目比,莲座叶完全展开,植株个体增大,开花期提前,花茎高度和数目增加。与Atwt相比,C35CG和C35C系莲座叶长宽比减小,叶柄缩短,开花期延迟,株高降低；CAPC系叶长宽比减小,叶面积和茎直径增加。C35CG系雄性不育,人工自花授粉也不育。C35C1系早期花不育,后期花人工自花授粉可育。CAPC1/2早期花不育,后期花正常可育,而CAPC3系完全可育。T3代C35C1和CAPC1/2/3表型与T1代基本相同,表型相对恢复程度的大小依次为Atcpd 2)C35C1和CAPC1系早期花不育与花丝细胞伸长受阻,导致花丝缩短,花药无法与柱头接触有关。
3)与Atcpd相比,C35C1、CAPC1和CAPC3系叶表皮细胞大小,细胞凸起(lobe)的数目与lobe长度增加,气孔密度降低。与Atwt相比,C35C1系叶表皮细胞大小,lobe数目与lobe长度减少(小),气孔密度增加；CAPC1和CAPC3系叶表皮细胞大小,lobe数目与lobe长度及气孔密度没有显著变化。
4)CAPC1和CAPC3系莲座叶海绵组织细胞数目减少,叶维管束木质部细胞较Atcpd和Atwt明显增加,茎维管束数目,维管束木质部、原形成层细胞数目也显著多于Atcpd和Atwt。
5)在黑暗和光照条件下C35C1、CAPC1和CAPC3系下胚轴伸长速率显著大于Atcpd,黑暗中子叶形状与野生型的相似,子叶不展开,呈钩状。
五、将35S::PeCPD-GFP.35S::PeCPD和AP::PeCPD分别导入拟南芥野生型(Atwt),分别各获得10个(035CG),7个(035C)和7个(OAPC)Atwt-PeCPD转基因系。对其中各1个转基因系(035CG24、OAPC4和035C7)的形态学、生理学和解剖学观察显示：
1)种子萌发期,035CG24、OAPC4和035C7系下胚轴和根伸长速率与Atwt相比显著增加,对盐的耐受力增强：营养生长期,035CG24系个体增大、叶面积增加、气孔密度减少；成熟期株高、果荚数、果荚长度和茎的直径也显著增加。OAPC4和035C7系无显著性变化。
2)将各Atwt-PeCPD转基因系((?))与生长素应答因子拟南芥转基因系(DR5-GUS)((?))杂交,发现F1代植株根中GUS活性与根伸长的速率成正相关,即根伸长的速率越快,根中GUS活性越强。
3)组织学观察发现,与Atwt相比,035CG24系叶片增厚,叶肉细胞变大,海绵组织细胞间的气腔增多,海绵细胞数目减少。茎维管束数目,维管束木质部和原形成层细胞数目增加。木质部细胞总数和原形成层细胞总数与茎的直径大小成正相关。
六、Real-Time qRT-PCR分析以上各转基因系以及Atcpd和Atwt之间PeCPD和其它BR相关基因的转录水平,结果显示：
1)C35C1、CAPC1和CAPC3系表型恢复程度大小与PeCPD和受BR正调节的BAS1的表达量成正相关,与BR负调节的DWF4和BR60X2的表达量成负相关；PeCPD的表达量与BAS1的表达量成正相关,与DWF4和BR60X2的表达量成负相关；外源BR正调节的TCH4和SAUR-AC1表达量与植株的恢复程度无明显关系。
2)035CG24,035C7和OAPC4系表型超Atwt程度与PeCPD和BAS1表达量成正相关,PeCPD表达量与BAS1表达量成正相关；DWF4表达量稍高于野生型,BR60X2表达量低于野生型；TCH4和SAUR-AC1表达量与植株表型也无显著关系。
七、采用ELISA方法分析了各Atcpd-PeCPD和Atwt-PeCPD转基因系内源BR的含量,结果显示,各转基因系及Atcpd和Atwt内源BR水平大小依次为O35CG24 八、采用双向电泳检测了CAPC3系和Atcpd之间蛋白表达谱的差异。ImageMaster2D Platinum Version5.0软件分析显示,CAPC3和Atcpd分别显示568和411个蛋白点,前者比后者多出157个蛋白点。其中可匹配的有236个(占41.5%),不匹配的有329个(57.9%)。在CAPC3中,有8个匹配点表达量显著上调,有3个匹配点表达量显著下调,另有19个非匹配点表达量甚高。对这19个非匹配点中最显著的7个点进行质谱鉴定发现,其中5个与光合作用有关,一个与植物抗逆性有关,另一个与叶绿体RNA的加工修饰有关。有文献报道,这几种蛋白均受BR正调节。
九、用35S::PeCPD-GFP转化烟草表皮细胞发现,PeCPD-GFP信号主要分布在质膜和细胞质内的一些网状结构上。这种分布模式与已报道的内质网标记蛋白的分布模式相同,这表明PeCPD蛋白可能主要定位于内质网。根据以上结果,主要得出以下结论：
1)胡杨CPD (PeCPD)基因与拟南芥CPD (AtCPD)基因的功能基本相似,都可能通过控制BL合成,调节植物的生长发育,因为异源PeCPD的表达明显地使Atcpd的表型得以恢复,并且导致其它BR合成相关基因的表达水平以及内源BR水平发生相应的变化。
2) PeCPD可能还存在其它功能,因为PeCPD异源表达并不能使Atcpd的表型完全恢复。恢复后的植株在形态学、解剖学、育性、组织和细胞学方面与野生型相比仍存在明显的差异,即使在恢复程度最好转基因系中。
3) PeCPD可能参与了叶和茎的发育,这一调节过程可能独立于BR信号通路,因为在BL水平并没有完全恢复的CAPC1系中,虽然株型大小与野生型一致,但叶变宽,茎分枝增多,茎增粗。
4) PeCPD功能也可能与光合作用有关,因为Real-Time qRT-PCR分析表明,PeCPD在成熟胡杨叶中的表达量显著高于在中、幼年叶中的表达量。此外,蛋白组学分析显示,在CAPC3系中,在所鉴定的7个特异蛋白点中有5个与光合作用有关。
5) PeCPD表达水平在杨树中具有种和组织器官的特异性。
6)在Atwt-PeCPD转基因系中,CaM35S启动子的效率显著地高于拟南芥CPD自身启动子的效率,而在Atcpd-PeCPD转基因系中,CaM35S启动子的效率出乎意外地显著低于拟南芥CPD自身启动子的效率,原因可能与CaM35S启动子在Atcpd中受抑制有关。
