超表达小麦基因TaSOD1.1和TaSOD1.2对烟草耐低温能力的影响

 【目的】研究超表达小麦基因TaSOD1.1和TaSOD1.2对烟草耐低温能力的影响。【方法】采用农杆菌介导的遗传转化技术,获得融合靶基因的转基因烟草植株;通过比较低温处理后对照和转基因系的表型和生理指标,鉴定超表达靶基因对烟草耐低温能力的调控效应。【结果】以分子检测鉴定的插入单拷贝基因的4个转基因系和对照为材料,低温处理后超表达外源基因的转基因植株叶片失绿缓慢,叶片超氧化物歧化酶（SOD）活性明显增加,反映细胞膜质过氧化程度的丙二醛（MDA）含量明显下降;叶片叶绿素a、b和类胡萝卜素含量、可溶性糖含量和可溶性蛋白含量均明显提高。【结论】超表达小麦TaSOD1.1和TaSOD1.2的烟草植株,具有增强SOD活性、明显缓解低温造成的细胞膜质过氧化程度、改善低温胁迫下植株的生理功能,可进而改善植株抵御低温胁迫的能力。     

华山新麦草蔗糖:蔗糖1-果糖基转移酶基因Ph-1-SST的克隆及序列分析

蔗糖:蔗糖1-果糖基转移酶基因1-SST在植物逆境胁迫反应中起重要作用。为了挖掘华山新麦草(Psathyrostachys huashanica Keng)抗非生物胁迫关键功能基因,以华山新麦草叶片为材料,利用RT-PCR结合RACE技术克隆1-SST基因的全长cDNA,命名为Ph-1-SST,登录号为KX761897。通过PCR法扩增其gDNA序列,测序结果表明该基因的gDNA和cDNA序列长度分别为3 344bp和2 001bp,编码666个氨基酸残基,序列结构分析结果表明该基因含4个外显子3个内含子,预测该蛋白相对分子质量为72.9ku,理论等电点为4.87。序列比对显示,该基因与大麦1-SST基因编码蛋白的相似性最高(90%),为糖基水解酶32家族成员。对1-SST氨基酸序列的系统进化树分析显示,Ph-1-SST及其同源蛋白位于不同分支,初步推断此全长cDNA是华山新麦草中编码蔗糖:蔗糖1-果糖基转移酶的一个新基因。结果为作物非生物胁迫改良提供重要基因资源。     

Metabolism of Reactive Oxygen Species in the Cytoplasmic Male-Sterile Cotton Anther

Reactive oxygen species （ROS） in plant cell, including superoxide （O2^-）, hydrogen peroxide （H2O2）, and malondialdehyde （MDA）, are thought to be important inducible factors of cell apoptosis if excessively accumulated in cells. To elucidate the metabolic mechanism of ROS production and scavenging in anthers of the cytoplasmic male-sterile （CMS） cotton, CMS line, maintainer, and hybrid F1 anthers, were employed for studying the relationship between CMS and metabolism of ROS, by comparing ROS changes in the sterile and fertile anthers at different developmental stages. The results showed that during the abortion preliminary stage （sporogenous cell division stage）, anthers of CMS line had higher contents of O2^-, H2O2, and MDA than those of maintainer or hybrid F1. Simultaneously, the higher activities of superoxide dismutase （SOD）, catalase （CAT）, and peroxidase （POD） in scavenging ROS were measured in the anthers of the CMS line, indicating that an increase of ROS in anthers of abortion preliminary stage had an inducible effect on the antioxidant enzymes. But during the abortion peak of CMS anther （pollen mother cell meiosis stage）, on the one hand, contents of O2^-, H2O2, and MDA were extraordinarily high in CMS anthers, on the other hand, the activities of SOD, CAT, and POD were excessively low, which disrupted the balance between the production and elimination of ROS and led to pollen mother cells apoptosis at this stage. In the following two stages （uninucleate microspore stage and mature pollen stage）, the contents of O2^- and H2O2 in the aborted anthers were approximated to contents in the fertile anthers of the maintainer and hybrid F1. However, MDA contents were continuously raised and enzymic activities of SOD, CAT, and POD were consistently decreased in sterile anthers, which indicated that ROS still had harmful effects on the anthers after the apoptosis of the male cells. Excessive accumulation of O2^-, H2O2, and MDA and significant reduction of ROS scavengingenzyme activities were coinstantaneous with male cells apoptosis in the anthers of the cotton CMS line. But when the restorer gene was transferred into the CMS line, excessive production of ROS could be eliminated in the anthers of hybrid F1.     

