【作者】 刘德春；

【导师】 孙中海；

【作者基本信息】 华中农业大学 ， 果树学， 2010， 博士

【摘要】 柑橘的周期性冻害在世界范围内时有发生,造成了巨大的经济损失。采用转基因技术将抗寒基因导入柑橘栽培品种中表达是缩短育种周期,快速获得柑橘抗寒品种的有效途径之一。但是目前有关柑橘抗寒基因克隆的研究很少,这种情况严重限制了柑橘抗寒分子育种的进展。本研究从柑橘的抗寒近缘种枳(Poncirus trifoliata (L.) Raf.)中克隆出了柑橘冷响应基因表达调控途径中的3个关键基因PtrICE1、PtrHOS1和PtrLOS2,同时对它们进行了详细的生物信息学分析,研究了它们在不同环境胁迫下在枳中的时空表达模式。通过转基因技术鉴定了其中一个编码上游转录因子的基因PtrICE1的功能,证明了在模式植物烟草中超量表达PtrICE1能够提高转基因烟草的抗寒性。然后克隆出了PtrICE1的上游启动子序列,并且通过转基因技术初步证明该启动子具有一定的冷响应和机械损伤响应活性。本研究为最终通过转基因技术获得抗寒性强的柑橘栽培品种提供了新的基因资源和理论基础。本研究获得的主要结果如下：1.从枳中克隆出了一个bHLH (basic Helix-Loop-Helix)家族基因PtrICE1,该基因长1933 bp,包含一个1464 bp的开放阅读框(ORF),编码487个氨基酸,蛋白质分子量为53.6 kDa,等电点为5.30。PtrICE1蛋白与其它植物ICE1家族蛋白的氨基酸序列一致性很高,并且具有保守的SUMO (small ubiquitin-related modifier)结合结构域、bHLH结构域和C-末端区域。系统进化树的分析结果表明,植物ICE1家族蛋白可分为双子叶植物类和单子叶植物类两大类,PtrICE1属于双子叶植物类。2.在正常的生长条件下,PtrICE1在枳根、茎、叶中都有一定程度的表达。冷处理之后,枳叶和茎中PtrICE1表达先上调后下调,在根中表达先下调后上调。ABA处理之后,枳叶、茎、根中PtrICE1表达都是先上调后下调。这些结果说明冷处理和ABA处理都能影响枳不同器官中PtrICE1的表达。3.构建了PtrICE1的超量表达载体pMVICE1,并通过根癌农杆菌介导的叶盘法将pMVICE1导入模式植物烟草中,使PtrICE1在烟草中超量表达。通过PCR检测获得了17个阳性T0代转基因烟草植株。T0代转基因烟草的生长速度比野生型烟草慢,开花时间比野生型烟草推迟10d左右,开花以后两者的表型差异不大。4.T0代转基因烟草果实成熟后收集种子,经Km抗性筛选后获得阳性T1代转基因烟草。通过比较T1代转基因和野生型烟草在冷处理后的相对电导率、成活率以及表型后发现,与野生型烟草相比,T1代转基因烟草的相对电导率更低,成活率更高,并且常温处理之后能够恢复生长,说明转基因烟草的抗寒性比野生型更强,因此我们认为在植物中超量表达PtrICE1能够提高植物的抗寒性。5.本研究克隆出了PtrICE1基因的DNA序列。通过比较PtrICE1编码区的DNA和cDNA序列发现,PtrICE1含有四个外显子和三个内含子。采用pMD18-T载体介导的接头PCR方法克隆了PtrICE1基因的上游启动子序列,并采用PLACE程序对PtrICE1启动子上可能存在的顺式作用元件进行了分析,结果找到一些与植物抗逆性相关的顺式作用元件,其中包括冷响应元件(CRT/DRE、MYC)、光响应元件(GT1、GATA)、脱水和ABA响应元件(ABRE、MYB)和各种激素响应元件(ARF、ASF、GARE、WBOX等)。6.本研究构建了能够检测PtrICE1启动子活性的表达载体pBIPROICE1:GUS.通过根癌农杆菌介导的叶盘法将pBIPROICE1:GUS导入模式植物烟草中,并通过PCR检测获得了阳性的To代转基因烟草。To代转基因烟草叶片的GUS组织化学染色结果表明,野生型烟草叶片未出现蓝色斑点,而未经冷处理时转基因烟草叶片表面只出现了少量的蓝色斑点,但在叶片切口处染色较深。4℃冷处理之后,转基因烟草叶片上蓝色斑点的数量增加,且在叶片伤口处染色较深,说明PtrICE1启动子具有冷响应和机械损伤响应活性。7.从枳中克隆出了PtrHOS1基因,该基因长3434 bp,包含一个2922 bp的开放阅读框,编码974个氨基酸,蛋白质分子量为110.2 kDa,等电点为5.55。PtrHOS1蛋白与其它植物HOS1类似蛋白的氨基酸序列一致性很高,并且在PtrHOS1蛋白的N末端含有一个高度保守的环指结构域(RING finger domain)。植物HOS1类似蛋白可分为双子叶植物类和单子叶植物类两大类,PtrHOS1属于双子叶植物类。在正常的生长条件下,PtrHOS1在枳根、茎、叶中表达强烈,冷处理和ABA处理之后,在枳的根、茎、叶中PtrHOS1的表达都出现了暂时的下调,说明在枳的根、茎、叶中PtrHOS1的表达都受冷处理和ABA处理的下调调控。8.从枳中克隆出了PtrLOS2基因,该基因长1662 bp,包含一个1338 bp的开放阅读框,编码445个氨基酸,蛋白质分子量为47.79 kDa,等电点为5.54。PtrLOS2蛋白与拟南芥LOS2蛋白及其它植物烯醇酶的氨基酸序列一致性很高(超过86%),说明PtrLOS2很可能编码一种烯醇酶。PtrLOS2含有两个高度保守的结构域,它们分别是DNA结合结构域和转录抑制结构域。在正常的生长条件下,PtrLOS2在枳根、茎、叶中都有表达,枳根和茎中PtrLOS2的表达量要高于叶中。从总体上来说,经过冷处理后,枳叶片和茎中PtrLOS2的表达是先上调后下调,而在根中PtrLOS2的表达是下调的。ABA处理之后,枳叶片中PtrLOS2的表达总体上是上调的,而在茎和根中PtrLOS2的表达是先下调后再上调。这些结果说明冷处理和ABA处理都能影响枳不同器官中PtrLOS2的表达。更多还原

【Abstract】 Periodic freezing injury occurred frequently in the worldwide, which brings about gigantic economic losses. Introduced cold tolerance genes into citrus through transgenic technique can shortened the breeding cycle, and obtain the cold tolerant citrus cultivars quickly. However, there were few cold tolerance genes had been cloned and identified from citrus, which limiting the development of breeding cold hardy citrus cultivars through genetic engineering.In this research, three important genes in cold responsive gene expression pathway were cloned from citrus cold hardy relative trifoliate orange (Poncirus trifoliata (L.) Raf.), which were named PtrICE1, PtrHOS1 and PtrLOS2 respectively. These genes were analyzed in detail by bioinformatics. The space-time expression patterns of these genes after different stress treatments were also