7) PeCPD异源表达可促进Atwt根的生长(伸长),这种促进作用可能是通过促进的生长素合成或分布实现的。
8) PeCPD是一个优良的可用来转化其它林木或农作物的基因,因为在本研究中发现一些Atwt-PeCPD转基因系植株个体变大、株高增加、种子产量、茎的直径增加和抗逆性增强。
Brassinosteroids (BRs) are new type of plant growth regulators, and are considered as the sixth class of phytohormone, which play critical roles in cell division and elongation, vascular differentiation, leaf and root development, photomorphogenesis, male fertility, senescence, and stress responsion. The mechanism underlying BR biosynthesis and its regulation are the "hot spot" among the current plant biological researches and many important progresses have been made. However, these studies mainly focused on the annual herbaceous plants, such as Arabidopsis, rice, tomato, pea, and so on, few is known in the perennial woody plants, such as trees. CPD (constitutive photomorphogenesis and dwarf) was initially isolated from an Arabidopsis cpd mutant. In BR biosynthesis pathway, CPD encodes a cytochrome P450enzyme functioning in the conversion of22-OH-CR to22OH-4-en-3-one. In present study, the homologous CPD genes were cloned from Populus euphratica Oliv with extremely high tolerance to drought, saline and alkaline and P. nigra var. thevesitina with relatively poor tolerance to stress. Then the corresponding expression vectors were constructed, with which the cpd mutant and wild type of Arabidopsis thaliana were transformed. Finally, the function of the CPD gene from P. euphratica was explored through morphology, genetics, physiology, anatomy and molecular biological analysis of the obtained transgenic lines. The main results are the follows:
1. The full-length cDNA homologous with Arabidopsis CPD, were isolated from P. nigra var. thevesitina and P. euphratica oliv, named PeCPD and PnCPD respectively, by homologous cloning combined with in silico cloning. Bioinformatics regarding PeCPD and PnCPD were analyzed. The results were the follows:
1) PeCPD and PnCPD contained equal length of coding regions, the proteins encoded by which shared97%amino acid (aa) homology.
2) The proteins deduced from the PeCPD and PnCPD shared67～92%aa identity to CYP90A family, were named PnCYP90A and PnCYP90A, respectively.
3) The phylogenctic relationship of PeCYP90A to PnCYP90A was closer than that of PeCYP90A/PnCYP90A to CYP90A6V1of P. trichocarpa.
4) The phylogenetic relationship of PeCYP90A/PnCYP90A to the CYP90As of Citrus sinensis, Carica papaya and Camellia japonica, were less close than that of PeCYP90A/PnCYP90A to the CYP90As of Vigna radiata and Pisum sativum.
