Construction of Flower-specific Chimeric Promoters and Analysis of Their Activities in Transgenic Torenia

Flower-specific promoters can enable transgenic enhancement of valuable ornamental traits, including flower shape and color. However, the identification of strong, tissue-specific promoters remains a limiting factor. To obtain enhanced flower-specific promoters, we constructed four chimeric promoters (p35S-PCHS-Ω, p35S-LCHS-Ω, pOCS-PCHS-Ω and pOCS-LCHS-Ω) combining the 35S or OCS enhancer fused to a 302 bp CHSA core promoter fragment from petunia (PCHS) or a 307 bp CHS core promoter fragment from lily (LCHS), and also containing an omega element (Ω). Each promoter was fused to the β-glucuronidase (GUS) reporter gene, and we examined the levels and tissue specificity of GUS expression in transgenic Torenia fournieri. p35S-PCHS-Ω and p35S-LCHS-Ω drove strong, constitutive GUS expression in all tissues, especially in colored corollas (p35S-PCHS-Ω) or in colored corollas and roots (p35S-LCHS-Ω). pOCS-PCHS-Ω drove stronger GUS expression in colored corollas than in other tissues but expression was weaker than that of p35S-PCHS-Ω. pOCS-LCHS-Ω drove GUS in colored corollas but also in roots. Among the four chimeric promoters, pOCS-PCHS-Ω exhibited stronger activity only in colored corollas, making it useful for transgenic enhancement of floral traits, such as expressing ‘blue genes’ in lily to produce new lines with blue flowers.     

过量表达GLOase导致转基因拟南芥中维生素C含量和抗逆能力提高

L-古洛糖酸-1,4-内酯氧化酶（L-gulono-1,4-lactone oxidase,GLOase）是维生素C合成途径中最后一步关键酶,小鼠（Musmusculus）编码GLOase的gulo基因转化拟南芥（Arabidopsis thaliana）的转基因株系中维生素c含量最高为5．74μmol·g^-1（FW）,是野生型的3．46倍、转p2301空载体对照的3．19倍。30％聚乙二醇（PEG-6000）模拟干旱胁迫的不同时间梯度中,幼苗期转基因拟南芥丙二醛含量低于同样处理下野生型和对照组拟南芥。不同NaCl浓度的盐胁迫下,转基因拟南芥在子叶期比野生型、对照组平均根长更长、侧根发育更好;幼苗期莲座叶长势更好、丙二醛含量更低。结果显示过量表达GLOase的转基因拟南芥在维生素c含量提高的同时,抗胁迫能力有所增强。     

拟南芥冷诱导型启动子CBF 3的克隆及活性检测

目的：构建冷诱导型启动子CBF3基因的植物表达载体,并将其转入烟草。方法：以拟南芥基因组DNA为模板,通过特异PCR扩增,克隆冷诱导表达启动子CBF3（C-repeat binding factor）。用CBF3启动子替换pBI121载体上的35S启动子构建新的载体pBC-GUS,通过农杆菌介导的叶盘法转化烟草。结果：获得了转基因烟草,转基因烟草的GUS组织化学染色及PCR分析结果表明,在低温诱导下,CBF3启动子可增强GUS基因表达。结论：CBF3启动子可应用于植物抗冷基因工程研究。     

人工合成的植物化猪α乳清蛋白基因在转基因拟兰芥的表达

将一个398bp的植物化的猪α乳清蛋白基因(Lactalbumin,LA)编码区克隆到带有花椰菜花叶病毒的35S启动子的质粒中。对它们进行PCR检测和序列分析,证实这些阳性克隆是实验预期的重组质粒。随即将35S启动子-α-乳清蛋白基因-终止子这一表达单元克隆到双元载体pCG-CB中,用该重组质粒双元载体pCG-CB-Lact转化农杆菌V3101后,以农杆菌法进行拟兰芥植物转化,用除草剂Finale对转化植物进行抗除草剂基因筛选,得到一些抗除草剂的转化植株。对这些抗除草剂植株进行猪α乳清蛋白基因PCR分析,成功筛选到带有猪α乳清蛋白基因并且可以在后代稳定遗传的转基因植物。Western blot蛋白质表达分析,表明猪α乳清蛋白在拟兰芥中成功表达,并且猪仅乳清蛋白被正确加工成天然蛋白。     

