拟南芥高迁移率族蛋白B族基因表达模式分析

为了解高迁移率族蛋白B族(HMGB)基因在拟南芥中的表达模式及作用方式,该研究克隆了拟南芥中5个编码HMGB的基因:AtHMGB1、AtHMGB2、AtHMGB3、AtHMGB4、AtHMGB5,并运用荧光实时定量PCR方法检测野生型拟南芥中以上5种基因在不同器官中的表达及在外源植物激素(ABA、2,4-D)处理前后的表达差异,选取AtHMGB2、AtHMGB4和AtHMGB5分别转化拟南芥并筛选出超表达株系,随即检测ABA诱导下超表达AtHMGB的转基因拟南芥的表型。研究证实:在野生型拟南芥中AtHMGB2在拟南芥各个器官中的表达量远高于其它家族成员,AtHMGB4和AtHMGB5在花、果荚和根中的表达略高于茎和叶;在ABA处理前后AtHMGB家族成员的表达水平有显著差异,其中AtHMGB2的表达被ABA显著负调控;ABA诱导下超表达AtHMGB2的转基因拟南芥与野生型相比出现萌发及生长迟缓现象,但超表达AtHMGB4与AtHMGB5的转基因拟南芥在ABA诱导下的种子萌发和幼苗生长与野生型相比差异不大。研究发现,AtHMGB家族成员在转录水平上响应ABA的方式各有不同,对理解AtHMGB家族成员的生物学功能提供了新的基础。     

蒙古冰草MwMYB4基因功能鉴定

MwMYB4基因是从蒙古冰草中克隆得到的MYB类转录因子家族成员之一。该研究以转MwMYB4基因的拟南芥后代为材料,通过在干旱和低温胁迫下对转基因植株进行表型分析、理化指标测试和分子鉴定,分析并验证MwMYB4基因的功能。结果显示:(1)蒙古冰草MwMYB4基因已成功整合到转基因拟南芥T_1代的基因组中并实现转录水平的表达。(2)转基因拟南芥T_2代植株在干旱胁迫条件下,转基因植株叶片枯黄程度较轻,相对电导率较野生型变化幅度低,脯氨酸含量明显高于野生型对照,且MwMYB4基因的表达量随干旱胁迫时间延长而增加。(3)在低温胁迫条件下,转基因拟南芥叶片的枯白程度明显低于野生型,且MwMYB4基因的表达量随低温胁迫时间增加而增加。研究表明,过量表达蒙古冰草MwMYB4基因能够提高转基因拟南芥对干旱和低温的耐受性,该基因可能在干旱胁迫和低温胁迫调控机制中发挥调控作用,可作为改良农作物和其他牧草抗旱、抗寒性的重要候选基因。     

陆地棉GhPYR1基因的克隆和功能分析

植物激素脱落酸(Abscisic acid,ABA)在植物应对干旱、盐碱等逆境胁迫以及植物种子萌发、根伸长、芽休眠等阶段发挥重要作用。PYR/PYL/RCAR蛋白家族是ABA受体,与ABA结合后能够启动ABA信号传导通路,诱导ABA应答基因的表达。利用电子克隆和RT-PCR技术从陆地棉中克隆了Gh PYR1基因,其编码的Gh PYR1蛋白与拟南芥中At PYR1蛋白相似度为73%。将Gh PYR1蛋白序列与拟南芥14个PYR/PYL/RCAR家族成员蛋白序列进行比对并构建进化树,发现它与拟南芥PYR/PYL/RCAR蛋白亚家族III亲缘关系最近。过表达Gh PYR1基因的T3代拟南芥在外源ABA处理下,其种子萌发和初期根生长均滞后于野生型,表现出对ABA更加敏感;高盐和干旱胁迫对转基因种子的萌发抑制更强烈,但苗期胁迫处理下转基因拟南芥的长势却明显优于野生型;同时在外源ABA诱导条件下ABA应答基因RD29A、RAB18的表达量较野生型有明显提高。以上结果说明Gh PYR1基因编码的蛋白是ABA的受体,过表达该基因能够提高植物对ABA的敏感性和增强应对逆境胁迫的能力。     

高效删除标记基因的Bhlea2植物表达载体的构建

为培育去除选择标记基因的耐旱转基因植物,同时利用Cre/Lox和FLP/frt系统,构建一个能够高效删除标记基因的Bhlea2基因植物表达载体.拟南芥rd29A启动子是在低温、干旱、高盐胁迫下的快速应答启动子,玉米ubiquitin启动子可有效驱动外源基因的转录,拟南芥pAB5启动子是花粉及胚胎等发育早期特异表达的启动子,利用上述启动子构建了表达Bhlea2基因并能够删除标记基因的植物表达载体.该表达载体包括重组酶表达元件pAB5-FLP、Bhlea2抗旱基因表达元件rd29A-Bhlea2和bar标记基因表达元件ubiquitin-bar.     