5) The subcellular location predicted according to signal peptide sequences was also distinct from each other among the CYP90As from different plant species.
2. The relative expression level of PeCPD in different poplar sections (species) and tissue/organ of P. euphratica were analyzed using Real-Time qRT-PCR.
1) The expression levels of PeCPD gene in P. cathayana (Tacamahaca section), P. euphratice(Turanga section), P. nigra var. thevesitina (Aigeiros section), P. tomentosa (Leucoides section) and P. bolleana (Leuce section) were1.0,1.4,1.1,0.8and1.1, respectively. The transcription level was the highest in P. euphratica and the lowest in P. tomentosa among them.
2) In P. euphratica, the transcription level of PeCPD in leaves was significantly higher than that in roots and stem cambium, and that in mature leaves was significantly higher than that in juvenile and intermediate leaves. In roots, PeCPD did not express.
3. Using PeCPD cDNA and plasmid pMDC43, three different expression vectors35S::PeCPDGFP,35S::PeCPD and AP::PeCPD were constructed,35S and AP represent the promoter of CaM35S and Arabidopsis CPD gene, respectively.
4. Using35S::PeCPDGFP,35S::PeCPD and AP::PeCPD to transform Arabidopsis cpd mutant (Atcpd), the following three group of Atcpd-PeCPD transgenic lines, named C35CG (3lines), C35C (2) and CAPC (3), were obtained. The morphology, genetics, physiology and anatomy analysis of the Atcpd-PeCPD lines showed:
1) Compared with Atcpd, the T1Atcpd-PeCPD lines showed expanded rosette leaves, increased plant size, ahead flowering as well as increased height, numbers and branches of stem. Compared with Arabidopsis wild type (Atwt), the ratio of leaf length to width, petiole length and plant height are significantly reduced in all of C35CG and C35C lines, but their flowering phase were delayed. In plant size and height as well as flowering phase, CAPC lines were similar with corresponding Atwt, but leave's width and area as well as stem's diameter significantly increased. C35CG1, C35CG2, C35CG3and C35C2lines were male sterile even in the case of illegitimate pollination. The early stage flowers of C35C1, CAPC1and CAPC2lines did not set seeds unless illegitimate pollination, but the late stage flowers normally set seeds. The CAPC3line was completely fertile. CAPC3, CAPC2, CAPC1and C35C1line of T3generation were basically similar with T1lines in phenotype. The degree of phenotypic restoration were Atcpd 2) The sterility of the early stage flowers of C35C1and CAPC1lines were related to the inhibited filament cell elongation, which made their anthers not touch with stigma.
3) In C35C1and CAPC lines, the size of epidermic cells as well as the length and number of lobes were significantly increased, whereas stomatal density relatively decreased, compared with Atcpd. In comparison with Atwt, the size of epidermic cells as well as the length and number of lobes of C35C1line were decreased, but stomatal density were increased. CAPC lines did not show visible differences from Atwt.
4) For the CAPC lines, the number of sponge cells was decreased, while air spaces increased. The number of xylem cell in leaf vascular bundle (VB) was greater than Atcpd and Atwt lines. And the numbers of stem VB, xylem and procambia cell in VB also increased relative to both Atcpd and Atwt.
5) The rate of hypocotyls elongation of C35C1, CAPC1and CAPC3lines was significantly increased in the light and dark compared with Atcpd. Phenotype of their cotyledons was similar to that of Atwt in the dark.
5. Using Vectors35S::PeCPDGFP,35S::PeCPD and AP::PeCPD to transform Atwt, the24Atwt-PeCPD transgenic lines, named O35CG (10),O35C (7) and OAPC (7), were obtained. From each group, one most promising line O35CG24, O35C7and OAPC4, was selected for further investigation. The results were as follows:
1) Compared to Atwt, the rate of hypocotyls and root elongation of O35CG24, O35C7and OAPC4all increased. However, for the O35CG24line, their leaf area and leaf epidermal cells increased, and stoma density decreased during vegetative growth phase. In mature phase, their plant height, silique mummer and stem diameter were also significantly increased. O35C7and OAPC4lines showed no significant change compared to Atwt.
2) For measurement of auxin activity, we crossed the Atwt-PeCPD lines with the transgenic plants expressing the DR5-GUS reporter. The result showed that highest GUS activity was detected in the O35CG24line with the longest root. The GUS activity was positively correlated to the root length.
3) Histology research showed that the leaf thickens, mesophyll cell size and spongy air spaces of O35CG24line were increased significantly, whereas, the number of sponge cells decreased. The numbers of VB, xylem and procambia cell in stem were significantly greater than Atwt lines. The number of xylem cell and procambia cell along the base of VB ring were positively correlated to the diameter of stem.
