【作者】 刘兆磊；

【导师】 章镇；

【作者基本信息】 南京农业大学 ， 观赏园艺， 2011， 博士

【摘要】 荷花(Nelumbo nucifera Gaertn),属睡莲科莲属大型挺水植物,是中国十大传统名花之一,花型花色丰富,抗(耐)污染能力强,具有极高的观赏和经济价值。本研究从荷花中克隆了与耐重金属和花发育相关的优异基因,分析其时空表达模式,通过转化模式植物拟南芥研究其功能,不仅能够从分子水平上探讨荷花重金属抗(耐)性和花发育的遗传变异机理,还可为利用植物基因工程技术改良荷花及它种花卉的观赏性状和提高抗逆能力提供理论依据和技术支持。主要结论为：1、利用RACE-PCR技术从荷花‘冬荷’的叶片中分离出植物络合素合酶基因NnPCSl,该基因全长2208bp,ORF区编码505个氨基酸,GenBank登录号为AB360381。推导的氨基酸序列由两个典型的植物络合素亚家族结构域组成,具有4个相邻的Cys-Cys元件(331-332、352-353、370-371和417-418位氨基酸)和植物络合素合酶蛋白的特征位点(Cys-56、Cys-90/91、Cys-109和Cys-113),与莴苣LsPCS1蛋白的同源性为70.41%。2、从荷花‘锦霞’的花瓣中分离出APETALA2同源基因NnAP2.该基因全长2048bp,ORF区编码511个氨基酸,GenBank登录号为AB622392. NnAP2除具备完全保守的核定位信号(KKSR)序列外,还具有AP2家族典型的结构域,与拟南芥AP2基因的两个AP2 domain (AP2-R1, AP2-R2)氨基酸同源性分别达到91.03%和85.29%。系统进化树分析表明,荷花NnAP2蛋白与双子叶植物葡萄亲缘关系最近。3、利用Real-Time PCR技术,分析了NnPCS1基因在荷花不同器官及不同重金属胁迫下的表达情况。结果显示NnPCSl基因为组成型表达,叶中CdCl2和CuSO4·5H2O处理后可明显提高基因的表达量,Zn离子一定程度上能有效缓解Cd离子毒害,但不同浓度Zn离子对Cd离子毒害的缓解作用不同。通过研究NnPCSl在根和叶中的节律性表达发现,24小时内NnPCSl在根和叶中均没有显著变化,但叶部的表达量高于根部。在400μmol/L CdCl2胁迫条件下,叶片处理1h和3h后的表达量分别是未处理的1.7倍和2.7倍；而根部初始略有上升,1h后下降,12h后检测不到表达。在500μmol/L CuSO4·5H2O处理下,1h后叶片的表达量是未处理的2.4倍；而根部在3小时内稳定表达,之后下降,6h后检测不到表达。两种金属离子诱导能力为：Cd2+>Cu2+。在400μmol/L CdCl2溶液处理48h后,NnPCS1基因检测不到有表达,但在400μmol/L CdCl2加入200μmol/L ZnS04溶液中,48h后该基因在叶片中恢复表达。对照为在400μmol/L CdCl2溶液和200μmol/L ZnS04·5H20胁迫48h后,叶片中的表达量,未检测到有NnPCS1的表达。4、利用Real-Time PCR技术分析了NnAP2基因在荷花不同组织、不同花器官、不同生育期中的表达情况。结果显示NnAP2基因皆有表达,从单瓣莲到重瓣莲到千瓣莲,NnAP2始终在花瓣中表达量最高,且非单瓣莲中NnAP2基因的表达量均高于单瓣莲。NnAP2基因在荷花的根、茎、叶、花等不同器官中都有表达,但花中的表达量最高,其次是叶。从蕾期到初开期到盛开期等不同生育期,NnAP2在花托、萼片、花瓣、雄蕊、雌蕊等不同部位都有表达。在三个生育期,花瓣表达量最高,其次是雄蕊,萼片最低。在五个代表性品种初开期之间,NnAP2在‘中国古代莲’、“(?)娘玉脚’和‘红台莲’的花瓣中表达强度最高,其次是雄蕊。在‘中国古代莲’和‘锦霞’的萼片中表达量均最低。在‘宜良千瓣’的花瓣中表达量最高,然后是萼片和花托。不论瓣型与否,NnAP2均在花瓣中表达量最高,除‘宜良千瓣’初开期雌雄蕊瓣化外,其余四个瓣型NnAP2相对表达量在雄蕊中仅次于花瓣。在5个品种的纵向对比中,NnAP2在花瓣中的表达量大小依次为：“(?)娘玉脚’(复瓣莲)>‘红台莲’(重台莲)>‘宜良千瓣’(千瓣莲)>‘锦霞’(重瓣莲)>‘中国古代莲’(单瓣莲)。5、构建了荷花NnPCS1正义表达载体,将NnPCS1基因转入拟南芥所得到的转基因植株具有对Cd离子毒害的不同抗性,在过量Cd离子胁迫下,能使植物缓解重金属离子的毒害。通过花序侵染法将NnPCS1基因转入拟南芥,经潮霉素抗性筛选,Gus染色和PCR检测,获得8株转基因株系(命名为pCSl-pCS8)。经Real-time PCR扩增发现,pCS2、 pCS5、pCS6表达量最高。在100μmol/L CdCl2处理3天后,三个转基因株系的GSH含量与野生型相比无显著变化,而NPT含量约是野生型的1.3到1.5倍,PC含量约是1.6到1.9倍,均显著高于野生型。NPT与PC两个指标值按大小依次为pCS2>pcs5> pcs6>WT(对照),但三个转基因株系之间并不存在显著差异。50μmol/L CdCl2和100μmol/L CdCl2处理1周,三个转基因株系地上部积累的Cd离子的量约为野生型的2-2.5倍,其中pcs2株系在两个浓度梯度下Cd离子的吸收值略高于pcs5和pcs6两个株系。NPT含量的增加促进了PC的生物合成,提高了植物对Cd离子的耐性。6、将NnAP2基因转入拟南芥所得到的转基因植株株高高于野生型,且转基因植株相对野生型普遍存在早花现象,但花瓣数量、雄蕊和雌蕊发育较野生型未有显著变化。构建荷花NnAP2正义表达载体,通过花序侵染法将该基因转入拟南芥。获得11个转基因株系(命名为AP2-1-AP2-11)。经Real-time PCR扩增发现,AP2-2和AP2-6表达量最高。2个转基因株系生长七周后株高显著高于野生型拟南芥。利用Real-timePCR分析了11个赤霉素相关基因的表达,发现野生型拟南芥中GA2ox3的表达量是转基因株系的2-3倍,GA2ox7的表达量是4-5倍,存在显著差异,其它基因的表达量并无显著差异。说明将外源NnAP2基因转入拟南芥中,转基因株系中GA2ox3和GA2ox7的表达量下调,促进了植株内源GA的生物合成,转基因植株表现出变高的表型。此外,转基因植株相对野生型普遍存在早花现象。分析了四个与开花相关的基因(LFY,FT,CO,FLC)在播种后12天与18天的相对表达量,发现播种后12天,AP2-2和AP2-6植株叶片中LFY和FT基因的表达量高于野生型。播种后18天,LFY和FT基因的表达量仍然高于野生型,但LFY基因的相对表达量迅速上升,CO基因的表达量无显著差异,而FLC基因在野生型植株中的表达量则显著高于两个转基因株系植株。说明将外源NnAP2基因转入拟南芥后调控开花相关基因的表达,转基因植株中FLC的下调、FT和LFY的上调起到了促进成花的作用,而CO基因的作用不显著。更多还原

