【作者】 高强；

【导师】 张举仁；

【作者基本信息】 山东大学 ， 细胞生物学， 2009， 博士

【摘要】 随着现代植物分子生物学技术的发展,人们对拟南芥和水稻等模式植物进行了广泛深入的研究,对高等植物非生物胁迫应答的分子机制,特别是在植物对胁迫信号的感知和转导、胁迫相关基因表达的调控、以及渗透保护物质合成、抗氧化防御系统、脱水保护等相关关键基因的克隆与功能鉴定等诸多方面都有了较为深入的了解。拟南芥（Arabidopsis thaliana）是一种典型的甜土植物,作为耐盐耐旱机制研究的模式植物有其自身的局限性。盐芥（Thellungiella halophila）是拟南芥的近缘物种,是一种高度耐盐耐早的盐生植物,该种植物在盐胁迫的前后既不产生盐腺也没有形态上的明显变化,表明其耐盐性在很大程度上取决于该物种的基本生理及其生化机制;而且盐芥还具有基因组小,cDNA序列与拟南芥相似程度高等优点,已逐渐成为人们研究植物非生物胁迫响应的模式植物。本研究以盐芥作为实验材料,对本实验室克隆的Na⁺/Pi转运体基因的表达模式及其生物学功能进行了深入分析,并首次克隆了盐芥受盐胁迫诱导的黄素结合单氧化酶FMO基因,且对其功能及表达模式进行了研究。盐芥Na⁺/Pi转运体基因功能的研究本实验室已克隆出盐芥Na⁺/Pi转运体（T.halophila Na⁺/Pi transpoter,ThNPT）的全长cDNA,之前的研究通过生物信息学预测及进一步的GFP融合蛋白亚细胞定位实验,证明该基因编码的蛋白定位于叶绿体上,并通过在酵母中异源表达证明该基因编码一个磷转运体。本工作首先结合RT-PCR技术和定量PCR技术对该基因的表达模式进行了分析,结果表明:（1）该基因在莲座叶中表达量最高,其次在苔生叶有较高的表达,在苔、荚果和花中也有表达,在根中表达量最低,这与ThNPT定位于叶绿体的结果相对应;（2）利用400mM NaCl、100μM ABA、100μM MeJA、18%（W/V）PEG6000、4℃低温和42℃高温对盐芥进行处理,检测该基因在根和叶片中的表达变化,发现该基因在叶中受高盐和高温胁迫诱导上调表达;而在低温条件下,根中该基因的转录水平显著下调;而已有的数据表明拟南芥中的同源基因在高盐和高温处理时表达没有明显变化;（3）该基因在叶中的转录水平受光照诱导。由以上结果可以得出:ThNPT是一个主要在叶中表达,并且其表达受到光照、高温和盐胁迫的诱导的基因。本工作利用盐芥完整叶绿体为材料,研究叶绿体在不同pH、不同一价阳离子和Pi浓度条件下的Pi吸收动力学。结果表明,pH6.5是叶绿体的磷吸收的最适pH,添加25mM Na⁺可以显著刺激叶绿体的磷转运能力,并且Na⁺是最适一价阳离子,Km值为613μmol Pi g_chlorophyll^-1min^-1。构建过表达载体和RNAi载体,并通过遗传转化获得了相应的盐芥及拟南芥的转基因株系,对所得株系材料的叶绿体磷吸收能力测定发现,在盐芥中过表达ThNPT基因对盐芥的表型没有显著影响,但可以显著提高盐芥叶绿体中Pi的含量及对其的吸收速率,而通过RNAi抑制该基因的表达却对叶绿体中Pi的含量和吸收速率未能产生影响。利用激光扫描共聚焦显微镜和荧光探针技术发现,正常生长条件下ThNPT表达水平最高的过表达盐芥株系的完整叶绿体中Na⁺含量显著比未转基因对照株系高;而RNAi干扰的转基因株系完整叶绿体中Na⁺含量与未转基因对照株系没有显著差别。这些实验证据表明在盐芥中过表达ThNPT,可以提高植物叶绿体中Na⁺和Pi含量。同时我们还发现,过表达该基因还可以显著提高盐芥的光合速率。将生长8周的盐芥进行水培,在分别含有Na⁺、磷、Na⁺和磷的Hoagland营养液中培养2周,结果表明,当采用Hoagland营养液进行培养时,ThNPT基因过表达盐芥株系叶片中Na⁺含量显著比野生对照株系高;在Hoagland营养液中添加100mM NaCl培养14天后过表达株系叶片中的Pi含量显著比对照高;当采用不含KH₂PO₄的Hoagland营养液培养后,过表达株系叶片中的Na⁺比对照显著高。同时对盐芥的光合参数进行测定发现,在Hoagland营养液中,过表达ThNPT株系的净光合速率显著高于野生型对照;在Hoagland营养液中添加100mM NaCl培养后,各株系的净光合速率均有提高,但过表达ThNPT株系盐芥的净光合速率显著比对照的高;当采用不含有KH₂PO₄的Hoagland营养液培养后,各株系盐芥中的净光合速率均降低,过表达ThNPT基因的盐芥的净光合速率比对照稍高,其差异没有达到显著水平。结果表明,（1）在水培条件下,ThNPT基因可以在过表达盐芥株系叶片积累更多的Na⁺;（2）Na⁺可以促进ThNPT基因过表达盐芥株系叶片的Pi吸收能力;（3）低浓度NaCl可以促进盐芥的净光合速率。我们推测,ThNPT可能通过改变了叶绿体内的离子平衡和磷的存在形式和分布,从而影响了盐芥的光合作用。盐芥是高度耐盐耐旱的盐生植物,不易受到非生物胁迫,故在盐芥中抑制和过量表达Na⁺/Pi转运体基因不能有效探知该基因与植物抗逆的关系,而盐芥ThNPT与拟南芥中的同源基因在cDNA水平有86%的相似性,所以我们在拟南芥中过表达ThNPT基因和RNAi拟南芥中的同源基因,分析转基因拟南芥株系的抗逆能力,力争明确Na⁺/Pi转运体在植物抗逆胁迫中的作用。实验结果发现:过表达ThNPT转基因拟南芥在加有150mM和200mM NaCl的MS培养基上萌发率显著低于对照株系;在含有150 mM和200mM NaCl的MS培养基上,无论生长10天还是20天时,过表达ThNPT转基因拟南芥的生物量和根长显著比对照低,RNAi干扰的转基因株系与未转基因对照株系没有显著差别。而在MS培养基中生长10天后,过表达ThNPT转基因拟南芥的生物量和根长显著比对照高,RNAi干扰的转基因株系与未转基因对照株系没有显著差别;在无磷MS培养基生长10天后过表达ThNPT转基因拟南芥生物量和根长与未转基因对照株系没有显著差别。我们推测,由于过表达ThNPT,在盐胁迫条件下拟南芥可能在体内较快积累了较多的Na⁺,从而增加了它对NaCl胁迫的敏感性。盐芥FMO1基因的克隆和功能分析根据拟南芥的黄素结合单氧化酶（Flavin-containing monooxygenase,At1G62570）的基因序列信息,通过RT-PCR和RACE的方法获得了盐芥中同源基因的cDNA全长,该基因被命名为TsFMO1。TsFMO1的cDNA序列全长1743bp（Accession no.AY436553）,包含84bp的5′非翻译区（5’UTR）和272bp的3′非翻译区（3′UTR）以及一个1386bp的开放读码框（open reading frame,ORF）。