6. Real-Time qRT-PCR analysis of expression level of PeCPD and other BR-related genes in the transgenic lines revealed:
1) The degree of phenotypic rescue of C35C1, CAPC1and CAPC3lines relative to Atcpd was positively correlated to the expression level of PeCPD and BAS1genes, and negatively to that of DWF4and BR6OX2gene. The expression level of PeCPD was positively correlated to that of BAS1and negatively to that of DWF4and BR6OX2. The expression level of TCH4and SAUR-AC1were not significantly associated with the degrees of their phenotypic rescue.
2) Phenotypic change degree of O35CG24/O35C7/OAPC4lines relative to Atwt was positively correlated to the expression level of PeCPD and BAS1. The expression level of PeCPD was positively correlated to that of BAS1. The expression levels of DWF4in O35CG24/O35C7/OAPC4lines were higher than that in Atwt. The expression level of TCH4and SAUR-AC1were not related to with the phenotypic changes of O35CG24/O35C7/OAPC4lines.
7. A analysis of total endogenous BR levels revealed that the endogenous BR level in the all transgenic lines, Atcpd and Atwt from lower to higher was O35CG24 8. Two-dimensional electrophoresis was used to analyze the protein difference between Atcpd and CAPC3lines. The result showed that there were568protein spots in CAPC3and411protein spots in Atcpd. In the CAPC3, there were eight significantly up-regulated proteins, three down-regulated proteins and nineteen new proteins compared to Atcpd. MALDI TOF/TOF-MS/MS identification of seven of the19new proteins found that five were involved in photosynthesis, one in RNA processing, and one in cell defense. It is reported that the7proteins all were positively regulated by BR.
9. The PeCPD-GFP fused protein were transient expression by agroinfiltration in Nicotiana benthamiana leaves. The result showed that the PeCPD-GFP protein mainly distributed on cytomembrane and cytoplasm with typical reticulate structure, which is similar to that of the marker protein for endoplasmic reticulum (ER), suggesting that PeCPD protein is possibly located in ER.
Based on the above results, the following conclusions are drawn:
1) P. euphratica CPD (PeCPD) and Arabidopsis CPD (AtCPD) possibly share similar function in BR biosynthesis, because the heterogeneous expression of PeCPD could apparently made the phenotype of Atcpd mutants to be recovered, and alter the level of BR-related genes expression and endogenous BR.
2) PeCPD may have other functions in the growth and development of plant, since the phenotype of Atcpd could not be completely restored by heterogeneous expression of PeCPD.
3) PeCPD may regulate BR-independent the leaf and stem development, because the CAPC1lines that has relative low BL level to Atwt showed broad leaves, much branched stem, increased stem diameter.
4) PeCPD possibly regulates photosynthesis because the transcripts of PeCPD in mature leaves of P. euphratica are significantly higher than that in juvenile and intermediate leaves. Moreover, proteomics analysis showed that five proteins were involved in photosynthesis in seven proteins identified by MALDI TOF/TOF-MS/MS.
5) The level of PeCPD expression was distinctively different in different poplar sections and tissues/organs of P. euphratice.
6) In the Atcpd-PeCPD transgenic lines, the efficiency of CaM35S promoter was significantly less than that of the promoter of AtCPD gene, while in the Atwt-PeCPD line, the efficiency of CaM35S promoter was significantly higher than that of the promoter of AtCPD gene, suggesting that the function of CaM35S promoter might be partly suppressed in Atcpd.
7) The phenomenon that rate of root elongation is enhanced in the Atwt-PeCPD transgenic lines indicated that PeCPD regulates the root growth possibly though improving the biosynthesis or distribution of auxin.
8) PeCPD is a promising gene that can be used to transform other crop or tree, since plant height, seed yield, stem diameter and resistance to salt are significantly increased in O35CG24lines due to heterogeneous expression of PeCPD.
