大豆诱导型启动子驱动类受体蛋白激酶GmSARK转基因植物分析

植物LRR型类受体蛋白激酶在植物生命活动中发挥着重要作用。前期研究发现, 大豆(Glycine max)LRR型类受体蛋白激酶基因GmSARK可能参与调控大豆叶片的衰老过程。利用CaMV 35S启动子驱动组成型过表达GmSARK基因可导致转基因植株出现致死表型, 据此构建了可诱导型启动子GVG驱动GmSARK基因过表达的双元表达载体, 转化野生型拟南芥(Arabidopsis thaliana)并获得了多株转基因植株。研究结果表明, 外源施加诱导物地塞米松可引起GmSARK基因在转基因植株中过表达, 并导致转基因植株出现叶片变黄下卷和生长受抑制等表型; 外源细胞分裂素处理可以抑制GmSARK的表达, 但是不能逆转GmSARK过表达所引起的上述变化。     

胁迫诱导型启动子在植物抗逆基因工程中的应用

在抗逆基因工程中,大多采用的是组成型表达启动子,组成型表达启动子驱动外源抗逆基因表达虽然可以提高转基因植物的抗逆性,但会导致转基因植株生长迟缓或不育;而胁迫诱导型启动子则可提高转基因植物的抗逆性,不影响其正常生长发育,所以,胁迫诱导型启动子已逐渐用于植物抗逆基因工程。本文介绍不同胁迫诱导型启动子在植物抗逆基因工程中的应用。     

异源过表达水稻OsSAPP3基因促进拟南芥叶片衰老

蛋白磷酸酶催化的蛋白质可逆磷酸化反应是叶片衰老的关键环节。该研究筛选并克隆了1个新的参与水稻(Oryza sativa)叶片衰老调控的PP2C基因OsSAPP3。研究表明, OsSAPP3的启动子在Pro_OsSAPP3-GUS转基因拟南芥(Arabidopsis thaliana)的莲座叶中有活性, 并且活性以依赖叶龄方式增加。利用CaMV 35S启动子驱动组成型异源过表达OsSAPP3导致转基因拟南芥无法正常生长。用可诱导型启动子GVG系统驱动OsSAPP3异源过表达导致转基因拟南芥出现莲座叶变小、数量增加、叶片早衰及抽薹开花提前等早衰表型。外源诱导OsSAPP3基因异源过表达后, 利用实时荧光定量PCR检测到SAG12、WRKY6和NAC2等衰老标志基因显著上调表达。研究结果表明, OsSAPP3是参与水稻叶片衰老的正向调控因子。     

大豆生长素响应因子GmARF16参与调节叶片衰老进程

生长素响应因子(auxin response factors,ARFs)通过调节下游靶基因广泛参与植物生长发育过程,但ARFs如何调控植物叶片衰老的分子机制还不清楚。该文首先利用实时荧光定量PCR(q PCR)技术,分析大豆生长素响应基因Gm ARF16在叶片自然衰老、人工黑暗诱导衰老、外源植物生长素IAA处理条件下的表达模式,结果表明,该基因与叶片衰老调控密切相关,并且属于生长素的原初响应基因。为了进一步验证Gm ARF16基因的功能,采用农杆菌转化方法分别获得基因敲减(Gm ARF16-RNAi)和抗降解表达(m Gm ARF16)的转基因大豆植株。与非转基因对照相比,Gm ARF16-RNAi转基因大豆植株的叶片叶绿素含量和最大光量子效率(Fv/Fm)显著提高,叶片衰老标记基因(Gm CYSP1)的表达受到抑制,而m Gm ARF16转基因大豆植株则呈现出与Gm ARF16-RNAi转基因大豆植株相反的叶片生理表型。结果表明大豆生长素响应因子Gm ARF16正调节叶片的衰老进程。该研究表明,Gm ARF16在植物生长发育进程中发挥着重要作用。     

高表达水稻WRKY72基因影响拟南芥生长素信号传导

植物转录调控因子WRKY基因家族是一个拥有众多成员的超家族,功能涵盖了植物生长发育的控制与抗病耐逆的调节。我们主要分析了OsWRKY72基因在外源植物拟南芥中的生物学功能。通过转基因拟南芥(Arabidopsis thaliana)的遗传学研究发现外源高表达该基因不单明显地抑制转基因植株的顶端优势,增强植株侧枝的生长,还改变了转基因植株叶片和角果的发育。进一步分析证实,高表达OsWRKY72基因所导致转基因拟南芥植株的表型和其它生理现象都与生长素信号通路改变所导致的表型和生理变化极其相近。这些结果说明OsWRKY72基因在外源植物拟南芥体内高表达后很可能改变了其正常的生长素信号通路。     