该读码框编码461个氨基酸,含有FMO基因所特有的FAD和NADP结合位点2个保守结构域。将盐芥TsFMO1基因在酵母FMO突变体YHR176W中进行异源表达,可以降低酵母对还原剂DTT的生长速率敏感性,这表明TsFMO1是一个有功能的FMO。表达谱分析表明该基因在盐芥的莲座叶、根、苔、苔生叶、花、果荚中都有表达,TsFMO1在莲座叶中表达量最高,其次为花,根和苔,在果荚和苔生叶也具有较低的表达。利用400mM NaCl、100μM ABA、100μM MeJA、15mM H₂O₂、18%（W/V）PEG6000、4℃低温和42℃高温对盐芥进行处理,检测该基因在盐芥根和叶片组织中的表达变化。结果表明NaCl、高温、ABA和低温处理条件下TsFMO1基因在叶中的转录水平明显上调;在PEG6000处理和H₂O₂处理条件下TsFMO1基因在根中的转录水平分别上调和下调;在其它情况下呈现组成型表达。利用激光扫描共聚焦显微镜和荧光探针技术发现,非逆境生长条件下外源TsFMO1基因表达水平最高的过表达盐芥株系叶原生质体中活性氧物质含量显著比未转基因对照株系高;而RNAi干扰转基因株系叶原生质体中活性氧物质含量与未转基因对照株系没有显著差别。这说明在盐芥中过表达TsFMO1基因,可以提高植物细胞中活性氧物质含量,而由于该基因是FMO基因家族的成员,家族中其它成员可能具有相似功能,以致于专一干扰TsFMO1基因的RNAi结构的表达未能明显影响细胞中活性氧物质含量。由于盐芥TsFMO1与拟南芥中的同源基因有83%的核酸序列相似性,所以我们以过表达和RNAi干扰的转基因拟南芥为材料,分析转基因拟南芥的抗逆性和TsFMO1在植物抗逆境胁迫中的作用。分别用50,100,150,200mM NaCl处理时,在种子萌发率、小苗根长、干重和鲜重指标上转RNAi基因拟南芥与对照无显著差异,而过表达TsFMO1的转基因拟南芥在100mM NaCl处理条件下,萌发率、干重和鲜重显著比对照高。说明过表达TsFMO1可以增强拟南芥对NaCl的耐受性。而在进行Mannitol和H₂O₂处理中,过表达TsFMO1的转基因拟南芥和对照在种子萌发率、小苗根长、干重和鲜重指标上没有显著差异。本工作还克隆了另外3个TsFMO基因的片段,并设计引物进行定量RT-PCR分析,发现这3个TsFMO基因的表达模式和对胁迫的应答反应不一致,这提示FMO基因家族成员可能参与了不同的代谢活动,具有更多的功能等待研究。总之,本工作分析了盐芥Na⁺/Pi转运体基因（ThNPT）在各种组织器官和不同胁迫处理条件下的表达模式,发现该基因的表达受高盐和高温胁迫诱导;研究了盐芥叶绿体的Na⁺、Pi转运动力学特性,验证了叶绿体中存在Na⁺/Pi转运功能。通过遗传转化获得了ThNPT基因过表达和抑制表达的转基因纯合的盐芥株系,发现在盐芥中过表达ThNPT基因可以显著提高叶绿体中Na⁺含量、Pi含量和吸收速率及光合速率,通讨RNAi抑制该基因表达对叶绿体中Pi含量和吸收速率并未产生明显影响,推测是由于ThNPT属多基因基因家族成员,该家族中各成员的功能可能相互重叠。本工作有助于揭示高盐和低磷胁迫条件下ThNPT基因对细胞质和叶绿体间Na⁺和Pi渗透平衡的影响以及该基因的生物学意义。本论文还根据拟南芥FMO1的序列信息,采用同源克隆的方法得到了盐芥TsFMO1基因的全长cDNA。盐芥TsFMO1基因可以互补酵母突变体的生长缺陷,与酵母FMO具有相似的基因功能。同时研究了TsFMO1基因在各种组织器官和不同胁迫处理条件下的表达模式,发现该基因的表达受到多种胁迫处理的调控。在盐芥中过表达TsFMO1基因可以显著增加细胞中活性氧物质含量,而在拟南芥中过表达该基因,可以提高拟南芥对低浓度下NaCl的耐受性。这些工作通过对盐芥相关基因的克隆,为了解拟南芥和盐芥在耐盐机制的差异提供了重要资料。更多还原

【Abstract】 With the development of modern techniques of plant molecular biology,great and widespread studies have been carried out in the mechanisms of plant adoptions to abiotic stress,especially in signal sensation and transduction,regulations of transcript, ion homeostasis,ROS scavenging systems and compatible solutes synthesis mechanisms in model plant Arabidopsis and Rice.As a typical glycophyte, Arabidopsis can’t reveal all salt tolerance mechanisms,especially some specialized mechanisms in halophytes.Salt cress（Thellungiella halophila）,a relative species of the Arabidopsis,is a classical halophyte with high salt tolerance.T.halophila produces neither salt glands nor other complex morphological alterations either before or after salt adaptation.It appears that the salt-tolerance is more dependent on its basic biochemical and physiological mechanisms,and salt cress has desirable genetic traits: high cDNA sequence similarity to that of Arabidopsis,small genome size and easy transformation,so it becomes the prospective model plant for studying salinity tolerance.In the present study,a novel Flavin-containing monooxygenase（FMO）-like gene, designated as TsFMO1,was cloned from