【Abstract】 Nelumbo nucifera is a large emergent aquatic plant belonging to Nelumbo of the Nympheaceae. And it is one of the top ten traditional Chinese flowers having highly ornamental and economic value, rich flower type and color and strong ability of anti-pollution. In our study, we cloned two valuable genes about anti-heavy metal and flower development, analyzed their temporal and spatial expression and further studied their function by transforming them into Arabidopsis thaliana. We can not only investigate genetic variation mechanism about anti-heavy metal and flower development but also provide a theoretical basis and technical support to improve amental characters and anti-pollution ability by using plant genetic engineering in Nelumbo nucifera.1. NnPCSl (GenBank accession AB360381) was cloned from Nelumbo nucifera leaf by RACE-PCR. We found that the length of NnPCSl was 2208bp and the putative protein contained 505 amino acids and two typical subfamily domains which had four adjacent Cys-Cys element in the site of 331-332,352-353,370-371,417-418 and four PCS protein feature sites in Cys-56, Cys-90/91, Cys-109, Cys-113. The most closely related amino acids sequence (70.41%identity) was Lactuca sativa PCS.2. NnAP2 (GenBank accession AB622392) was cloned from Nelumbo nucifera petal finding that the length of NnAP2 was 2048bp and the putative protein contained 511 amino acids. Addition to a completely conserved nuclear localization signal (KKSR) sequences, NnAP2 had the typical AP2 domain belonging to AP2 subfamily which was the character of AP2 family, the identity of it compared to Arabidopsis thaliana was 91.03%and 85.29% respectively. The phylogeny of plant AP2 polypeptides suggested that NnAP2 had the the closest orthologs relationship with VvAP2.3. The NnPCS1 expression was analyzed by Real-Time PCR in different organs and under different heavy metal stress in Nelumbo nucifera. NnPCS1 was constitutively expressing and the transcription level of NnPCS1 was induced by the presence of either CdCl2 or CUSO4·5H2O in the leaf. Zn can effectively relieve Cd toxicity to a certain extent, but different concentrations of Zn were different on the alleviation of Cd toxicity.From our results, NnPCS1 expression was not diurnally regulated in root and leaf within 24 h, but the expression in leaf was higher than in root. The leaf expression level increased 1.7 fold after exposure to 400μmol/L CdCl2 for 1h, and by 2.7 fold after 3h; and by 2.4 fold after being exposed to 500μmol/L CUSO4·5H2O for 1h. These enhanced levels of transcription fell subsequently, although transcript abundance increased again between 6h and 12h of the CdCl2 treatment and between 3h and 12h of the CUSO4·5H2O treatment. In the root, NnPCS1 expression initially rose slightly after exposure to CdCl2, but fell back after 1h and was no