苹果矮化砧木T337 MdCBB1基因的克隆与表达分析

为研究油菜素内酯(BR)对苹果矮化砧木T337株高调控机制的影响,以T337盆栽苗为试材,利用RT-PCR技术从T337中分离出BR合成关键基因MdCBB1.1和MdCBB1.2,二者的开放阅读框均为1 707bp,分别编码一个568个氨基酸的蛋白,分子质量分别为6.624 1×104 ku和6.622 8×104 ku。生物信息学分析结果显示,MdCBB1.1和MdCBB1.2基因编码的蛋白含有FAD_binding_4和GlcD 2个明显的结构域,为非核蛋白,属于植物CBB1蛋白家族。表达分析结果显示,MdCBB1.1和MdCBB1.2基因在T337不同组织均有表达,但是顶梢和根中的表达量明显高于其他组织(P0.05)。并且外源芸苔素内酯(BL)处理能够上调二者在木质部中的表达(P0.05)。     

番茄一个褪黑素合成基因的cDNA克隆与表达分析

为深入研究褪黑素的分子生物学功能,根据番茄基因组数据库的SlSNAT基因序列信息设计引物,以耐盐番茄材LA1401(PI365967)叶片RNA反转录得到的cDNA为模板,利用高保真酶克隆了番茄的褪黑素合成酶基因SlSNAT,基因CDS(coding sequence)序列全长为768 bp,共编码255个氨基酸。利用酶切连接的方法,构建了该基因的超表达载体。实时定量PCR结果表明,SlSNAT基因在叶片中的表达量最高,显著高于其在花、果实、根、种子和萼片中的表达量。在不同非生物逆境处理下的表达结果显示,在干旱处理条件下的表达量最高,其次是在甘露醇的渗透胁迫逆境,在这2种胁迫条件下的表达量均显著高于其在过氧化氢、氯化钠、低温和褪黑素诱导下的表达量。另外,在盐胁迫条件下,SlSNAT基因在根部的表达量受到明显抑制。     

陆地棉盐胁迫应答基因GhWRKY41的克隆与分析

为研究转录因子GhWRKY41在陆地棉盐胁迫应答过程中的作用,基于差减文库分析结果,利用RT-PCR和RACE技术,克隆了GhWRKY41基因(GenBank登录号为HM002635)。该基因cDNA长度为1 630bp,含有ORF(Open reading frame)为1 068bp,编码355个氨基酸的多肽,包含2个内含子。通过瞬时表达分析亚细胞定位,结果表明,转录因子GhWRKY41定位于细胞核,符合转录因子特性。转基因株系发芽试验结果表明,过量表达GhWRKY41基因,可显著提高转基因棉花在干旱、盐和低温胁迫下的发芽率;利用Real-time PCR技术,证明在盐和干旱胁迫条件下,转基因株系中GhWRKY41基因的表达量显著上升。GhWRKY41基因在根、茎和叶片中表达存在差异,根系中胁迫6h上调达到最高,茎中则胁迫48h达到最高,而叶片中仅6和24h上调表达。进一步比较转基因棉花与野生型棉花的纤维品质性状,结果表明GhWRKY41的过表达可以提高转基因棉花的衣分。因此,GhWRKY41参与了棉花响应盐和干旱胁迫应答过程,且过表达可提高转基因棉花耐盐性和耐旱性。     