一、采用电子克隆和3'RACE策略,从胡杨和箭杆杨中克隆出了与拟南芥CPD同源的cDNA全序列(命名为PeCPD和PnCPD);对PeCPD和PnPCD的生物信息学分析显示：
1) PeCPD和PnCPD cDNA分别长1746bp和1553bp,二者总长不同,但ORF大小完全相同,都编码一个由476个氨基酸组成的蛋白质,氨基酸序列一致性为97%。
2) PeCPD和PnCPD编码的蛋白与CYP90A蛋白家族具有67-92%的同源性,因此确定该蛋白属于CYP90A家族(分别命名为PeCYP90A和PnCYP90A)。
3) PeCYP90A与PnCYP90A间的同源性显著高于PeCYP90A/PnCYP90A与毛果杨CYP90A之间的同源性。
4) PeCYP90A/PnCYP90A与木本植物甜橙、番木瓜和山茶CYP90A的进化关系相反地小于PeCYP90A/PnCYP90A与草本植物豌豆和豇豆CYP90A的进化关系。
5)不同CYP90A之间,根据信号肽序列预测的亚细胞定位也有所不同。
二、采用Real-Time qRT-PCR法分析了不同派(section)杨树及胡杨不同组织(器官)之间PeCPD基因表达水平,结果显示：
1)在青杨(P. cathayana,青杨派)、胡杨(P. euphratice,胡杨派)、箭杆杨(P. nigra var. thevesitina,黑杨派)、大叶杨(P. tomentosa,大叶杨派)和新疆杨(P. euphratice,白杨派)中的相对表达量分别为1.0,1.4,1.1,0.8和1.1,胡杨中的表达量最高,大叶杨中的表达量最低。
2)在胡杨中,成熟叶中的表达量最高,中间叶次之,幼叶最低。PeCPD在根中完全不表达,在叶脉和枝形成层中低表达。
三、将PeCPD cDNA插入植物表达载体pMDC43,构建了由花椰菜花叶病毒启动子(CaM35S)驱动的PeCPD表达载体35S::PeCPD,由CaM35S驱动的PeCPD-GFP融合表达载体35S::PeCPD-GFP,以及由拟南芥CPD基因启动子(AP)驱动的PeCPD表达载体AP::PeCPD.
四、将35S::PeCPD-GFP、35S::PeCPD和AP::PeCPD分别导入拟南芥cpd突变体(Atcpd),分别得到3(C35CG1/2/3)、2(C35C1/2)和3个(CAPC1/2/3)Atcpd-PeCPD转基因系。对以上各转基因系的形态学、遗传学、生理学和解剖学研究显示：
1)T1代C35CG1/2/3、C35C1/2和CAPC1/2/3系与Atcpd(?)目比,莲座叶完全展开,植株个体增大,开花期提前,花茎高度和数目增加。与Atwt相比,C35CG和C35C系莲座叶长宽比减小,叶柄缩短,开花期延迟,株高降低；CAPC系叶长宽比减小,叶面积和茎直径增加。C35CG系雄性不育,人工自花授粉也不育。C35C1系早期花不育,后期花人工自花授粉可育。CAPC1/2早期花不育,后期花正常可育,而CAPC3系完全可育。T3代C35C1和CAPC1/2/3表型与T1代基本相同,表型相对恢复程度的大小依次为Atcpd
3)与Atcpd相比,C35C1、CAPC1和CAPC3系叶表皮细胞大小,细胞凸起(lobe)的数目与lobe长度增加,气孔密度降低。与Atwt相比,C35C1系叶表皮细胞大小,lobe数目与lobe长度减少(小),气孔密度增加；CAPC1和CAPC3系叶表皮细胞大小,lobe数目与lobe长度及气孔密度没有显著变化。
4)CAPC1和CAPC3系莲座叶海绵组织细胞数目减少,叶维管束木质部细胞较Atcpd和Atwt明显增加,茎维管束数目,维管束木质部、原形成层细胞数目也显著多于Atcpd和Atwt。
5)在黑暗和光照条件下C35C1、CAPC1和CAPC3系下胚轴伸长速率显著大于Atcpd,黑暗中子叶形状与野生型的相似,子叶不展开,呈钩状。
五、将35S::PeCPD-GFP.35S::PeCPD和AP::PeCPD分别导入拟南芥野生型(Atwt),分别各获得10个(035CG),7个(035C)和7个(OAPC)Atwt-PeCPD转基因系。对其中各1个转基因系(035CG24、OAPC4和035C7)的形态学、生理学和解剖学观察显示：
1)种子萌发期,035CG24、OAPC4和035C7系下胚轴和根伸长速率与Atwt相比显著增加,对盐的耐受力增强：营养生长期,035CG24系个体增大、叶面积增加、气孔密度减少；成熟期株高、果荚数、果荚长度和茎的直径也显著增加。OAPC4和035C7系无显著性变化。
2)将各Atwt-PeCPD转基因系((?))与生长素应答因子拟南芥转基因系(DR5-GUS)((?))杂交,发现F1代植株根中GUS活性与根伸长的速率成正相关,即根伸长的速率越快,根中GUS活性越强。
3)组织学观察发现,与Atwt相比,035CG24系叶片增厚,叶肉细胞变大,海绵组织细胞间的气腔增多,海绵细胞数目减少。茎维管束数目,维管束木质部和原形成层细胞数目增加。木质部细胞总数和原形成层细胞总数与茎的直径大小成正相关。
六、Real-Time qRT-PCR分析以上各转基因系以及Atcpd和Atwt之间PeCPD和其它BR相关基因的转录水平,结果显示：
1)C35C1、CAPC1和CAPC3系表型恢复程度大小与PeCPD和受BR正调节的BAS1的表达量成正相关,与BR负调节的DWF4和BR60X2的表达量成负相关；PeCPD的表达量与BAS1的表达量成正相关,与DWF4和BR60X2的表达量成负相关；外源BR正调节的TCH4和SAUR-AC1表达量与植株的恢复程度无明显关系。
2)035CG24,035C7和OAPC4系表型超Atwt程度与PeCPD和BAS1表达量成正相关,PeCPD表达量与BAS1表达量成正相关；DWF4表达量稍高于野生型,BR60X2表达量低于野生型；TCH4和SAUR-AC1表达量与植株表型也无显著关系。
七、采用ELISA方法分析了各Atcpd-PeCPD和Atwt-PeCPD转基因系内源BR的含量,结果显示,各转基因系及Atcpd和Atwt内源BR水平大小依次为O35CG24
九、用35S::PeCPD-GFP转化烟草表皮细胞发现,PeCPD-GFP信号主要分布在质膜和细胞质内的一些网状结构上。这种分布模式与已报道的内质网标记蛋白的分布模式相同,这表明PeCPD蛋白可能主要定位于内质网。根据以上结果,主要得出以下结论：