longer detectable by 12h. When exposed to CUSO4·5H2O, its expression was stable over the first 3h, thereafter decreasing to below the level of detection by 6h. These showed that Cd2+ had the higher regulation capacity in regulating NnPCS1 transcription than Cu2+. The NnPCS1 expression was inhibited that can not be detected in the leaf by exposure to 400 μmol/L CdCl2 after 48h, but the NnPCS1 expressed after exposure to 400 μmol/L CdCl2 plus 200 μmol/L ZnSO4 with the control of no expression under 400μmol/L CdCl2 and 200μmol/L ZnSO4 exposure.4. The NnAP2 expression was analyzed in different organizations, flower organs and growth stages. From simple petal flower cultivar to double-petalled flower cultivar and then to thousand petal flower cultivar, the NnAP2 expression was always the highest in petal, however, the expression in non-simple petal flower cultivar was higher than in simple petal flower cultivar.NnAP2 expression was detected in root, stem, leaf and flower, and the expression was the highest in flower followed by leaf. In bud, early blossoming and full bloom stages. NnAP2 was expressed in all floral organ:receptacle, sepal, petal, stamen and gynoecium. During the three phases of flowering, the expression level of NnAP2 was highest in petal, followed by stamen and lowest in sepal. Between five representative species in the early blossoming stage, the expression level of NnAP2 was also the highest in petal followed by stamen in’Zhongguogudailian’,’Yaoniangyujiao’and’Hongtailian’. In’Yiliangqianban’, the expression in petal was the highest followed by sepal and receptacle. We can concluded that regardless of flower type, the NnAP2 expression was always the highest in petal. In early blossoming stage, the stamen and gynoecium are degenerated to petals. In other four flower type cultivars, the NnAP2 expression in stamen was only lower than in petal. In the longitudinal comparison of five representative cultivars, order of the expression in petal was that double-petalled flower ’Yaoniangyujiao’> proliferation flower ’Hongtailian’> thousand petal flower’Yiliangqianban’> multiplicate flower’Jinxia’> simple petal flower ’Zhongguogudailian’.5. We constructed the NnPCSl heterologous expression vector and transformed it to Arabidopsis thaliana. The transgenic plants had differently tolerant to Cd toxicity. Under excessive Cd stress, they can mitigate the heavy metal ions toxicity.The NnPCSl was transformed to Arabidopsis thaliana by the floral dip method. Eight plants were selected as being carriers of the transgene on the basis of the Tl hygromycm selection, GUS assay and PCR and these were designated pcsl through pcs8. The NnPCS1 expression in pcs2, pcs5, pcs6 was higher than in other five lines and there is no expression in wild-type by Real-time PCR. At 100μmol/L CdCl2, there was no significant difference with respect to the content of GSH between