蒺藜苜蓿MtROP10基因的克隆及其功能初探

目的:克隆并研究蒺藜苜蓿ROP基因的功能,为研究该基因家族在共生途径中的作用提供依据.方法:采用RACE方法,从蒺藜苜蓿中克隆MtROP基因,利用生物信息软件比对同源性及ROP蛋白特征结构分析,利用RT-PCR方法分析该基因的组织特异性表达,构建该基因的过表达载体并转化拟南芥.结果:获得了蒺藜苜蓿ROP家族中与拟南芥ROP10高度同源的MtROP10全长序列.氨基酸编码序列具有明显的ROP家族蛋白的结构域特征.该基因在花中高表达,根中低表达.拟南芥中过表达MtROP10,可导致根毛变粗、变短、分叉.结论:MtROP10属于植物ROP家族蛋白,可能在根毛的极性生长方面具有较为重要的功能.     

pib基因启动子及其诱导启动性初探

将pib基因上游5.7 kb区段取代pCAMBIA1301中gus基因上游的35S启动子构建了pib拟启动区-GUS+ 35S-hpt 基因表达载体pNAR604。经农杆菌介导转化水稻成熟胚愈伤,获得了转基因抗潮霉素愈伤和36株转基因水稻植株。 转基因抗性愈伤和转基因植株根的组织化学GUS活性检测表明,光照培养下的抗性愈伤和转基因植株根不能使X-gluc显色,而暗处理24 h后的抗性愈伤和定植后转基因植株的根能使X-gluc显色。转基因植株GUS荧光定量分析结果表明,GUS表达具有器官特异性,黑暗处理前根的GUS活性最高、茎次之,分别是是叶片的7倍和3倍,叶片中仅有痕量本底。24 h黑暗处理后根、茎、叶中GUS活性都有增加,且叶片中的增加比例最大,其活性仅次于根。5 mmol/L水杨酸和0.3 mol/L NaCl叶面喷施转基因植株24 h后叶片中GUS活性分别为处理前的2.7和3.6倍。初步确定pib拟启动区是一个诱导型启动子。黑暗、水杨酸和NaCl能诱导该启动子启动活性。     

启动子诱捕在棉花基因组中的功能分析

利用根癌农杆菌介导的遗传转化,将启动子诱捕（Promoter trapping）元件插入到棉花基因组,获得141个独立的转化子,其中97％的转化子经PCR扩增为阳性。不同组织中GUS基因的表达频率为：根部48％,茎的微管组织9．2％,叶5．2％,花51％;同时检测了不同植株中GUS基因的表达模式,发现GUS基因在不同株系的植株间的表达模式呈现较大的差异,有些植株中GUS基因是组织特异表达,有些则是器官特异表达,有些则在多个器官中均有表达。所建立的启动子诱捕系统中的GUS基因高频率、多模式和时空特异性表达为分离基因及其调节序列、开展功能基因组研究奠定了坚实的基础。     

西瓜wml1 5'端启动子区域对番茄的遗传转化及其转录调控功能研究

根据wml1 5’端启动子区域内部的限制性酶切位点,分离得到长度分别为1573bp、1197bp、896bp、795bp的片段,并与GUS基因融合构成转录融合体。用农杆菌介导法将这些片段转入番茄中,对转基因植株进行GUS活性分析,发现1573bp、1197bp、896bp的片段都能诱导GUS在授粉后15天、30天、45天的番茄果实中表达,且表达强度随果实发育而增强,而在叶片、茎、根中未检测到GUS基因表达。而795bp的片段转化的植株中则未检测到GUS基因表达。推定857bp至957bp之间的序列中包含了启动子行使正常功能必需的元件。     

西瓜wml1 5＇端启动子区域对番茄的遗传转化及其转录调控功能研究

根据wml1 5‘端启动子区域内部的限制性酶切位点,分离得到长度分别为1573bp、1197bp、896bp、795bp的片段,并与GUS基因融合构成转录融合体。用农杆菌介导法将这些片段转入番茄中,对转基因植株进行GUS活性分析,发现1573bp、1197bp、896bp的片段都能诱导GUS在授粉后15天、30天、45天的番茄果实中表达,且表达强度随果实发育而增强,而在叶片、茎、根中未检测到GUS基因表达。而795bp的片段转化的植株中则未检测到GUS基因表达。推定857bp至957bp之间的序列中包含了启动子行使正常功能必需的元件。     