T.halophila,and the functions and expression models of TsFMO1 and a Na⁺/Pi transporter gene（ThNPT） cloned in our lab before were further studied.Function analysis of T.halophila Na⁺/Pi transporter gene（ThNPT）A full-length cDNA sequence encoding T.halophila Na⁺/Pi transporter gene was cloned by our lab.The deduced 510 amino acid sequence contains 10 trans-membrane domains and it was located in chloroplast by expressing N terminal GFP fusion protein in tobacco cells.Heterologous expression of truncated ThNPT cDNA in yeast mutant PAM2 can transport Pi from medium into yeast cells and Na⁺ at low concentration（500μM） could activate the Pi transport activity.In this work,expression pattern of ThNPT was analyzed by RT-PCR and Real-time RT-PCR.The results showed that:（1） the expression level of ThNPT in rosette leaves was the highest,cauline leaf also had high expression,the stems,silique and open flowers also had gene expression and the expression in the root was the lowest;（2） the gene was significantly up-regulated in the leaves when exposed to NaCl or 42℃stress and down-regulated under 4℃stress;（3） the expression in leaf was induced by light.We could conclude that ThNPT was a gene mainly expressed in leaf and induced by light,heat and salt stress.Intact chloroplasts of T.halophila were isolated to determine the Pi transport activity under different pH,and different concentrations of Na⁺ and Pi,respectively. The results showed that the optimum pH of the Pi transport activity of chloroplasts is pH6.5,25mM Na⁺ could activate the Pi transport activity most significantly.The ThNPT over-expression and RNAi vectors were constructed,and transgenic T.halophila and Arabidopsis were got,separately.The Pi transport activity and Pi content of chloroplasts in over-expression transgenic T.halophila plants were higher than those wild types（WT）,but there were no significant differences between RNAi transgenic plants and WT in Pi transport activity and Pi content of chloroplasts.The accumulation of Na⁺ in chloroplasts was measured using Sodium Green fluorescent indicator and laser scanning confocal microscopy.Higher fluorescent signals（i.e.higher Na⁺ concentration） were observed in chloroplasts of over-expression transgenic T.halophila plants.It was found that the over-expression transgenic T.halophila plants accumulated significantly higher Na⁺ than WT in chloroplasts,but there was no obvious difference on Na⁺ accumulation between the WT and RNAi transgenic plants chloroplasts.These results suggested that the over expression of ThNPT enhanced the accumilations of Na⁺ and Pi in chloroplasts.We found that the photosynthesis rates of over-expression transgenic T.halophila plants were enhanced.Eight week-old T.halophila seedlings were hydrophilic cultured for 2 weeks in normal,Pi deprived Hoagland solutions or Hoagland solutions