亚热带地区水稻(Oryza sativa L.)气孔臭氧通量和产量的响应关系

基于开放式臭氧浓度升高O_3-FACE(Free-Air Concentration Elevation of O_3)实验平台,利用前期水稻O_3-FACE试验的基础数据,通过建立水稻产量与不同评价指标(累积气孔O_3吸收通量PODY和O_3浓度指标AOTX)的响应关系,比较了水稻产量损失与各评价指标的相关性差异,通过对暴露剂量、吸收通量相关参数取值与产量损失的观察和分析结果的比较,找出更为合理的农作物臭氧风险评估阈值。结果表明:随着通量阈值Y[0~11 nmol O_3·m~(-2)PLA·s~(-1)(PLA:projected leaf area,投影叶面积)]和暴露浓度阈值X(0~50 n L·L~(-1))的增加,回归分析R~2值逐渐增加,当Y为11 nmol O_3m~(-2)PLA·s~(-1)和X为50 n L·L~(-1)时,气孔臭氧吸收通量POD11和累积暴露剂量AOT50与水稻相对产量的相关性最大,当通量阈值Y为8~13 nmol O_3·m~(-2)PLA·s~(-1)和暴露阈值X为46~58 n L·L~(-1)时,可获得较高的R~2值取值范围,分别为0.70~0.75和0.70~0.745。参考文献发现,目前地表臭氧污染可能引起的水稻产量损失范围为5%~8%,对照圈中POD9~10和AOT40~45产量损失的预测值亦在这区间,但前者R~2值(0.73~0.74)明显高于后者R~2值(0.64~0.69),表明基于气孔臭氧通量的评价指标能更好地反映水稻产量的变化。通过进一步分析发现,当通量阈值Y为9 nmol O_3·m~(-2)PLA·s~(-1)时,能更准确地评估水稻产量损失,且其R~2值(0.73)高于通量指标POD6(0.57)。以上研究结果表明,通量指标POD9更适合评估亚热带地区O_3污染对水稻作物的影响。     

Reactive oxygen species activity in the interaction of rice with Erwinia chrysanthemi pv. zeae

Activities of reactive oxygen species (ROS) were investigated in the interaction between rice and Erwinia chrysanthemi pv. zeae. Results showed that O₂·⁻, H₂O₂ and malondialdehyde (MDA) in resistant variety (128) had higher increases in activity compared to those in the susceptible variety (Texian 13) 24 hours after bacteria inoculation. The activities of superoxide dismutase (SOD) increased in 128 and Texian 13 twenty-four hours after inoculation and then decreased, but the SOD activity in 128 was found to be usually lower than that in Texian 13. The CAT activity in Texian 13 had two peaks at 24 h and 96 h after inoculation, while little change was seen in 128. In conclusion, ROS and its related enzymes could be correlated to rice resistance against E. chrysanthemi pv. zeae. __________ Translated from Journal of Huazhong Agricultural University, 2007, 26(4): 451–455 [译自: 华中农业大学学报]     

马铃薯StTrxF基因的克隆与功能分析

为探究马铃薯硫氧还蛋白基因(StTrxF)的耐盐生理机制,通过RT-PCR方法从马铃薯品种中薯5号中克隆得到全长549bp硫氧还蛋白基因(StTrxF)。该基因编码182个氨基酸,预测蛋白质分子量为44.17ku,理论等电点(pI)为5.23,具有典型的Thioredoxin结构域。由StTrxF基因推导的氨基酸序列与番茄、拟南芥、芝麻和赤霞珠等TrxF蛋白质氨基酸序列的同源性为96.02%～59.28%。构建植物过表达载体,导入拟南芥中,获得转基因拟南芥纯系植株。通过对转基因拟南芥植株的耐盐离体和盆栽鉴定,表明转基因植株的耐盐性显著提高。同时盐胁迫下,转基因植株的超氧化物歧化酶(SOD)活性和脯氨酸含量显著提高,丙二醛(MDA)含量显著降低。结果表明,表达StTtxF基因通过增加转基因植株脯氨酸含量,提高SOD活性,降低MDA含量,以维持细胞渗透平衡并激活ROS清除系统,具有提高转基因拟南芥植株耐盐性的显著效果。     

气调对蕨菜采后活性氧代谢的影响

为了研究气调对蕨菜贮藏期间活性氧代谢的影响,设置不同CO2和O2比例,对蕨菜采后、H2O2、MDA含量和SOD、POD活性及维生素C含量进行了研究.结果表明:适宜的CO2和O2浓度有利于采后蕨菜维持较高的SOD和POD活性,缓解蕨菜体内活性氧自由基和H2O2的积累,减少MDA含量的积累,降低活性氧自由基对细胞膜的伤害,利于蕨菜采后vC的保存和干鲜物质量的损失.试验中最好的气体组合为CO2 2%和O2 6%,其次为CO2 6%和O2 10%,这些组合能很好地保存蕨菜中vC含量,可使蕨菜保鲜15 d以上.     