水稻rbcS启动子控制的外源基因在转基因水稻中的特异性表达

为将不同启动子用于转基因水稻的研究,从武运粳8号水稻中克隆了Rubisco小亚基基因(rbcS)的5'上游调控区,构建了由rbcS启动子引导的GUS融合基因,并经农杆菌介导导入到水稻中.对转基因水稻植株中GUS活性的定性与定量测定结果表明,rbcS启动子可驱动GUS报告基因在转基因水稻植株叶片和叶鞘内的叶肉细胞中特异性高效表达,而在茎、根和种子等器官中不表达或表达活性极弱,表现出明显的组织与细胞特异性.结果还表明,光诱导处理可明显提高rbcS启动子启动的外源基因的表达量.     

拟南芥AtNCED2基因启动子区域序列克隆及其活性分析

目的:克隆拟南芥AtNCED2基因启动子区域序列,并分析其组织器官特异性及对外界刺激的响应.方法:通过PCR从拟南芥基因组中克隆AtNCED2基因5'侧翼2295bp启动子区域序列(AtNCED2p),并进行生物信息学分析.构建AtNCED2p驱动GUS的植物双元表达载体pAtNCED2p::GUS,通过根癌农杆菌介导法将其转化野生型拟南芥,检测转基因植侏中GUS表达的组织器官特异性.结果:该启动子序列中存在TATA-box、CAAT-box、根器官特异性元件、ABA响应元件、低温响应元件、昼夜节律响应元件等顺式作用元件.GUS活性主要集中在转基因拟南芥根尖及侧根发生部位.外源ABA处理的转基因植株根中GUS活性为174.8nmol 4-MU min-1 mg-1蛋白,明显高于对照值91.7nmol 4-MU min-1mg-1蛋白.结论:AtNCED2基因可能在根的生长和发育中起作用,且外源ABA处理增强其在根中的表达.     

番茄Lehsp23.8启动子的热诱导表达调控序列鉴定

番茄线粒体小分子热激蛋白(Lehsp23.8)启动子是典型的热诱导启动子。为了研究热激条件下该启动子的调控序列,本研究将不同长度的Lehsp23.8启动子序列与gus基因融合,构建5′缺失植物表达载体。然后用农杆菌介导法转化烟草,PCR及Southern blotting结果表明融合基因已经整合到烟草基因组中。GUS组织化学染色结果表明:不同长度Lehsp23.8启动子转基因植株热激处理后,在幼苗根、茎、叶以及花和果实中均表现出GUS活性,只是染色强弱有差异。叶片中GUS荧光活性测定结果表明:在热激处理条件下,565bp的Lehsp23.8启动子介导的GUS表达最强;而255bp的Lehsp23.8启动子介导的GUS表达最弱。说明Lehsp23.8启动子中255bp的序列即能满足该启动子的热激表达,-565bp～-255bp之间存在明显的增强子元件,而-871bp～-565bp之间的片段具有一定的抑制作用。     

中间锦鸡儿CiNAC038启动子的克隆及对激素响应分析

NAC转录因子家族是植物中特有的、家族数目较多的一类转录因子家族,对植物生长发育起重要作用。了解CiNAC038的表达调控分子机制,为中间锦鸡儿CiNAC038功能研究奠定基础。以中间锦鸡儿为植物材料,通过染色体步移法克隆启动子序列,并对启动子序列上的响应元件进行分析。构建GUS表达载体,并转化拟南芥,对拟南芥的组织特异性进行表达分析,用ABA诱导转基因植株,研究ABA与CiNAC038的关系。结果显示,克隆了1 800 bp的CiNAC038启动子序列,该启动子包含多种顺式元件。成功构建植物表达载体ProCiNAC038∶GUS,通过浸花法转化至野生型拟南芥。GUS组织化学染色结果显示,转基因拟南芥幼苗根部染色较深,胚轴无染色;成熟期转基因拟南芥的叶脉、果荚两端、花瓣、花药等组织染色较深,茎无染色。CiNAC038启动子驱动的GUS报告基因主要在植物叶片、根和花的组织器官表达。进一步ABA诱导表达分析发现,GUS染色随着浓度增加颜色越浅。CiNAC038启动子是ABA抑制型启动子。     