added with 100mM NaCl,respectively.The results indicated that:In Hoagland solutions,the content of Na⁺ in leaves of the over-expression transgenic T.halophila plants was significantly higher than that of WT;in Hoagland solutions added with 100mM NaCl,the content of Pi in leaves of the over-expression transgenic T.halophila plants was significantly higher than that of WT;while in Pi deprived Hoagland solutions,the content of Na⁺ in leaves of the over-expression transgenic T.halophila plants was significantly higher than that of WT.The photosynthesis rates were also measured and the results showed that:In Hoagland solutions,the photosynthesis rates of the over-expression transgenic T.halophila plants was significantly higher than that of WT;in Hoagland solutions added with 100mM NaCl,the photosynthesis rates of all plants were enhanced and the photosynthesis rates of the over-expression transgenic T.halophila plants were significantly higher than that of WT;while in Pi deprived Hoagland solutions,the photosynthesis rates of all plants were decreased,and there was no obvious difference on the photosynthesis rates between the WT and over-expression transgenic plants.It could be concluded that:（1） the over-expression transgenic T.halophila plants can improve the photosynthesis rates and accumulate more Na⁺ in leaves than WT;（2） the content of Pi in leaves of the over-expression transgenic T.halophila plants was increased when 100 mM NaCl was added in the Hoagland solution;（3） the photosynthesis rates of T.halophila can be enhanced by low concentration of NaCl. We inferred that ThNPT might change the ion balance and Pi content of the chloroplast,which influenced the photosynthesis rate.The expression of ThNPT was significantly up-regulated in the leaves when exposed to NaCl stress.T.halophila is a classical halophyte with high salt tolerance, over-expression and RNAi ThNPT in T.halophila can not find the function of ThNPT in plant stress tolerance.Because ThNPT and its homolog from Arabidopsis have high similarity up to 86%at cDNA level,ThNPT was over expressed and its homolog from Arabidopsis was suppressed in Arabidopsis.The stress tolerance of transgenic Arabidopsis was analyzed to confer the putative function of ThNPT in plant stress tolerance.The results showed that:On the MS medium containing 150mM or 200 mM NaCl,the ThNPT over-expression transgenic Arabidopsis plants showed the lower percentage of seed germination than WT;the growth,root length and fresh weight of ThNPT over-expression transgenic Arabidopsis plants were lower than those of WT; after cultured on MS medium containing the 150mM or 200 mM NaCl,there was no obvious difference on seed germination and seedling growth between the WT and RNAi transgenic plants.The ThNPT over-expression transgenic Arabidopsis plants showed the higher root