detected in this research. In addtion, the function of PtrICE1 which encodes an upstream transcription factor was identified through transgenic technique. Overexpressed PtrICE1 in tabacco could improve the cold tolerance of transgenic tabacco seedings. Moreover, the upstream promoter sequence of PtrICE1 was also cloned from trifoliate orange, and the cold and wounding responsive activity of PtrICE1 promoter was proved through transgenic technique. This research provided novel genes and theoretic bases for obtaining cold hardy citrus cultivated varieties through genetic engineering and transgenic technique. The main results of this research were as follows:1. A bHLH (basic Helix-Loop-Helix) family gene PtrICE1 was cloned from trifoliate orange. The length of PtrICE1 was 1933 bp with an open reading frame of 1464 bp, encoding a protein of 487 amino acids with a molecular weight of 53.6 kDa and a theoretical isoelectric point of 5.30. PtrICE1 protein shared high identity with other plants ICE1 family proteins and had conserved SUMO (small ubiquitin-related modifier) protein conjugation motif, bHLH domain and C-terminal region. The analysis results of phylogenetic tree revealed that the plants ICE1 famliy proteins could be divided to dicotyledonous plant group and monocotyledonous plant group. PtrICE1 protein belonged to dicotyledonous plant group.2. The PtrICE1 was constitutively expressed in leaves, stems and roots of trifoliate orange under normal growth condition. After cold treatments, the PtrICE1 expression in leaves and stems was up-regulated first and down-regulated afterwards. However, the PtrICE1 expression in roots was down-regulated first and up-regulated afterwards after cold treatments. After ABA treatments, the PtrICE1 expression in leaves, stems and roots was up-regulated first and down-regulated afterwards. These results suggested that the PtrICE1 expression in different organs of trifoliate orange was influenced by cold and ABA treatments.3. The overexpression vector pMVICE1 of PtrICE1 was constructed and transformed to tabacco through Agrobacterium tumefaciens mediated leaf-disc method. The transgentic tabacco seedings overexpressed PtrICE1 were detected by PCR and 17 positive T0 generation transgentic tabacco seedings were obtained in this research. The T0 generation transgentic tabacco growed slower and flower later (delayed about 10 d) than wild type tabacco. After flowering, there were no obvious phenotype difference between T0 generation transgentic and wild type tabacco seedings.4. T0 generation transgentic tabacco seeds were collected to produce T1 generation transgentic tabacco seedings by Km resistance selection. After cold treatments, the T1 generation transgentic tabacco seedings have lower relative electrical conductivity and higher survival rate compared to the wild type. Moreover, the growth of T1 generation transgentic tabacco could recover after normal temperature treatment. These results suggested that the transgentic tabacco seedings had stronger cold tolerance than wild type. Thus, we concluded that overexpressed PtrICE1 in plants could improve plants cold tolerance.5. The PtrICE1 DNA sequence was also cloned in this research. Four exons and three introns were found in PtrICE1 DNA sequence. The PtrICE1 promoter sequence was cloned by pMD18-T mediated adapter PCR. Several