转铁蛋白基因烟草中内源铁蛋白基因的差异表达及含铁量的变化

铁蛋白是一种由24个亚基组成的高分子贮藏蛋白质,可以储存多达4500个铁原子,在动植物及微生物的新陈代谢中起着非常重要的作用。有研究表明,外源铁蛋白的大量表达可以提高植物储存铁离子的能力。为了明确外源铁蛋白基因转化植物中内源铁蛋白基因差异表达与植物含铁量的关系,本研究在成功获得2个烟草铁蛋白基因的全长cDNA克隆NtFerl（登录号：ay083924）和NtFer2（登录号：ay141105）的基础上,以烟草品种SR-1（Nicotiana tabacum cv．Petit Havana SR-1）为受体,培育了转铁蛋白基因烟草。将双元载体pBI121中的GUS基因用来自大豆的铁蛋白基因SoyFer1（登录号：m64337）置换,利用农杆菌介导法转化烟草叶盘,获得在CaMV35S启动子驱动表达的大豆铁蛋白基因转化烟草植株。Northern杂交和Western杂交分析表明外源铁蛋白基因在转基因烟草中得到了正确表达。比较转基因烟草和非转基因烟草的内源铁蛋白基因表达强度、叶片铁含量、根系铁还原酶活性、株高和鲜重表明,外源铁蛋白基因不但促进了NtFer1的表达,提高转基因植株的储存铁的能力和根系铁还原酶活性,而且促进植株的生长速度。以上结果说明,外源铁蛋白基因转化烟草中内源铁蛋白基因的表达、铁离子的还原吸收及光和作用都得到了进一步的提高。     

植物逆境相关启动子及功能

启动子是调控基因表达的重要顺式元件, 在植物基因表达调控过程中起着重要作用。目前植物抗逆基因工程中, 人们大多使用组成型表达启动子驱动目的基因的表达。组成型表达启动子虽然能提高转基因植株的抗逆性, 但是其持续过量地表达转化的外源基因会阻碍植物的生长且减少其产量。因此, 只在胁迫条件下才会驱动外源基因表达的诱导型启动子的研究显得尤其重要, 已成为目前研究的热点。文章综述了受非生物逆境和生物逆境胁迫诱导的植物基因启动子的种类和功能, 并展望了植物逆境诱导启动子的研究方向和前景。     

大豆类受体蛋白激酶基因(rlpk2)RNAi双元表达载体的构建及其转基因

植物类受体蛋白激酶(plant receptor-like kinases RLKs)以其特有的结构在植物的生长、发育和防御等多种生理生化过程中发挥着重要的作用。利用RNA干扰技术(RNA interference RNAi)来研究RLKs的功能已日趋成熟。本文根据植物中hpRNA(hairpin RNA)的原理,以大豆类受体蛋白激酶基因rlpk2为靶基因,在rlpk2-cDNA序列3'端选择312bp作为构建RNAi的序列,借助中间克隆载体,经过三次亚克隆,最后形成含rlpk2-RNAi表达盒的双元表达载体pART27-R2,并转入农杆菌LBA4404。采用农杆菌介导大豆子叶节转化方法,共获得了三株转基因植株。转基因植株 RT-PCR分析表明rlpk2基因已被成功敲减(knock-down),并且发现敲减大豆叶片中的rlpk2基因表达明显改善大豆叶片的光合能力,结合前期研究结果,表明rlpk2基因可能在维持叶绿体的结构及保护叶绿体膜系统的完整性方面起负调节作用。     

内源茉莉酸甲酯的积累改变了大豆叶片与根的发育

茉莉酸甲酯是一种调节植物形态发生、诱导防御相关基因的植物信号转导分子。为了解内源茉莉酸甲酯在植物发育中的作用,将编码茉莉酸甲基转移酶的NTR1基因与CaMV 35S启动子连接并导入大豆植株。PCR及Northern杂交结果表明,NTR1基因稳定整合在大豆基因组并得到过量表达。与野生型植株相比,转基因大豆叶片与根的形态发生了显著的变化。大部分转基因大豆叶片变得细长,初生根生长受到抑制而侧根的生长却受到促进。定量分析结果表明,转基因大豆植株叶片中茉莉酸甲酯的含量比对照高出 2～2.5 倍。这些结果表明,内源茉莉酸甲酯的积累参与了大豆形态发生的调控。     

外源基因在烟草绿色组织中的高效表达研究

研究外源基因在受体绿色组织中的特异表达情况,分别构建由棉花PsbP启动子驱动的GUS基因及CP4 epsps 基因的植物表达载体pBI121-P、pBI121-PE.农杆菌介导法转化到烟草中,获得20株转pBI121栽体、40株转pBI121-P栽体和32株转pBI121-PE载体的烟草阳性植株.组织化学染色分析表明,GhPsbP启动子驱动的GUS基因只在转基因烟草的叶片和茎中表达,在根中不表达;Real-time PCR分析表明,PsbP启动子驱动CP4 epsps基因在叶和茎中的表达量分别是根中的15倍和10倍;草甘膦抗性试验证明,PsbP启动子驱动的CP4 epsps基因在烟草叶及茎的绿色组织中的表达量足以忍受1%浓度草甘膦的毒害.证明GhPsbp启动子可以有效地驱动外源基因在烟草的绿色组织中高效特异的表达.     