length and fresh weight than WT after 10 day on MS medium plates,while there was no obvious difference on root length and fresh weight between the WT and over-expression transgenic plants on Pi deprived MS medium plates.We inferred that the over-expression transgenic Arabidopsis plants might accumulate more Na⁺ in leaves than WT,and their sensitivity of salt stress was increased.Cloning and function analysis of T.halophila Flavin-containing monooxygenase, TsFMO1A novel Flavin-containing monooxygenase（FMO）-like gene,designated as TsFMO1,was cloned from Thellungiella halophila by reverse transcriptase PCR （RT-PCR） coupled with rapid amplification of cDNA ends（RACE） approach according to the sequence of putative Arabidopsis Flavin-containing monooxygenase （At1G62570）.This cDNA Sequence included 1743bp（Accession no.EF011646 in genbank） containing an open reading frame（ORF） of 1386 bp,a 5′untranslated region （UTR） of 84 bp and a 3′untranslated region of 272 bp.The deduced amino acid sequence was 461 amino acids in length which contained the putative FAD and NADP binding motifs.In order to determine whether TsFMO1 has similar functions as Yeast FMO, TsFMO1 was used to rescue the yeast FMO mutant YHR176W which exhibited retarded growth in the presence of DTT.The growth rate of YHR176W （pYES-TsFMO1） was similar with that of the BY4741（pYES）,which suggested that TsFMO1 could rescue the retardant growth of yeast FMO mutant to DTT.This result indicated that TsFMO1 was a functional flavin-containing monooxygenase like Yeast FMO.RT-PCR analysis indicated that the TsFMO1 was constitutively expressed in the roots and rosette leaves,stems and open flowers under normal growth conditions,the expression level of TsFMO1 in rosette leaves was the highest,moderate in flower, root and stem and the lowest in silique and cauline leaf.The expressions of the TsFMO1 under different environmental stress conditions were analyzed by quantitative real-time RT-PCR.The results showed that the transcript level of TsFMO1 in leaf was quickly and significantly（more than two folds） up-regulated by high salt stress,ABA,4℃and 42℃treatment,respectively;the transcript level of TsFMO1 in root was down-regulated under PEG and H₂O₂ treatment,respectively. The over-expression and RNAi vectors were constructed,and transgenic T.halophila and Arabidopsis were got,respectively.The accumulation of Reactive Oxygen Species（ROS） in cells was measured using H₂DCFDA fluorescent indicator and laser scanning confocal microscopy.Higher fluorescent signals（i.e.higher ROS concentration） were observed in protoplasts of over-expression transgenic T.halophila plants.It was found that the over-expression transgenic T.halophila plants accumulated significantly higher ROS than WT protoplasts,but there was no obvious difference on ROS accumulation between the WT