1)胡杨CPD (PeCPD)基因与拟南芥CPD (AtCPD)基因的功能基本相似,都可能通过控制BL合成,调节植物的生长发育,因为异源PeCPD的表达明显地使Atcpd的表型得以恢复,并且导致其它BR合成相关基因的表达水平以及内源BR水平发生相应的变化。
2) PeCPD可能还存在其它功能,因为PeCPD异源表达并不能使Atcpd的表型完全恢复。恢复后的植株在形态学、解剖学、育性、组织和细胞学方面与野生型相比仍存在明显的差异,即使在恢复程度最好转基因系中。
3) PeCPD可能参与了叶和茎的发育,这一调节过程可能独立于BR信号通路,因为在BL水平并没有完全恢复的CAPC1系中,虽然株型大小与野生型一致,但叶变宽,茎分枝增多,茎增粗。
4) PeCPD功能也可能与光合作用有关,因为Real-Time qRT-PCR分析表明,PeCPD在成熟胡杨叶中的表达量显著高于在中、幼年叶中的表达量。此外,蛋白组学分析显示,在CAPC3系中,在所鉴定的7个特异蛋白点中有5个与光合作用有关。
5) PeCPD表达水平在杨树中具有种和组织器官的特异性。
6)在Atwt-PeCPD转基因系中,CaM35S启动子的效率显著地高于拟南芥CPD自身启动子的效率,而在Atcpd-PeCPD转基因系中,CaM35S启动子的效率出乎意外地显著低于拟南芥CPD自身启动子的效率,原因可能与CaM35S启动子在Atcpd中受抑制有关。
7) PeCPD异源表达可促进Atwt根的生长(伸长),这种促进作用可能是通过促进的生长素合成或分布实现的。
8) PeCPD是一个优良的可用来转化其它林木或农作物的基因,因为在本研究中发现一些Atwt-PeCPD转基因系植株个体变大、株高增加、种子产量、茎的直径增加和抗逆性增强。
Brassinosteroids (BRs) are new type of plant growth regulators, and are considered as the sixth class of phytohormone, which play critical roles in cell division and elongation, vascular differentiation, leaf and root development, photomorphogenesis, male fertility, senescence, and stress responsion. The mechanism underlying BR biosynthesis and its regulation are the "hot spot" among the current plant biological researches and many important progresses have been made. However, these studies mainly focused on the annual herbaceous plants, such as Arabidopsis, rice, tomato, pea, and so on, few is known in the perennial woody plants, such as trees. CPD (constitutive photomorphogenesis and dwarf) was initially isolated from an Arabidopsis cpd mutant. In BR biosynthesis pathway, CPD encodes a cytochrome P450enzyme functioning in the conversion of22-OH-CR to22OH-4-en-3-one. In present study, the homologous CPD genes were cloned from Populus euphratica Oliv with extremely high tolerance to drought, saline and alkaline and P. nigra var. thevesitina with relatively poor tolerance to stress. Then the corresponding expression vectors were constructed, with which the cpd mutant and wild type of Arabidopsis thaliana were transformed. Finally, the function of the CPD gene from P. euphratica was explored through morphology, genetics, physiology, anatomy and molecular biological analysis of the obtained transgenic lines. The main results are the follows:
1. The full-length cDNA homologous with Arabidopsis CPD, were isolated from P. nigra var. thevesitina and P. euphratica oliv, named PeCPD and PnCPD respectively, by homologous cloning combined with in silico cloning. Bioinformatics regarding PeCPD and PnCPD were analyzed. The results were the follows:
1) PeCPD and PnCPD contained equal length of coding regions, the proteins encoded by which shared97%amino acid (aa) homology.