拟南芥中光信号系统中关键基因CRY1、CRY2和COP1启动子的表达模式分析

通过构建表达光信号系统关键基因CRY1、CRY2和COP1启动子与GUS融合基因的拟南芥转基因植株,并对转基因植株进行GUS组织化学染色的结果表明,CRY1、CRY2和COP1的表达模式不受光条件的调控,并且在各器官有广泛的表达。分别分析CRY1基因启动子在cop1突变体以及COP1基因启动子在cry1突变体遗传背景中表达模式的结果表明,CRY1和COP1在转录水平上不存在明显的相互调控关系。     

人工合成启动子SAR在拟南芥中的表达分析

利用植物防御基因中的病原诱导响应元件和最小35S启动子(-62～+1),人工合成了启动子SAP,并以GUS基因为报告基因,在转基因拟南芥中分析了合成启动子的表达特性.通过对转基因拟南芥GUS组织染色的分析表明:SAR启动子在子叶、毛刺、根茎交接处和根系中优势表达,在老叶中的表达量高于幼叶,说明SAR启动子具有组织和发育表达特异性.     

转基因PnDof30拟南芥非生物胁迫下的抗性分析

Dof(DNA-binding with one finger)转录因子是植物中特有的一类转录因子,是锌指蛋白家族中的一个具有众多成员的家族,氨基酸长度一般在200~400,含有非常保守的N端和较为多变的C端。已有研究表明,Dof转录因子家族在参与植物发育的多种生理途径和调节碳氮代谢、增加氮素的吸收与利用,提高植株抗逆能力中起着重要作用。为了探究小黑杨(Populus simonii×P.nigra)中Dof30基因的抗逆能力,本研究以转基因PnDof30拟南芥为研究对象,对干旱、盐和渗透胁迫后过表达PnDof30拟南芥株系L2和野生型拟南芥WT的生理指标进行比较。发现胁迫后拟南芥株系L2的种子萌发率、根长和鲜重等指标均高于WT;同时SOD、POD、脯氨酸含量高于WT,叶绿素和MDA含量下降;胁迫后L2中的PnDof30基因表达量显著提高。这些结果表明了PnDof30基因具有抗旱、耐盐和渗透胁迫的能力,对全面了解Dof转录因子的抗逆胁迫功能具有重要的意义。     

AhAO2转基因拟南芥植株抗旱生理分析

目的:研究AhAO2基因对拟南芥抗旱生理的影响.方法:以转AhAO2基因拟南芥(AO2 -1 -4、AO2 -3 -7、AO2 -8 -1)为实验材料,通过植物根长、相对含水量、气孔开度和抗氧化酶比活力指标的综合评定,分析超表达AhAO2基因对拟南芥抗旱生理的影响.结果:在PEG胁迫下,AO2 -1 -4、AO2 -3 -7、AO2 -8 -1植株体内相对含水量分别比野生型高2.1％、1.3％和1.6％;超表达植株AO2 -1 -4、AO2 -3 -7、AO2 -8 -1抗氧化酶POD酶比活力较野生型提高73.1％、66.2％和74.4％,SOD酶比活力较野生型提高64.6％、80.5％和43.1％.而野生型的气孔开度在正常生长和PEG胁迫的条件下均低于超表达拟南芥株系.AhAO2转基因植株可能通过提高抗氧化酶活性提升了抗旱能力.     

Simultaneous Over-Expression of PaSOD and RaAPX in Transgenic Arabidopsis thaliana Confers Cold Stress Tolerance through Increase in Vascular Lignifications

Antioxidant enzymes play a significant role in eliminating toxic levels of reactive oxygen species (ROS), generated during stress from living cells. In the present study, two different antioxidant enzymes namely copper-zinc superoxide dismutase derived from Potentilla astrisanguinea (PaSOD) and ascorbate peroxidase (RaAPX) from Rheum austral both of which are high altitude cold niche area plants of Himalaya were cloned and simultaneously over-expressed in Arabidopsis thaliana to alleviate cold stress. It was found that the transgenic plants over-expressing both the genes were more tolerant to cold stress than either of the single gene expressing transgenic plants during growth and development. In both single (PaSOD, RaAPX) and double (PaSOD + RaAPX) transgenic plants higher levels of total antioxidant enzyme activities, chlorophyll content, total soluble sugars, proline content and lower levels of ROS, ion leakage were recorded when compared to the WT during cold stress (4°C), besides increase in yield. In the present study, Confocal and SEM analysis in conjunction with qPCR data on the expression pattern of lignin biosynthetic pathway genes revealed that the cold stress tolerance of the transgenic plants might be because of the peroxide induced up-regulation of lignin by antioxidant genes mediated triggering.     