potential stress tolerance related cis-elements were predicted to be involved in the PtrICE1 promoter using the PLACE Program, including cold responsive elements (CRT/DRE, MYC), light responsive elements (GT1, GATA), dehydration and ABA responsive elements (ABRE, MYB) and several auxin responsive elements (ARF, ASF, GARE, WBOX).6. The pBIPROICE1:GUS expression vector used to detected the PtrICE1 promoter activity was constructed in this research. The pBIPROICE1:GUS vector was transformed to tabacco through Agrobacterium tumefaciens mediated leaf-disc method and obtained positive T0 generation transgentic tabacco seedings by PCR. The results of GUS histochemical staining revealed that the leaves of wild type tabacco could not be blue stained. Before cold treatment, a little of blue spots were observed in the leaves of T0 generation transgentic tabacco but the cut of which were deep blue stained. After cold treatments, more blue spots were observed in the leaves of T0 generation transgentic tabacco and the cut of which was also deep blue stained. These results suggested that the PtrICE1 promoter has cold and wounding responsive activities.7. The PtrHOSl gene was cloned from trifoliate orange. The length of PtrHOSl was 3434 bp with an open reading frame of 2922 bp, encoding a protein of 974 amino acids with a molecular weight of 110.2 kDa and a theoretical isoelectric point of 5.55. The amino acid sequence of PtrHOS1 protein shared high identity with that of other plants HOS1-like proteins and had a conserved RING finger domain in its N terminal. The plants HOS1-like proteins could be divided to dicotyledonous plant group and monocotyledonous plant group. PtrHOS1 protein belonged to dicotyledonous plant group. The PtrHOS1 was constitutively expressed at high level in leaves, stems and roots of trifoliate orange under normal growth condition. After cold and ABA treatments, the PtrHOS1 expression had several declined periods in leaves, stems and roots, which suggested that the PtrHOS1 expression in different organs of trifoliate orange were down-regulated both by cold and ABA.8. The PtrLOS2 gene was cloned from trifoliate orange. The PtrLOS2 cDNA is 1662 bp in length with a 1338 bp open reading frame, encoding a deduced 445 amino acid residue protein with a predicted molecular mass of 47.79 kDa and an isoelectric point of 5.54. The amino acid sequence of the PtrLOS2 protein shared high identity (over 86%) with Arabidopsis LOS2 protein and other plants enolase, which suggested that the PtrLOS2 probably encodes an enolase. Moreover, PtrLOS2 protein had a conserved DNA-binding and a repression domain. PtrLOS2 was constitutively expressed in leaves, stems and roots of trifoliate orange under normal growth condition. In addition, PtrLOS2 expression in roots and stems was much higher than that in leaves under normal growth condition. As a whole, the expression of PtrLOS2 was up-regulated first and down-regulated afterwards in leaves and stems, but down-regulated in roots after cold treatments. After ABA treatment, the expression of PtrLOS2 was up-regulated in leaves. However, PtrLOS2 expression in stems and roots was down-regulated first and up-regulated afterwards. These results suggested that the PtrLOS2 expression in different organs of trifoliate orange was influenced by cold and ABA treatments.更多还原