拟南芥非特异性磷脂酶C4基因表达模式的研究

拟南芥非特异性磷脂酶C4（AtNPC4）具有降解磷脂酰胆碱（PC）,产生二酰甘油（DAG）和磷酸胆碱的活性。本研究从拟南芥基因组中分离了NPC4基因起始密码子上游1 379bp的启动子序列,与GUS报告基因融合后转化拟南芥,获得转基因植株。GUS组织化学染色表明,AtNPC4基因主要在处于衰老过程中的叶片中高水平表达,在根、茎、种荚和花中也有一定程度的表达,这种表达模式与RT-PCR结果相一致。另外,通过RT-PCR发现,AtNPC4基因在转录水平上受脱落酸的诱导,但不受水杨酸和茉莉素诱导。     

Silencing of the Rac1 GTPase MtROP9 in Medicago truncatula stimulates early mycorrhizal and oomycete root colonizations but negatively affects rhizobial infection

RAC/ROP proteins (ρ-related GTPases of plants) are plant-specific small G proteins that function as molecular switches within elementary signal transduction pathways, including the regulation of reactive oxygen species (ROS) generation during early microbial infection via the activation of NADPH oxidase homologs of plants termed RBOH (for respiratory burst oxidase homolog). We investigated the role of Medicago truncatula Jemalong A17 small GTPase MtROP9, orthologous to Medicago sativa Rac1, via an RNA interference silencing approach. Composite M. truncatula plants (MtROP9i) whose roots have been transformed by Agrobacterium rhizogenes carrying the RNA interference vector were generated and infected with the symbiotic arbuscular mycorrhiza fungus Glomus intraradices and the rhizobial bacterium Sinorhizobium meliloti as well as with the pathogenic oomycete Aphanomyces euteiches. MtROP9i transgenic lines showed a clear growth-reduced phenotype and revealed neither ROS generation nor MtROP9 and MtRBOH gene expression after microbial infection. Coincidently, antioxidative compounds were not induced in infected MtROP9i roots, as documented by differential proteomics (two-dimensional differential gel electrophoresis). Furthermore, MtROP9 knockdown clearly promoted mycorrhizal and A. euteiches early hyphal root colonization, while rhizobial infection was clearly impaired. Infected MtROP9i roots showed, in part, extremely swollen noninfected root hairs and reduced numbers of deformed nodules. S. meliloti nodulation factor treatments of MtROP9i led to deformed root hairs showing progressed swelling of its upper regions or even of the entire root hair and spontaneous constrictions but reduced branching effects occurring only at swollen root hairs. These results suggest a key role of Rac1 GTPase MtROP9 in ROS-mediated early infection signaling.     

Isolation and characterization of root-specific phosphate transporter promoters from Medicago trunatula

Promoters of phosphate transporter genes MtPT1 and MtPT2 of Medicago truncatula were isolated by utilizing the gene-space sequence information and by screening of a genomic library, respectively. Two reporter genes, beta-glucuronidase (GUS) and green fluorescent protein (GFP) were placed under the control of the MtPT1 and MtPT2 promoters. These chimeric transgenes were introduced into Arabidopsis thaliana and transgenic roots of M. truncatula, and expression patterns of the reporter genes were assayed in plants grown under different phosphate (Pi) concentrations. The expression of GUS and GFP was only observed in root tissues, and the levels of expression decreased with increasing concentrations of Pi. GUS activities in roots of transgenic plants decreased 10-fold when the plants were transferred from 10 microM to 2 mM Pi conditions, however, when the plants were transferred back to 10 microM Pi conditions, GUS expression reversed back to the original level. The two promoters lead to different expression patterns inside root tissues. The MtPT1 promoter leads to preferential expression in root epidermal and cortex cells, while MtPT2 promoter results in strong expression in the vascular cylinder in the center of roots. Promoter deletion analyses revealed possible sequences involved in root specificity and Pi responsiveness. The promoters are valuable tools for defined engineering of plants, particularly for root-specific expression of transgenes.     

