过表达海洋微生物宏基因组MbCSP提高转基因拟南芥的抗旱和耐寒性

干旱和低温是影响农作物生长发育的重要因素,培育转基因作物是解决此问题的有效途径。冷激蛋白（Cold Shock Proteins,CSPs）是一类高度保守的核酸结合蛋白,参与非生物胁迫应答等细胞生理活动,转CSP基因可增强作物抗逆能力。以海洋微生物宏基因组DNA为模板,采用锚定PCR的方法克隆得到了MbCSP基因,其ORF为216 bp,编码一个由71个氨基酸构成的蛋白;对其进行同源性分析,显示该氨基酸序列与EcCSPG、EcCSPA（大肠杆菌 Escherichia coli）,BsCspB（枯草芽孢杆菌Bacillus subtilis）和BcCspA（蜡样芽孢杆菌 Bacillus cereus）等冷休克蛋白氨基酸序列同源性在60%~90%;对该氨基酸序列进行多重序列比对和系统发育树分析,结果发现MbCSP蛋白包含RNP1（KGFGFI）和RNP2（VFVHF）等CSP蛋白经典的保守结构域,其与EcCspG（大肠杆菌）和CmCspG、CmCspB（堆肥宏基因组）等生物的冷休克蛋白亲缘关系较近。为进一步探讨冷休克蛋白MbCSP的功能,构建了植物表达载体pTF101-MbCSP,采用花序浸染法转化拟南芥,经过除草剂筛选和PCR检测,获得转基因植株。进行半定量RT-PCR分析,选择表达量最高的阳性株系进行后续的生理检测。结果表明：在干旱胁迫及低温胁迫下,转基因拟南芥的生长状况明显优于野生型,其生物量显著高于野生型植株;转基因拟南芥的叶片相对含水量、叶绿素含量和超氧化物歧化酶活性均高于野生型拟南芥,而丙二醛含量则低于野生型拟南芥。上述结果表明,过表达海洋微生物宏基因组MbCSP能够提高转基因拟南芥的抗旱和耐寒能力,为培育转基因作物新品种奠定了基础。     

光敏启动子控制的酵母Hem1基因在烟草中表达

拟南芥hemA1基因启动子是一种光响应型启动子,它控制着植物体内5-氨基乙酰丙酸(ALA)昼夜节律型合成.将该启动子与酿酒酵母Hem1基因构建的二价载体转入烟草中,获得转基因植株.以转二价基因的烟草T0代种子为材料,用不同浓度卡那霉素(Km)溶液浸种,结果显示,种子发芽率和子叶绿化率随着Km浓度升高而降低.将1 000 mg·L-1Km溶液中长出的162株抗性植株移植至盆钵中培养,GUS检测出的阳性比率为92%.用特异引物PCR法检测Km抗性-GUS阳性植株,发现含有拟南芥HemA1基因启动子的比率为92.5%,含有酿酒酵母Hem1基因的比率为88%,含有二价重组基因的比率为84.2%.RT-PCR检测表明,Hem1基因在黑暗中的表达量明显低于光照下,证明光敏启动子有效地控制了结构基因Hem1的表达.生理指标分析表明,与野生型相比,转基因植株ALA合成速率、叶绿素含量明显增加,叶绿素b/a比值提高.野生型植株基部叶片SPAD值显著降低,而转基因植株基部叶片SPAD值保持较高水平.以上结果说明,外源二价基因已经成功整合到烟草基因组中,并且能够在光照条件下过量表达,合成过量ALA.     