2) The proteins deduced from the PeCPD and PnCPD shared67～92%aa identity to CYP90A family, were named PnCYP90A and PnCYP90A, respectively.
3) The phylogenctic relationship of PeCYP90A to PnCYP90A was closer than that of PeCYP90A/PnCYP90A to CYP90A6V1of P. trichocarpa.
4) The phylogenetic relationship of PeCYP90A/PnCYP90A to the CYP90As of Citrus sinensis, Carica papaya and Camellia japonica, were less close than that of PeCYP90A/PnCYP90A to the CYP90As of Vigna radiata and Pisum sativum.
5) The subcellular location predicted according to signal peptide sequences was also distinct from each other among the CYP90As from different plant species.
2. The relative expression level of PeCPD in different poplar sections (species) and tissue/organ of P. euphratica were analyzed using Real-Time qRT-PCR.
1) The expression levels of PeCPD gene in P. cathayana (Tacamahaca section), P. euphratice(Turanga section), P. nigra var. thevesitina (Aigeiros section), P. tomentosa (Leucoides section) and P. bolleana (Leuce section) were1.0,1.4,1.1,0.8and1.1, respectively. The transcription level was the highest in P. euphratica and the lowest in P. tomentosa among them.
2) In P. euphratica, the transcription level of PeCPD in leaves was significantly higher than that in roots and stem cambium, and that in mature leaves was significantly higher than that in juvenile and intermediate leaves. In roots, PeCPD did not express.
3. Using PeCPD cDNA and plasmid pMDC43, three different expression vectors35S::PeCPDGFP,35S::PeCPD and AP::PeCPD were constructed,35S and AP represent the promoter of CaM35S and Arabidopsis CPD gene, respectively.
4. Using35S::PeCPDGFP,35S::PeCPD and AP::PeCPD to transform Arabidopsis cpd mutant (Atcpd), the following three group of Atcpd-PeCPD transgenic lines, named C35CG (3lines), C35C (2) and CAPC (3), were obtained. The morphology, genetics, physiology and anatomy analysis of the Atcpd-PeCPD lines showed:
1) Compared with Atcpd, the T1Atcpd-PeCPD lines showed expanded rosette leaves, increased plant size, ahead flowering as well as increased height, numbers and branches of stem. Compared with Arabidopsis wild type (Atwt), the ratio of leaf length to width, petiole length and plant height are significantly reduced in all of C35CG and C35C lines, but their flowering phase were delayed. In plant size and height as well as flowering phase, CAPC lines were similar with corresponding Atwt, but leave's width and area as well as stem's diameter significantly increased. C35CG1, C35CG2, C35CG3and C35C2lines were male sterile even in the case of illegitimate pollination. The early stage flowers of C35C1, CAPC1and CAPC2lines did not set seeds unless illegitimate pollination, but the late stage flowers normally set seeds. The CAPC3line was completely fertile. CAPC3, CAPC2, CAPC1and C35C1line of T3generation were basically similar with T1lines in phenotype. The degree of phenotypic restoration were Atcpd
3) In C35C1and CAPC lines, the size of epidermic cells as well as the length and number of lobes were significantly increased, whereas stomatal density relatively decreased, compared with Atcpd. In comparison with Atwt, the size of epidermic cells as well as the length and number of lobes of C35C1line were decreased, but stomatal density were increased. CAPC lines did not show visible differences from Atwt.
4) For the CAPC lines, the number of sponge cells was decreased, while air spaces increased. The number of xylem cell in leaf vascular bundle (VB) was greater than Atcpd and Atwt lines. And the numbers of stem VB, xylem and procambia cell in VB also increased relative to both Atcpd and Atwt.
5) The rate of hypocotyls elongation of C35C1, CAPC1and CAPC3lines was significantly increased in the light and dark compared with Atcpd. Phenotype of their cotyledons was similar to that of Atwt in the dark.