the transgenic and the wild type plants, but the level of NPT was 1.3 to 1.5 fold higher than than of wild type and the level of PC was 1.6 to 1.9 fold higher. The size of the two indicator values was pcs2> pcs5> pcs6>WT (control), however, the difference between the transgenic plants was not significant. After one week exposure of 50μmol/L CdCl2 and 100μmol/L CdCl2, the Cd content of three transgenic lines in shoot was 2 to 2.5 fold higher than that in wild type. The pcs2 Cd content was slightly higher than in pcs5 and pcs6 under two CdCl2 concentration. Increased NPT contents promoted the biosynthesis of PC and increased Cd tolerance.6. NnAP2 gene was transformed into Arabidopsis thaliana. The transgenic plants had higher stem height than wild type and were early flowering. However, we can not see any change in the petal number, stamen and pistil of transgenic plants.We constructed the heterologous expression vector and Arabidopsis thaliana Col-0 was transformed by the floral dip method. Eleven plants were selected and these were designated AP2-1 through AP2-11. The NnPCSl expression in AP2-2, AP2-6 was higher than in other nine lines and there was no expression in wild-type by Real-time PCR. The stem height of transgenic plants AP2-2 and AP2-6 was higher than that in the wild type after growth for seven weeks. Moreover we analyzed eleven GA related genes finding that the expression of GA2ox3 in transgenic plants was 2-3 fold greater than that of wild type, while that in the expression of GA2ox7, was 4-5 fold higher. The GA2ox3 and GA2ox7 had significant differences compared to wild type, while the other nine genes had no significant difference. These showed that the decrease of GA2ox3 and GA2ox7 promoted the biosynthesis of endogenous GA when NnAP2 was transformed to Arabidopsis thaliana and that may be why the transgenic plants had higher stem height. In addition, two transgenic lines were early flowering compared to wild-type. We analyzed the expression of four flowering relative genes (LFY, FT, CO, FLC) after sowing for 12 and 18 days. The transcription level of LFY and FT in AP2-2 and AP2-6 plants was higher than that in wild type after sowing for 12 days, however, there was no significant difference in CO and FLC expression. After sowing for 18 days, the transcription level of LFY and FT in AP2-2 and AP2-6 plants was still higher than that in wild type. But the expression of LFY increased rapidly, there was no significant difference in CO gene expression between transgenic lines and wild type, however, the expression of FLC in wild type was higher that in two transgenic lines. Results showed that the expression of flowering genes were regulated when endogenous GA when NnAP2 was transformed to A. thaliana. The decrease of FLC and the increase of FT and LFY promoted the flowering, however, in this process CO had no significant effect.更多还原