糙皮侧耳Pleurotus ostreatus子实体凝集素基因polectin2及其启动子的克隆及功能鉴定

凝集素在食用菌的生长发育、抗病虫等逆境及抗肿瘤等方面发挥着重要的作用,但是关于糙皮侧耳凝集素基因的抗逆功能研究还比较薄弱。因此,本文利用PCR技术克隆得到糙皮侧耳子实体凝集素polectin2基因及其启动子序列,测序和生物信息学结果显示该基因不含内含子,完整开放阅读框为408bp,编码136个氨基酸,预测蛋白分子量15.3k Da;启动子polectinpro序列长度为927bp,生物数据库PLACE分析结果表明polectinpro含有低温、干旱胁迫诱导和病程相关等抗逆元件。然后,构建含有GFP融合蛋白的重组植物表达载体pCAMBIA1302-polectinpro-gfp,并进行烟草遗传转化,结果证明该启动子能够在低温诱导下驱动gfp基因表达。同时成功构建了含有polectin2的重组植物表达载体pCAMBIA1301-polectin2并进行了烟草遗传转化;对阳性转化植株T1代和野生型种子进行低温胁迫实验,结果表明转基因种子萌发率明显高于野生型。综上所述,推测polectin2基因具有耐低温功能,且其启动子具有低温诱导活性。上述结果为进一步利用子实体凝集素polectin2基因提高食用菌的耐低温等抗逆功能提供了理论依据和技术支持。     

过量表达棉花CBF2基因提高转基因拟南芥抗旱耐盐能力

CBF/DREB是一类植物中特有的转录因子,在植物抵抗逆境胁迫过程中发挥重要功能。本研究从陆地棉(Gossypium hirsutum L.)Coker 312中克隆获得1个棉花CBF/DREB基因,命名为Gh CBF2,该基因编码一个由216个氨基酸组成的CBF蛋白。序列分析结果显示,Gh CBF2与其他植物的CBF蛋白类似,含有AP2转录因子典型的保守结构域。干旱或高盐胁迫处理明显增加了Gh CBF2基因的表达量。亚细胞定位分析结果发现Gh CBF2定位在细胞核中。将Gh CBF2基因构建到由35S启动子调控的植物表达载体p MD上并转化拟南芥(Arabidopsis thaliana L.),结果表明,在干旱和盐胁迫条件下,过量表达Gh CBF2基因拟南芥的成活率显著高于野生型,并且游离脯氨酸和可溶性糖含量也高于野生型,说明转Gh CBF2基因提高了拟南芥的耐盐抗旱能力。采用实时荧光定量PCR方法分析胁迫相关标记基因COR15A、RD29A和ERD6的表达情况,结果显示转基因株系中的表达量显著高于野生型,说明Gh CBF2参与调控拟南芥干旱和盐胁迫相关基因的表达。     

拟南芥CBF2基因的抗旱性研究

以拟南芥为材料,根据已报道的序列设计引物克隆了CBF2基因及rd29A基因启动子,并构建了植物表达载体pBI-rd-cbf,用以转化烟草。转基因烟草的Northern杂交检测显示,30%PEG诱导条件下CBF2基因在诱导30 min之后持续较高的表达水平,且表达水平受胁迫诱导程度的影响。抗旱生理指标测定显示,干旱条件下转基因烟草植株的脯氨酸含量迅速增高,远超于野生型;而丙二醛含量的增长幅度要比野生型植株小很多,且随着胁迫时间的延长,转基因植株体内的丙二醛含量逐步趋于稳定。试验证明,CBF2基因在提高农作物的抗旱性方面具有极大的利用价值。     

水稻含有B-box锌指结构域的OsBBX25蛋白参与植物对非生物胁迫的响应

锌指蛋白在调控植物生长发育和应对逆境过程中发挥着重要作用.为进一步研究锌指类蛋白参与植物非生物胁迫响应的分子机制,对水稻(Oryza sativa)中一个编码含有B-box锌指结构域蛋白的OsBBX25基因进行了功能分析.OsBBX25受盐、干旱和ABA诱导表达.异源表达OsBBX25的转基因拟南芥(Arabidopsis thaliana)与野生型相比对盐和干旱的耐受性增强,且盐胁迫条件下转基因植物中KIN1、RD29A和COR15的表达上调,干旱胁迫下KIN1、RD29A和RD22的表达上调.外源施加ABA时,转基因植物的萌发率与野生型之间没有明显差异.OsBBX25可能作为转录调控的辅助因子调节胁迫应答相关基因的表达,进而参与植物对非生物胁迫的响应.     