二穗短柄草FRUITFULL1基因表达分析及RNA干扰和过表达载体的构建

为了揭示BdFUL1的生物学功能,利用生物信息学和分子生物学方法,分析二穗短柄草转录因子FRUITFULL1-2(BdFUL1-2)的结构、序列特征以及与不同植物AP1/FUL的亲缘关系,构建进化树。利用定量RT-PCR等分子生物学方法,分析非生物逆境和外源激素处理条件下两个转录本BdFUL1-1和BdFUL1-2的表达水平。结果表明:BdFUL1属于典型的植物MADS-box基因,与小麦的WFUL1有很近的亲缘关系;在高温、低温、干旱胁迫以及不同激素ABA、6-BA和SA处理下,BdFUL1-1的表达水平显著升高,暗示BdFUL1可能通过激素信号传导而参与响应非生物胁迫。同时,构建BdFUL1-1和BdFUL1-2的RNA干扰和过表达载体。     

异源表达玉米ZmWRKY114基因增强拟南芥对盐胁迫的敏感性

WRKY转录因子参与调控植物对各种非生物胁迫的应答反应。从玉米中分离得到一个响应高盐胁迫的WRKY基因,将其命名为ZmWRKY114。亚细胞定位实验表明ZmWRKY114定位于细胞核内且和ZmCaM2可以发生相互作用。为进一步研究ZmWRKY114基因的生物学功能,将ZmWRKY114基因构建到植物表达载体pCAMBIA1301上,并采用农杆菌转化法转化拟南芥,扩繁后对T3代转基因拟南芥进行了耐盐性试验。结果表明,转基因株系的绿苗率在高盐胁迫条件下显著低于野生型拟南芥,并且高盐胁迫对转基因拟南芥的主根抑制程度更大。同时,ZmWRKY114在拟南芥中的异源过表达还可以导致丙二醛的积累和相对电解质渗透率的增加,从而使转基因植物对盐胁迫的耐受性显著降低。上述结果表明ZmWRKY 114基因可能是盐胁迫反应的负调控因子。     

Overexpression of AmDUF1517 enhanced tolerance to salinity,drought, and cold stress in transgenic cotton

As abiotic stresses become more severe as a result of global climate changes, the growth and development of plants are restricted. In the development of agricultural crops with greater stress tolerance, AmDUF1517 had been isolated from the highly stress-tolerant shrub Ammopiptanthus mongolicus, and can significantly enhance stress tolerance when inserted in Arabidopsis thaliana. In this study, we inserted this gene into cotton to analyze its potential for conferring stress tolerance. Two independent transgenic cotton lines were used. Southern blot analyses indicated that AmDUF1517 was integrated into the cotton genome. Physiological analysis demonstrated that AmDUF1517-transgenic cotton had stronger resistance than the control when treated with salt, drought, and cold stresses. Further analysis showed that trans-AmDUF1517 cotton displayed significantly higher antioxidant enzyme (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione S-transferase (GST)) activity and less reactive oxygen species (ROS) accumulation, which suggests that overexpression of AmDUF1517 can improve cotton resistance to stress by maintaining ROS homeostasis, as well as by alleviating cell membrane injury. These results imply that AmDUF1517 is a candidate gene in improving cotton resistance to abiotic stress.     

二穗短柄草DREB1G基因表达模式分析和在拟南芥中的异源过量表达

CBF/DREB基因是一类植物特异性转录因子,通过调控下游抗逆相关基因的表达,在植物逆境胁迫应答过程中发挥重要作用。为了研究新型的禾本科模式植物二穗短柄草DREB基因的功能,克隆BdDREB1G的cDNA序列,分析该基因在各种非生物胁迫条件下的表达,构建过表达载体,获得转基因拟南芥。结果表明:高温、低温和水杨酸胁迫处理显著诱导BdDREB1G的表达,暗示该基因响应高温和低温胁迫。初步观察结果说明,该与野生型拟南芥相比,转基因植株生长发育迟缓。这些为研究该基因响应非生物胁迫的功能奠定基础。     

甘蓝型油菜种子特异表达油体蛋白启动子P_(BnOA03)的克隆及功能分析

为了改良油菜种子的性状,常常需要研究种子特异表达目的基因的情况。在分析油菜油体蛋白表达模式的基础上,选择种子特异高表达的油体蛋白,根据甘蓝型油菜基因组序列设计引物,利用PCR技术从基因组中克隆到一段715bp的启动子序列。启动子顺式元件分析表明,这一克隆序列具有CAAT-box和TATA-box启动子基本元件,另外含有ABA响应元件等多种顺式元件。进一步利用GUS报告基因在拟南芥中对该启动子的功能进行鉴定,结果表明,在转基因株系的幼苗、根、茎、叶、花和种子发育前期及成熟种子中没有检测到GUS基因明显表达,在种子发育的中后期检测到GUS基因的表达逐渐增强。说明这个启动子的表达调控具有一定的种子特异性。