5. Using Vectors35S::PeCPDGFP,35S::PeCPD and AP::PeCPD to transform Atwt, the24Atwt-PeCPD transgenic lines, named O35CG (10),O35C (7) and OAPC (7), were obtained. From each group, one most promising line O35CG24, O35C7and OAPC4, was selected for further investigation. The results were as follows:
1) Compared to Atwt, the rate of hypocotyls and root elongation of O35CG24, O35C7and OAPC4all increased. However, for the O35CG24line, their leaf area and leaf epidermal cells increased, and stoma density decreased during vegetative growth phase. In mature phase, their plant height, silique mummer and stem diameter were also significantly increased. O35C7and OAPC4lines showed no significant change compared to Atwt.
2) For measurement of auxin activity, we crossed the Atwt-PeCPD lines with the transgenic plants expressing the DR5-GUS reporter. The result showed that highest GUS activity was detected in the O35CG24line with the longest root. The GUS activity was positively correlated to the root length.
3) Histology research showed that the leaf thickens, mesophyll cell size and spongy air spaces of O35CG24line were increased significantly, whereas, the number of sponge cells decreased. The numbers of VB, xylem and procambia cell in stem were significantly greater than Atwt lines. The number of xylem cell and procambia cell along the base of VB ring were positively correlated to the diameter of stem.
6. Real-Time qRT-PCR analysis of expression level of PeCPD and other BR-related genes in the transgenic lines revealed:
1) The degree of phenotypic rescue of C35C1, CAPC1and CAPC3lines relative to Atcpd was positively correlated to the expression level of PeCPD and BAS1genes, and negatively to that of DWF4and BR6OX2gene. The expression level of PeCPD was positively correlated to that of BAS1and negatively to that of DWF4and BR6OX2. The expression level of TCH4and SAUR-AC1were not significantly associated with the degrees of their phenotypic rescue.
2) Phenotypic change degree of O35CG24/O35C7/OAPC4lines relative to Atwt was positively correlated to the expression level of PeCPD and BAS1. The expression level of PeCPD was positively correlated to that of BAS1. The expression levels of DWF4in O35CG24/O35C7/OAPC4lines were higher than that in Atwt. The expression level of TCH4and SAUR-AC1were not related to with the phenotypic changes of O35CG24/O35C7/OAPC4lines.
7. A analysis of total endogenous BR levels revealed that the endogenous BR level in the all transgenic lines, Atcpd and Atwt from lower to higher was O35CG24
9. The PeCPD-GFP fused protein were transient expression by agroinfiltration in Nicotiana benthamiana leaves. The result showed that the PeCPD-GFP protein mainly distributed on cytomembrane and cytoplasm with typical reticulate structure, which is similar to that of the marker protein for endoplasmic reticulum (ER), suggesting that PeCPD protein is possibly located in ER.
Based on the above results, the following conclusions are drawn:
1) P. euphratica CPD (PeCPD) and Arabidopsis CPD (AtCPD) possibly share similar function in BR biosynthesis, because the heterogeneous expression of PeCPD could apparently made the phenotype of Atcpd mutants to be recovered, and alter the level of BR-related genes expression and endogenous BR.
2) PeCPD may have other functions in the growth and development of plant, since the phenotype of Atcpd could not be completely restored by heterogeneous expression of PeCPD.
3) PeCPD may regulate BR-independent the leaf and stem development, because the CAPC1lines that has relative low BL level to Atwt showed broad leaves, much branched stem, increased stem diameter.
4) PeCPD possibly regulates photosynthesis because the transcripts of PeCPD in mature leaves of P. euphratica are significantly higher than that in juvenile and intermediate leaves. Moreover, proteomics analysis showed that five proteins were involved in photosynthesis in seven proteins identified by MALDI TOF/TOF-MS/MS.
5) The level of PeCPD expression was distinctively different in different poplar sections and tissues/organs of P. euphratice.
6) In the Atcpd-PeCPD transgenic lines, the efficiency of CaM35S promoter was significantly less than that of the promoter of AtCPD gene, while in the Atwt-PeCPD line, the efficiency of CaM35S promoter was significantly higher than that of the promoter of AtCPD gene, suggesting that the function of CaM35S promoter might be partly suppressed in Atcpd.
7) The phenomenon that rate of root elongation is enhanced in the Atwt-PeCPD transgenic lines indicated that PeCPD regulates the root growth possibly though improving the biosynthesis or distribution of auxin.
8) PeCPD is a promising gene that can be used to transform other crop or tree, since plant height, seed yield, stem diameter and resistance to salt are significantly increased in O35CG24lines due to heterogeneous expression of PeCPD.
引文
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