转ZjAPX基因拟南芥对NaCl、干旱胁迫的耐性研究

旨在探讨枣树抗坏血酸过氧化物酶基因ZjAPX在植物渗透胁迫中的作用。将ZjAPX基因转入到模式植物拟南芥,以野生型(WT)、转ZjAPX拟南芥株系T2为试材,进行不同浓度NaCl胁迫和干旱胁迫。结果表明,转基因株系的种子萌发、植株生长均优于野生型株系;荧光定量PCR检测转基因拟南芥植株在干旱和盐胁迫处理10 d后目的基因ZjAPX的表达量显著高于野生拟南芥,表明ZjAPX的高表达明显提高了植株的抗旱和耐盐性。     

胡杨PeECT8基因的克隆及功能分析

ECT基因家族已在拟南芥中被发现并报道,然而它们是否参与植物在逆境胁迫下的响应过程却鲜有报道。为研究胡杨PeECT8基因的功能,从胡杨叶片cDNA中克隆出PeECT8基因,构建CaMV 35S::Pe ECT8植物表达载体,利用花序浸染法转化拟南芥,经GUS组织化学染色和PCR检测,获得转基因植株,进而测定不同浓度盐(Na Cl)和甘露醇胁迫下转基因拟南芥种子的萌发率、生长势和根长。测序结果表明,该基因编码区长度为1821 bp,可编码606个氨基酸。蛋白序列比对发现,PeECT8基因与毛果杨PtrECT8同源基因所编码的氨基酸一致性达93.42%,PeECT8蛋白包含一个YT521-B-like保守结构域。相比于野生型,过量表达PeECT8的拟南芥在不同浓度NaCl胁迫下萌发率降低,在D-甘露醇胁迫下萌发率没有明显变化。在NaCl和D-甘露醇胁迫下,转基因拟南芥根的伸长长度小于野生型。这表明PeECT8基因在盐胁迫和渗透胁迫下发挥负调控的作用。     

Isolation and characterization of two sorbitol transporter gene promoters in micropropagated apple plants (Malus?×?domestica) regulated by drought stress

The role of polyol transporters in stress tolerance in plants have been elucidated by many studies. Sorbitol transporter genes MdSOT3, MdSOT4 and MdSOT5 in apple plants, which are important for sorbitol loading and unloading, are regulated by drought stress. To further confirm the role of sorbitol transporters in stress tolerance, the constructs harboring MdSOT3 and MdSOT5 genes were introduced into wild type Arabidopsis plants (Col-0) and the Arabidopsis transformed with MdSOT3 or MdSOT5 performed higher drought stress tolerance compared to WT. In order to further understand how sorbitol transporters are involved in drought tolerance in apple plants, upstream regions of sorbitol transporter genes were isolated from apple plant source leaves by Anchored PCR from genomic DNA obtained, and then were used to drive expression of the GUS reporter in tobacco plants. The results showed that the longest fragments of MdSOT3 and MdSOT5 promoters induced the highest GUS activity under drought stress conditions. Additionally, fragments of these promoters that contain cis-acting elements known to be involved in stress response also induced GUS activity under drought stress. Taken together, our data suggest that increased MdSOT3 and MdSOT5 activity, through cis-acting elements in the promoters of these genes, play important roles in imparting tolerance to drought in micropropagated apple plants.     

Arabidopsis and tobacco plants ectopically expressing the soybean antiquitin-like ALDH7 gene display enhanced tolerance to drought, salinity, and oxidative stress

Despite extensive studies in eukaryotic aldehyde dehydrogenases, functional information about the ALDH7 antiquitin-like proteins is lacking. A soybean antiquitin homologue gene, designated GmTP55, has been isolated which encodes a dehydrogenase motif-containing 55 kDa protein induced by dehydration and salt stress. GmTP55 is closely related to the stress-induced plant antiquitin-like proteins that belong to the ALDH7 family. Transgenic tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana) plants constitutively expressing GmTP55 have been obtained in order to examine the physiological role of this enzyme under a variety of stress conditions. Ectopic expression of GmTP55 in both Arabidopsis and tobacco conferred tolerance to salinity during germination and to water deficit during plant growth. Under salt stress, the germination efficiency of both transgenic tobacco and Arabidopsis seeds was significantly higher than that of their control counterparts. Likewise, under progressive drought, the transgenic tobacco lines apparently kept the shoot turgidity to a normal level, which contrasted with the leaf wilt phenotype of control plants. The transgenic plants also exhibited an enhanced tolerance to H(2)O(2)- and paraquat-induced oxidative stress. Both GmTP55-expressing Arabidopsis and tobacco seeds germinated efficiently in medium supplemented with H(2)O(2), whereas the germination of control seeds was drastically impaired. Similarly, transgenic tobacco leaf discs treated with paraquat displayed a significant reduction in the necrotic lesions as compared with control leaves. These transgenic lines also exhibited a lower concentration of lipid peroxidation-derived reactive aldehydes under oxidative stress. These results suggest that antiquitin may be involved in adaptive responses mediated by a physiologically relevant detoxification pathway in plants.