转拟南芥P5CS1基因增强羽衣甘蓝的耐旱性

为提高羽衣甘蓝的耐旱性,本文将拟南芥Δ1-吡咯啉-5-羧酸合成酶（P5CS1）基因经农杆菌介导转入羽衣甘蓝植株中,检测转基因株系与野生型植株在干旱胁迫下P5CS1 mRNA表达量、幼苗脯氨酸含量、株系根系性状、整株干重、鲜重和整株存活率。结果表明,在15%PEG6000渗透胁迫下,转基因植株的P5CS1基因mRNA表达量明显增加,转基因植株脯氨酸含量是野生型的2.4倍;主根长、最长侧根长、侧根数目、整株干重和鲜重均高于野生型,干重/鲜重则低于野生型,转基因植株的平均存活率为78%,极显著高于野生型。数据显示,AtP5CS1基因在羽衣甘蓝中的表达明显改善了转基因植株的耐旱性。     

超表达AVP1基因提高甜菜的耐低磷和耐盐抗旱性

为探讨H⁺-焦磷酸酶编码基因对甜菜磷吸收和抗性的影响,实现优良基因在甜菜基因工程中的利用,研究在甜菜中超表达拟南芥液泡膜H⁺-焦磷酸酶编码基因AVP1,对转基因甜菜分析其耐低磷、耐盐性和抗旱性。结果显示,AVP1基因在甜菜植株的叶片和块根中表达,且在逆境胁迫下增强表达量响应胁迫;低磷处理条件下,转基因甜菜与野生型甜菜相比具有更高的含磷量,可提高甜菜对磷的吸收利用效率;干旱、盐胁迫处理条件下,AVP1基因在转基因甜菜中显著上升,在盐胁迫或干旱处理条件下,转基因植株的生长受抑程度相对较轻。随着盐和干旱胁迫的加剧,转基因植株体内MDA含量与野生型植株相比较低而脯氨酸含量显著增加,AVP1基因可通过减轻逆境对甜菜细胞膜的损伤及提高甜菜细胞的渗透调节能力,进而增强甜菜对高盐和干旱胁迫的抗性。     

Transgenic tobacco plants expressingBoRS1 gene fromBrassica oleracea var.acephala show enhanced tolerance to water stress

Water stress is by far the leading environmental stress limiting crop yields worldwide. Genetic engineering techniques hold great promise for developing crop cultivars with high tolerance to water stress. In this study, theBrassica oleracea var.acephala BoRS1 gene was transferred into tobacco throughAgrobacterium- mediated leaf disc transformation. The transgenic status and transgene expression of the transgenic plants was confirmed by polymerase chain reaction (PCR) analysis, Southern hybridization and semi-quantitative one step RT-PCR analysis respectively. Subsequently, the growth status under water stress, and physiological responses to water stress of transgenic tobacco were studied. The results showed that the transgenic plants exhibited better growth status under water stress condition compared to the untransformed control plants. In physiological assessment of water tolerance, transgenic plants showed more dry matter accumulation and maintained significantly higher levels of leaf chlorophyll content along with increasing levels of water stress than the untransformed control plants. This study shows thatBoRS1 is a candidate gene in the engineering of crops for enhanced water stress tolerance.     

pFGC5941的改造及拟南芥NAC1和SIP1双基因反义表达载体的构建和遗传转化

拟南芥中的SIP1基因编码的蛋白与拟南芥盐胁迫应答中的关键蛋白SOS2存在互作关系,而NAC1为拟南芥中介导生长素信号促进其侧根发生的蛋白。本研究中我们将SIP1基因和NAC1正义基因以及SIP1基因和NAC1反义基因分别整合到一个经改造的具有2个35S启动子的可用于双基因表达的载体pFGC5941S中,构建了两个双基因表达载体pFGC5941S SIP1 NAC1 sense和pFGC5941S SIP1 NAC1 anti。并将这两个载体通过农杆菌介导的方法转化到野生型拟南芥中,共获得15株转基因植株。对这些转基因植株进行盐胁迫实验发现,在含75mmol·L-1 NaCl的MS培养基上,相比于野生型,pFGC5941S SIP1 NAC1 sense转基因植株主根增长,侧根数量明显增多,而pFGC5941S SIP1 NAC1 anti转基因植株长势与野生型苗相似。由此我们推测可能只有当SIP1和NAC1同时过表达时,才会促进盐胁迫下拟南芥侧根的发育。     

Overexpression of osmotin gene confers tolerance to salt and drought stresses in transgenic tomato (Solanum lycopersicum L.)