Analysis of Dihydrodipicolinate Synthase Promoter Activity in Arabidopsis thaliana by In-Planta Transformation

The present study was carried out to analyze the dihydrodipicolinate synthase (dhdps) gene promoter activity by tracing the GUS expression in tissues and in organs of Arabidopsis thaliana by in planta transformation. The Agrobacterium construct pBI101 used in the studies consists of the reporter gene gus under the control of Arabidopsis thaliana dhdps promoter with 3’ nos controlling sequences and nptll gene under the control of nos promoter and nos terminator. GUS expression in transformed Arabidopsis thaliana was found to be cell type-specific and expressed mainly in the fast growing tissues, where the protein synthesis is high. The histochemical analysis results indicate that the GUS expression was mainly observed in root meristem (elongation zone), emerging lateral roots and in the leaf vascular tissues. In reproductive organs, the GUS expression was observed in anthers, pollen grains and young immature embryos. Southern blot analysis results of T₂, progeny showed the presence of a single integration locus for both the nptll and dhdps promoter.The segregation analysis results showed that the kanamycin resistance gene has not followed the normal Mendelian inheritance.This might be due to the methylation of the nptll gene in some of the transformants.     

Aluminum Induces Oxidative Stress Genes in Arabidopsis thaliana

Changes in gene expression induced by toxic levels of Al were characterized to investigate the nature of Al stress. A cDNA library was constructed from Arabidopsis thaliana seedlings treated with Al for 2 h. We identified five cDNA clones that showed a transient induction of their mRNA levels, four cDNA clones that showed a longer induction period, and two down-regulated genes. Expression of the four long-term-induced genes remained at elevated levels for at least 48 h. The genes encoded peroxidase, glutathione-S-transferase, blue copper-binding protein, and a protein homologous to the reticuline:oxygen oxidoreductase enzyme. Three of these genes are known to be induced by oxidative stresses and the fourth is induced by pathogen treatment. Another oxidative stress gene, superoxide dismutase, and a gene for Bowman-Birk protease inhibitor were also induced by Al in A. thaliana. These results suggested that Al treatment of Arabidopsis induces oxidative stress. In confirmation of this hypothesis, three of four genes induced by Al stress in A. thaliana were also shown to be induced by ozone. Our results demonstrate that oxidative stress is an important component of the plant's reaction to toxic levels of Al.     

报春PFsHSP17-1基因转化拟南芥及耐热鉴定

根据灰岩皱叶报春PF sHSP 17-1热激蛋白基因序列,采用PCR法从差减文库中克隆得到该基因,并构建pCAMBIA1301-PF sHSP 17-1植物表达载体,通过根癌农杆菌介导的花器浸染法转化拟南芥,经潮霉素筛选、PCR和RT-PCR法鉴定后,对转基因植株和野生型对照进行高温胁迫,分析其耐热性差异.结果显示,在40℃、42℃、44℃处理下,转基因拟南芥的相对电导率和丙二醛含量升高的幅度均显著低于野生型对照,而脯氨酸含量的积累明显高于对照,可溶性蛋白的含量在热胁迫下也比对照稳定,仅有轻微下降.试验证明,PF sHSP 17-1基因对植物的耐热性有重要作用.     

拟南芥受Cd2+诱导表达基因的筛选及其在Cd2+胁迫下的功能

镉是一种毒性很大的重金属。土壤溶液中即使存在极低浓度Cd2+也能对植物造成伤害。早在植物做出结构和代谢的调整以适应逆境之前,由于Cd2+的刺激,植物的基因表达已经发生了变化。这里我们采用一种新的基于引物退火控制技术的差异显示方法来筛选受镉离子诱导表达的基因。获得的19条差异条带代表着18个基因。经过RT-PCR方法验证,其中6个基因确实是受Cd2+诱导表达,包括LEA(胚胎发育晚期丰富蛋白), AtGSTF2(谷胱甘肽-S-转移酶2), AtGSTF6(谷胱甘肽-S-转移酶6), HSP70(热激蛋白70), sHSP17.6B-CI(17.6 kDa 类型 I小分子热激蛋白)和sHSP17.6-CII(17.6 kDa类型II 小分子热激蛋白)。 这些结果有助于研究植物对镉离子胁迫的解毒机制。其中的三个热激蛋白基因的启动子也能考虑用于植物修复。     

