导　　师： 王学臣

学科专业： G1001

授予学位： 博士

作　　者： ;

机构地区： 中国农业大学

摘　　要： 气孔是植物与外界环境进行气体交换的门户.全球植物每年约耗掉65﹪的淡水资源,而这些水分大部分是通过气孔的蒸腾作用丧失掉的.因此有必要对气孔的开关进行调控.气孔运动的分子调控离不开高效的基因开关系统,保卫细胞特异性表达的启动子近几年倍受关注.我们的设想是:当水分供应充足时,气孔尽最大限度开放,吸收CO<,2>,增加光合产量;当干旱来临时,气孔迅速关闭,减少水分丧失,提高作物的水分高效利用率.这一目标的实现,需要诱导型的保卫细胞特异性表达或强表达启动子的创建,使目的基因在特定的条件下特定的部位表达.鉴于此,该文成功地构建了两个启动子,即DGP1和SIGP1.受干旱诱导的保卫细胞特异性表达启动子(DGP1,DROUGHT INDUCED GUARD CELL SPECIFIC PROMOTER)的构建:利用PCR的方法,扩增出干旱应答元件和保卫细胞特异性元件,然后组装而成.转基因烟草的组织化学定位表明,DGP1驱动的GUS基因在受到干旱诱导的情况下,在保卫细胞中特异性表达,而在未经干旱处理植株的保卫细胞和干旱处理的根、茎和花中均不表达;对转基因烟草GUS活性的定量分析表明,GUS活性明显地受干旱诱导,并且随着处理时间的延长而增强,其中干旱诱导8HR后GUS的活性是诱导前的179倍.而根、茎和叶肉组织在干旱诱导后GUS活性虽然有所提高,但变化不大.这些结果证明,在植物遭遇干旱胁迫时,DGP1启动子可以驱动目的基因在保卫细胞中特异性表达.构建了一个受多种胁迫诱导的保卫细胞强表达启动子(SIGP1,STRESS INDUCED GUARD CELL PROMOTER).该启动子全长746BP,包括多个TCGT基序、三个5'-TAAAG-3'核心序列和一个TATA框.通过对T1代转基因苗的组织化学定位表明,该启动子驱动的GUS基因在逆境(如干旱、ABA、高盐和冷)胁迫的叶片中表达,根中不表达,且老叶中的表达量高于幼叶中的表达量,说明其表达受发育阶段的调控.对下表皮的GUS染色发现,胁迫处理前保卫细胞的染色较浅,而处理后染色明显加深,说明SIGP1启动子可以驱动目的基因在处理后的保卫细胞中强表达.GUS活性荧光定量分析表明,SIGP1启动子驱动的GUS活性在干旱、ABA、高盐和冷等逆境处理下,在根的表达很微弱,且随着处理时间的延长变化不大,但在叶片和表皮中的GUS活性都有增加的趋势.为了深入理解调控气孔运动的分子机制,除了对保卫细胞特异性表达启动子进行研究外,还对与气孔运动相关的转录因子进行了研究.我们利用RT-PCR的方法,从拟南芥中克隆了与在气孔中表达较多的STDOF1基因的同源基因ATDOF1.7,构建了该基因的超表达载体,获得了T1代转基因烟草苗.通过对气孔运动的观察发现,该基因的超表达使叶片表面气孔密度降低,并且促进了气孔的开关.为我们将来从调控转录因子的表达入手,人工干预气孔的开关运动提供理论上的依据. Stomatal pores on the surface of leaf are gateways of gas exchange between plants and atmosphere. It was estimated that plants account for around 65/% fresh water use every year, which was mainly lost through stomata. So it is necessary to regulate the opening and closing of stomata in order to save water and improve plant drought resistance. The genetic regulation of stomatal movement mainly depends on an efficient control system of gene expression, and guard cell-specific promoter is becoming one of the best choices. Then a tentative plan came to our brains, is it possible for us to assure guard cells to open when water supply is enough, and close rapidly when plants come across drought stress by regulating expressional pattern of genes? Inducible guard cell-specific promoters can play an important role in driving target gene expression at specific position in given conditions such as drought stress. In this study we constructed two promoters /(DGP1 and SIGP1/) and analyzed their function in transgenic tobacc os. We combined the dehydration responsive element /(DRE/) with guard cell specific element /(GCSE/) to construct a novel drought-induced guard cell-specific promoter -DGP1. Histochemical assays in transgenic tobacco carrying B -glucuronidase /(gus/) gene fused to DGP1 showed that GUS activity was found to be highly inducible by drought treatment and specifically restricted to guard cells. No GUS activity was detected in roots, stems or flowers after treatment. Further quantitative analysis showed that GUS activity in the epidermal strips was apparently induced by dehydration and increased dramatically with the prolongation of treatment time. The GUS activity after 8 hours' treatment was 179 folds compared with that of those without treatment and no great changes were observed in roots, stems or mesophyll. These results suggested that DGP1 could drive target gene expressed mainly in guard cells when plant is subjected to drought stress. We constructed a multi stress induced promoter /(SIGP1/) strongly expressed in guard cells. The full length of S1GP1 is 746 bp, containing a number of TCGT motifs, three 5'-TAAAG-3'core sequence and a TATA box. Histochemical staining in Tl transgenic tobaccos showed that SIGP1 promoter was not only organ-specific /(especially expressed in leaves and guard cells/), but also highly inducible by multi stress treatment such as drought, ABA, high salinity and low temperature. Quantitative analysis showed that GUS activity in roots was very weak and no great changes were observed after treatment. However, GUS activity in leaves and epidermal strips increased with the prolongation of treatment time. In order to further understand the regulatory mechanism of stomatal movement, we studied one of transcription factors that might be related to stomatal movement. Using RT-PCR we isolated Atdofl.7 from Arabidopsis, which had a higher homology with StDOFl whose expression was very high in guard cells. However there was little knowledge about the function of Atdofl.7, especially to stomatal movement. We constructed plant expression vector driven by CaMV35S promoter and obtained Tl transgenic tobaccos. The results represented upon observation of stomatal movement showed that the density of stoma in the epidermal of transgenic tobaccos was much lower than that of wild type and the rate of opening and closing of stoma increased because of strong expression oi Atdofl. 7. It can provide the theoretic basis to study the stomatal movement by regulating the expression of transcription factors.

关 键 词： 保卫细胞特异性启动子 诱导启动子 转录因子 烟草 拟南芥

分 类 号： [Q943.2 Q945.17]

领　　域： [生物学] [生物学]