Ectopic expression of MgSM1, a Cerato-platanin family protein from Magnaporthe grisea, confers broad-spectrum disease resistance in Arabidopsis

Proteins belonging to the newly identified Cerato-platanin (CP) family have been shown to have elicitor activity in inducing disease resistance responses in various plants. In this study, we characterized a gene, MgSM1 , from Magnaporthe grisea , encoding a putative small protein belonging to the CP family. MgSM1 was constitutively expressed not only in different fungal growth stages but also during its infection process in rice plants. Agrobacterium-mediated transient expression of MgSM1 in Arabidopsis resulted in hypersensitive response in the infiltrated local leaves and enhanced disease resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato ( Pst ) DC3000 in upper leaves of plants, accompanyed by up-regulated expression of defense genes ( PR-1 , PR-5 and PDF1.2 ). Transgenic Arabidopsis plants expressing MgSM1 under control of a dexamethasone (DEX)-inducible promoter were generated. Expression of MgSM1 in transgenic plants was induced by exogenous application of DEX. MgSM1- expressing plants showed normal growth with application of <10 μ m DEX. After DEX induction, the MgSM1 -expressing plants showed enhanced disease resistance against B. cinerea , Alternaria brassicicola and Psto DC3000 as well as up-regulated expression of some of defense genes. Moreover, accumulation of reactive oxygen species was observed in MgSM1 -expressing plants. These results collectively suggest that ectopic expression of MgSM1 in transgenic plants confers broad-spectrum resistance against different types of pathogens. Our study also provides a novel strategy to generate environment-friendly crops with enhanced broad-spectrum resistance through ectopic expression of microbe-derived disease resistance-inducing proteins.     

Overexpression of a 9-cis-epoxycarotenoid dioxygenase gene in Nicotiana plumbaginifolia increases abscisic acid and phaseic acid levels and enhances drought tolerance

The plant hormone abscisic acid (ABA) plays important roles in seed maturation and dormancy and in adaptation to a variety of environmental stresses. An effort to engineer plants with elevated ABA levels and subsequent stress tolerance is focused on the genetic manipulation of the cleavage reaction. It has been shown in bean (Phaseolus vulgaris) that the gene encoding the cleavage enzyme (PvNCED1) is up-regulated by water stress, preceding accumulation of ABA. Transgenic wild tobacco (Nicotiana plumbaginifolia Viv.) plants were produced that overexpress the PvNCED1 gene either constitutively or in an inducible manner. The constitutive expression of PvNCED1 resulted in an increase in ABA and its catabolite, phaseic acid (PA). When the PvNCED1 gene was driven by the dexamethasone (DEX)-inducible promoter, a transient induction of PvNCED1 message and accumulation of ABA and PA were observed in different lines after application of DEX. Accumulation of ABA started to level off after 6 h, whereas the PA level continued to increase. In the presence of DEX, seeds from homozygous transgenic line TN1 showed a 4-d delay in germination. After spraying with DEX, the detached leaves from line TN1 had a drastic decrease in their water loss relative to control leaves. These plants also showed a marked increase in their tolerance to drought stress. These results indicate that it is possible to manipulate ABA levels in plants by overexpressing the key regulatory gene in ABA biosynthesis and that stress tolerance can be improved by increasing ABA levels.     

An efficient Agrobacterium-mediated transient transformation of Arabidopsis

Agrobacterium tumefaciens-mediated transient transformation has been a useful procedure for characterization of proteins and their functions in plants, including analysis of protein-protein interactions. Agrobacterium-mediated transient transformation of Nicotiana benthamiana by leaf infiltration has been widely used due to its ease and high efficiency. However, in Arabidopsis this procedure has been challenging. Previous studies suggested that this difficulty was caused by plant immune responses triggered by perception of Agrobacterium. Here, we report a simple and robust method for Agrobacterium-mediated transient transformation in Arabidopsis. AvrPto is an effector protein from the bacterial plant pathogen Pseudomonas syringae that suppresses plant immunity by interfering with plant immune receptors. We used transgenic Arabidopsis plants that conditionally express AvrPto under the control of a dexamethasone (DEX)-inducible promoter. When the transgenic plants were pretreated with DEX prior to infection with Agrobacterium carrying a β-glucuronidase (GUS, uidA) gene with an artificial intron and driven by the CaMV 35S promoter, transient GUS expression was dramatically enhanced compared to that in mock-pretreated plants. This transient expression system was successfully applied to analysis of the subcellular localization of a cyan fluorescent protein (CFP) fusion and a protein-protein interaction in Arabidopsis. Our findings enable efficient use of Agrobacterium-mediated transient transformation in Arabidopsis thaliana.