The Commelina Yellow Mottle Virus Promoter Is a Strong Promoter in Vascular Tissue of Transgenic Gossypium hirsutum Plants

Explants of cotton (Gossypium hirsutum L. cv. Jingmian 7) were transformed with Agrobacterium tumefaciens (Smith et Townsend ) Conn LBA4404 harboring an expression cassette composed of CoYMV (Commelina Yellow Mottle Virus) promoter-gus-nos terminator on the plant expression vector pBcopd2. Transgenic plants were regenerated and selected on a medium containing kanamycin. GUS (β-glucuronidase) activity assays and Southern blot analysis confirmed that the chimerical gus gene was integrated into and expressed in the regenerated cotton plants. Plant expression vector pBI121 was also transferred into the same cotton variety and the regenerated transgenic plants were used as a positive control in GUS activity analysis. Evidences from histochemical analysis of GUS activity demonstrated that under the control of a 597 bp CoYMV promoter the gus gene was highly expressed in the vascular tissues of leaves, petioles, stems, roots, hypocotyls, bracteal leaves and most of the flower parts while GUS activity could not be detected in stigma, anther sac and developing cotton fibers of the transgenic cotton plants. GUS specific activity in various organs and tissues from transgenic cotton lines was determined and the results indicated that the CoYMV promoter-gus activities were at the same level or higher than that of CaMV 35S promoter-gus in leaf veins and roots where the vascular tissues occupy a relatively larger part of the organs, but in other organs like leaves, cotyledons and hypocotyls where the vascular tissues occupy a smaller part of the organs the CoYMV promoter-gus activity was only 1/3-1/5 of the CaMV 35S promoter-gus activity. The GUS activity ratio between veins and leaves was averaged 0.5 for 35S-GUS plants and about 2.0 for CoYMV promoter-gus transgenic plants. These results further demonstrated the vascular specific property of the promoter in transgenic cotton plants. An increasing trend of GUS activity in leaf vascular tissues of transgenic cotton plants developing from young to older was observed.     

Visualization of somatic deletions mediated by R/<Emphasis Type="Italic">RS</Emphasis> site-specific recombination and induction of germinal deletions caused by callus differentiation and regeneration in rice

A transgenic rice plant expressing the recombinase of Zygosaccharomyces rouxii under the control of the CaMV 35S promoter was crossed with a transgenic plant carrying a cryptic (beta-glucuronidase) GUS reporter gene, which was activated by recombinase-mediated deletions between two specific recombination sites ( RSs). In F(1) plants, GUS activity was observed as blue spots and stripes in vascular bundles in several parts of the leaves. GUS expression was detected in all of the calli induced from F(1) seeds and throughout the regenerated plants. DNA analysis using the polymerase chain reaction and Southern blotting showed that R/ RS-mediated deletions occurred in all of the cells of the regenerated plants. Stable GUS expression was confirmed in the progeny resulting from self-pollination. Thus, the deletions obtained in the regenerated plants were genetically equivalent to the germinal deletions. These results indicate that the induction of callus differentiation and shoot regeneration is an effective manner to activate the R/ RS system and to produce plants with chromosomal deletions.     

Development of a virus induced gene silencing vector from a legumes infecting tobamovirus

Medicago truncatula, the model plant of legumes, is well characterized, but there is only a little knowledge about it as a viral host. Viral vectors can be used for expressing foreign genes or for virus-induced gene silencing (VIGS), what is a fast and powerful tool to determine gene functions in plants. Viral vectors effective on Nicotiana benthamiana have been constructed from a number of viruses, however, only few of them were effective in other plants. A Tobamovirus, Sunnhemp mosaic virus (SHMV) systemically infects Medicago truncatula without causing severe symptoms. To set up a viral vector for Medicago truncatula, we prepared an infectious cDNA clone of SHMV. We constructed two VIGS vectors differing in the promoter element to drive foreign gene expression. The vectors were effective both in the expression and in the silencing of a transgene Green Fluorescent Protein (GFP) and in silencing of an endogenous gene Phytoene desaturase (PDS) on N. benthamiana. Still only one of the vectors was able to successfully silence the endogenous Chlorata 42 gene in M. truncatula.