Abiotic stresses, especially salinity and drought, are major limiting factors for plant growth and crop productivity. In an attempt to develop salt and drought tolerant tomato, a DNA cassette containing tobacco osmotin gene driven by a cauliflower mosaic virus 35S promoter was transferred to tomato (Solanum lycopersicum) via Agrobacterium-mediated transformation. Putative T0 transgenic plants were screened by PCR analysis. The selected transformants were evaluated for salt and drought stress tolerance by physiological analysis at T1 and T2 generations. Integration of the osmotin gene in transgenic T1 plants was verified by Southern blot hybridization. Transgenic expression of the osmotin gene was verified by RT-PCR and northern blotting in T1 plants. T1 progenies from both transformed and untransformed plants were tested for salt and drought tolerance by subjecting them to different levels of NaCl stress and by withholding water supply, respectively. Results from different physiological tests demonstrated enhanced tolerance to salt and drought stresses in transgenic plants harboring the osmotin gene as compared to the wild-type plants. The transgenic lines showed significantly higher relative water content, chlorophyll content, proline content, and leaf expansion than the wild-type plants under stress conditions. The present investigation clearly shows that overexpression of osmotin gene enhances salt and drought stress tolerance in transgenic tomato plants.     

过表达番茄GDP-L-半乳糖磷酸酶基因提高烟草抗MV诱导的氧化胁迫能力

为了探讨番茄GDP—L-半乳糖磷酸酶对烟草抗坏血酸（AsA）含量及抗氧化能力的影响,从番茄叶片中分离了GDP-L-半乳糖磷酸酶基因（LeGGP）,并转入到烟草中。以野生型（WT）和转正义LeGGP烟草株系T1-3和T1-15为试材,测定了甲基紫精（MV）处理下AsA、脱氢抗坏血酸（DHA）、H2O2、O2-和叶绿素含量、抗坏血酸过氧化物酶（APX）活性、光合速率和叶绿素荧光参数等。Northem杂交分析表明LeGGP的表达受MV的诱导,在MV处理下,野生型烟草的离体叶圆片发生比转基因烟草更严重的光漂白,转基因烟草的AsA含量及清除H2O2和O2-的能力明显强于野生型,过表达LePGG胀高了烟草的生长量。并且转基因烟草比野生型具有更高的净光合效率（Pn）和光系统Ⅱ（PSII）最大光化学效率（眠）。结果表明,LeGGP的过表达有助于提高烟草AsA含量及抗氧化胁迫能力。     

pFGC5941的改造及拟南芥NAC1和SIP1双基因反义表达载体的构建和遗传转化

拟南芥中的SIP1基因编码的蛋白与拟南芥盐胁迫应答中的关键蛋白SOS2存在互作关系,而NACl为拟南芥中介导生长素信号促进其侧根发生的蛋白。本研究中我们将SIPl基因和NAC1正义基因以及SIP1基因和NAC1反义基因分别整合到一个经改造的具有2个35S启动子的可用于双基因表达的载体pF—GC5941S中,构建了两个双基因表达载体pFGC5941S-SIP1-NAC1-sense和pFGC5941S-SIP1-NACl-anti。并将这两个载体通过农杆菌介导的方法转化到野生型拟南芥中,共获得15株转基因植株。对这些转基因植株进行盐胁迫实验发现,在含75mnlol·L-1 NaCl的MS培养基上,相比于野生型,pFGC5941S-SIP1-NAC1-sense转基因植株主根增长,侧根数量明显增多,而pFGC5941S-SIP1-NAC1-anti转基因植株长势与野生型苗相似。由此我们推测可能只有当SIP1和NAC1同时过表达时,才会促进盐胁迫下拟南芥侧根的发育。     