拟南芥信号肽AtRALF1的表达、纯化及活性分析

利用RT-PCR扩增获得拟南芥At RALF1基因的全长cDNA序列,将其构建入携带His标签的原核表达载体p ET28b中,获得重组表达载体p ET28b-At RALF1,并将其转入到大肠杆菌BL21(DE3)中。随后在不同的IPTG浓度、温度和诱导时间条件下,进行At RALF1蛋白的表达研究,建立了At RALF1融合蛋白的高效表达体系。结果表明,在30℃、1 mmol/L IPTG的条件下诱导4 h,At RALF1融合蛋白的表达量最大。进一步用获得的At RALF1融合蛋白处理苗龄5 d的野生型拟南芥(Col-0)植株,发现其根的生长受到了抑制,表明我们获得了具有活性的At RALF1小肽,为进一步研究该小肽奠定了基础。     

Transgenic Nicotiana tabacum and Arabidopsis thaliana plants overexpressing allene oxide synthase

 Allene oxide synthase (AOS), encoded by a single gene in Arabidopsis thaliana (L.) Heynh., catalyzes the first step specific to the octadecanoid pathway. Enzyme activity is very low in control plants, but is upregulated by wounding, octadecanoids, ethylene, salicylate and coronatine (D. Laudert and E.W. Weiler, 1998, Plant J 15: 675–684). In order to study the consequences of constitutive expression of AOS on the level of jasmonates, a complete cDNA encoding the enzyme from A. thaliana was constitutively expressed in both  A. thaliana and tobacco (Nicotiana tabacum L.). Overexpression of AOS did not alter the basal level of jasmonic acid; thus, output of the jasmonate pathway in the unchallenged plant appears to be strictly limited by substrate availability. In wounded plants overexpressing AOS, peak jasmonate levels were 2- to 3-fold higher compared to untransformed plants. More importantly, the transgenic plants reached the maximum jasmonate levels significantly earlier than wounded untransformed control plants. These findings suggest that overexpression of AOS might be a way of controlling defense dynamics in higher plants. Received: 10 February 2000 / Accepted: 11 March 2000     

Activity and crystal structure of Arabidopsis thaliana UDP-N-acetylglucosamine acyltransferase

The UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase, encoded by lpxA, catalyzes the first step of lipid A biosynthesis in Gram-negative bacteria, the (R)-3-hydroxyacyl-ACP-dependent acylation of the 3-OH group of UDP-GlcNAc. Recently, we demonstrated that the Arabidopsis thaliana orthologs of six enzymes of the bacterial lipid A pathway produce lipid A precursors with structures similar to those of Escherichia coli lipid A precursors [Li, C., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 11387-11392]. To build upon this finding, we have cloned, purified, and determined the crystal structure of the A. thaliana LpxA ortholog (AtLpxA) to 2.1 ? resolution. The overall structure of AtLpxA is very similar to that of E. coli LpxA (EcLpxA) with an α-helical-rich C-terminus and characteristic N-terminal left-handed parallel β-helix (LβH). All key catalytic and chain length-determining residues of EcLpxA are conserved in AtLpxA; however, AtLpxA has an additional coil and loop added to the LβH not seen in EcLpxA. Consistent with the similarities between the two structures, purified AtLpxA catalyzes the same reaction as EcLpxA. In addition, A. thaliana lpxA complements an E. coli mutant lacking the chromosomal lpxA and promotes the synthesis of lipid A in vivo similar to the lipid A produced in the presence of E. coli lpxA. This work shows that AtLpxA is a functional UDP-GlcNAc acyltransferase that is able to catalyze the same reaction as EcLpxA and supports the hypothesis that lipid A molecules are biosynthesized in Arabidopsis and other plants.     

CfGST转基因拟南芥抗寒性和叶肉细胞超微结构研究

CfGSr基因克隆于加拿大森林害虫云杉蚜虫的幼虫基因组。研究CfGST转基因拟南芥表型特征,以及低温条件下叶肉细胞超微结构与存活率。结果表明,与野生型拟南芥相比,CfGSr转基因拟南芥的茎粗、叶宽,植株高度、分枝数、荚果数皆降低,生长速度减慢。低温（5±1）℃处理后,转基因拟南芥叶片的叶绿体和线粒体膜结构完整清晰,存活率提高25．14％。