and RNAi transgenic plants protoplasts.The results indicated that over expression of TsFMO1 enhanced the accumilations of ROS in protoplasts,and provided direct evidence for the relationship between TsFMO1 and ROS generation.As TsFMO1 and its homolog from Arabidopsis have high similarity up to 83%at cDNA level,TsFMO1 was over expressed and its homolog from Arabidopsis was suppressed in Arabidopsis.The stress tolerance of transgenic Arabidopsis was analyzed to confer the putative function of TsFMO1 in plant stress tolerance.On the MS medium containing 0,50,100,150 and 200mM NaCl,there was no significant difference on seed germination and seedling growth between the WT and RNAi transgenic plants,while the TsFMO1 over-expression transgenic Arabidopsis plants showed higher percentage of seed germination and fresh weight biomass than WT. There was no significant difference on seed germination and seedling growth between the WT and over-expression transgenic plants when exposed to Mannitol and H₂O₂ stress.Based on the results above we concluded that ThNPT was mainly expressed in leaves,and the expression was up-regulated by light,NaCl and 42℃,respectively.Pi transport activity of intact chloroplasts was analyzed and Na⁺ stimulated Pi transport activity was detected.The Pi transport activity,Pi content of chloroplasts and net photosynthesis rates in over-expression transgenic T.halophila plants were higher than that of wild types（WT）,but there were no significant differences between RNAi transgenie plants and WT in Pi transport activity,Pi content of chloroplasts and net photosynthesis rates.We inferred that ThNPT might be a multiply member gene family,and the Pi transport activity of each member might overlap.Our work may help reveal the biological role of the Na⁺ and Pi homeostasis between cytoplasm and chloroplast under high salinity and low Pi stress. A novel Flavin-containing monooxygenase（FMO）-like gene,designated as TsFMO1,was cloned from Thellungiella halophila according to the sequence of putative Arabidopsis Flavin-containing monooxygenase（At1G62570）.TsFMO1 was found to complement and rescue the retardant growth to DTT of yeast fmo mutant strain,indicating that TsFMO1 was a functional flavin-containing monooxygenase like Yeast FMO.This gene was mainly constructively expressed in many tissues and regulated by salt stress,ABA,4℃,42℃,PEG and H₂O₂.Over expression of TsFMO1 in T.halophila enhanced the accumilations of ROS in protoplasts,and over expression of TsFMO1 in Arabidopsis improved the salt tolerance under low concentration of NaCl.These works may help us understand the difference in the salt tolerance mechanisms between nonhalophytes（Arabidopsis） and halophytes（T.halophila）,and provide the valuable information for establishing the experiment designs of crop salt tolerance improvement.更多还原