Modification of pFGC5941 Construct and Transfer of Double Antisense Genes of NAC1 and SIP1 into Arabidopsis thaliana

SIP1 encodes the protein which show interact with SOS2, the protein involving in plant responding to saline stress while NAC1 encodes the protein which involves in auxin signal and promoting the development of lateral root of Arabidopsis thaliana. In present study SIP1 and NAC1-sense and NAC1-anti were inserted into pFGC5941S constructs, making the expression vectors pFGC5941S-SIP1-NAC1-sense and pFGC5941S-SIP1-NAC1-anti, respectively. Fifteen transgenic A.thaliana harboring these two constructs (pFGC5941S-SIP1-NAC1-sense and pFGC5941S-SIP1-NAC1-anti) were then generated via Agrobacterium tumefaciens-mediated transformation. The phenotypes of homozygous transformants which were grown on MS medium with 75mmol·L-1 NaCl showed that compared with wild-type A.thaliana, pFGC5941S-SIP1-NAC1-sense transgenic plants exhibited longer main root and increasing amounts of lateral roots, while no obvious differences were observed in pFGC5941S-SIP1-NAC1-anti transgenic plants. These results indicated that the development of A.thaliana lateral roots under salt stress was specifically promoted by both overexpression of SIP1 gene and NAC1 gene.     

Co-expression of Pennisetum glaucum vacuolar Na⁺/H⁺ antiporter and Arabidopsis H⁺-pyrophosphatase enhances salt tolerance in transgenic tomato

Salinity is one of the major abiotic stresses affecting plant productivity. Tomato (Solanum lycopersicum L.), an important and widespread crop in the world, is sensitive to moderate levels of salt in the soil. To generate tomato plants that can adapt to saline soil, AVP1, a vacuolar H(+)-pyrophosphatase gene from Arabidopsis thaliana, and PgNHX1, a vacuolar Na(+)/H(+) antiporter gene from Pennisetum glaucum, were co-expressed by Agrobacterium tumefaciens-mediated transformation. A sample of transformants was self-pollinated, and progeny were evaluated for salt tolerance in vitro and in vivo. It is reported here that co-expression of AVP1 and PgNHX1 confers enhanced salt tolerance to the transformed tomato compared with the AVP1 and PgNHX1 single gene transgenic plants and the wild-type. These transgenic plants grew well in the presence of 200 mM NaCl while wild-type plants exhibited chlorosis and died within 3 weeks. The transgenic line co-expressing AVP1 and PgNHX1 retained more chlorophyll and accumulated 1.4 times more proline as a response to stress than single gene transformants. Moreover, these transgenic plants accumulated a 1.5 times higher Na(+) content in their leaf tissue than the single gene transformants. The toxic effect of Na(+) accumulation in the cytosol is reduced by its sequestration into the vacuole. The physiological analysis of the transgenic lines clearly demonstrates that co-expression of AVP1 and PgNHX1 improved the osmoregulatory capacity of double transgenic lines by enhanced sequestration of ions into the vacuole by increasing the availability of protons and thus alleviating the toxic effect of Na(+).     

Over-expression of heat shock protein gene <Emphasis Type="Italic">hsp26</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> enhances heat tolerance

In the yeast Saccharomyces cerevisiae, the molecular chaperone HSP26 has the remarkable ability to sense increases in temperature directly and can switch from an inactive to a chaperone-active state. In this report, we analyzed the effect of expression of HSP26 in Arabidopsis thaliana plants and their response to high temperature stress. The hsp26 transgenic plants exhibited stronger growth than wild type plants at 45 °C for 16 h. The chlorophyll content and chlorophyll fluorescence decreased much more in wild type than in transgenic plants. Moreover, the transgenic plants had higher proline and soluble sugar contents, and lower relative electrical conductivity and malondialdehyde contents after high temperature stress. Furthermore, we found that over-expression of HSP26 in Arabidopsis increased the amount of free proline, elevated the expression of proline biosynthetic pathway genes and therefore enhanced Arabidopsis tolerance to heat stress.     

小麦耐逆基因-TaLEA2转化拟南芥的研究

研究小麦第3组LEA基因中T aLEA2对耐旱和耐盐性能的影响.将小麦第3组LEA基因T aLEA2连接在双元表达载体pB I121 C aM V 35S启动子下游,构建了能在植物中高效表达的载体pB I121-T aLEA2.通过农杆菌介导的真空渗透法,将其转入野生拟南芥中,经抗性筛选及PCR验证,获得T0代转基因植株,并用不同浓度的PEG 4000和N aC l对转基因拟南芥的耐逆性进行检测.结果表明,这些转基因植株可明显改进拟南芥在10%PEG及0.8%N aC l培养基上的生长状态.在实验条件下,转基因拟南芥的耐旱性及耐盐性均有所提高,提示T aLEA2基因在植物水分调节方面有重要作用.     

高粱ATP合成酶E亚基基因(SbATPase-E)的克隆及其抗逆功能研究

高粱是一种抗旱性较强的禾谷类作物。本研究在高粱中克隆到一个全长为693 bp的编码ATP合成酶E亚基的基因(SbATPase-E)。在高粱幼苗期,SbATPase-E基因受Na Cl和脱落酸(ABA)处理诱导上调表达。该基因在拟南芥中过量表达可提高转基因植株的耐旱性和耐盐性,在逆境胁迫条件下转基因拟南芥植株较野生型植株根系发达,可能是转基因植株耐旱性和耐盐性提高的主要原因。在干旱胁迫条件下,转基因植株中DREB2A、P5CS1、RD29A、RAB18和ABI1基因的表达量相对于野生型植株中的表达量提高更为显著;在高盐处理条件下,转基因植株中SOS1和SOS2基因的表达量也较野生型植株中的表达量明显提高。这些抗逆相关基因的上调表达可能是转基因植株抗逆性提高的主要分子机制。     

超量表达MrCN基因提高烟草植株对TMV的抗性

利用农杆菌介导的遗传转化法将含有普通烟草Ubi．U4启动子驱动MrCN基因表达的元件导入TMV敏感烟草品种K326中,对筛选鉴定出的T0代转基因植株接种TMV,测定其接种前后不同时期的理化指标,以接种TMV的野生型植株为对照。结果显示,转基因植株和野生型植株接种TMV前叶绿素（Chl）和MDA含量YLSOD、POD和CAT酶活力均无显著差异,但接种TMV后野生型植株Chl含量逐渐降低,转基因植株Chl含量先增后降,在接种TMV后5d时转基因植株叶片Chl含量显著高于野生型植株。接种前期（0-3d）转基因植株MDA含量略低于野生型植株,但差异不显著;但接种后期（3～5d）前者的MDA含量显著低于后者。接种TMV后转基因植株中SOD、POD及CAT酶活性变化幅度较野生型植株高,以CAT的变幅最为显著。另外,Real-timePCR分析结果表明,接种TMV后3d时转基因植株删蹦、PR-1α及NrCN表达量均显著高于野生型植株。以上结果表明,通过转基因技术提高烟草抗病基NNrCN的表达量,能提高防御酶活性及病程相关基因的表达量,从而延缓植株感染TMV的发病时间,增强敏感植株对TMV的抗性。     

HAL1 mediate salt adaptation in Arabidopsis thaliana

INTRODUCTIONSalinity is a major environmental stress that isa substantial constraint to crop production both fordry land and irrigated agriculture. The detrimental impact of this stress is perpetuated and exacerbated by management practices used to facilitatehigh-output crop production. To overcome theselimitations and improve production efficiency in theface of a burgeoning world population, more salt tolerant crops must be developed. In contrast with traditional breeding, the direct ill…