转果聚糖蔗糖转移酶基因(Sac B)美丽胡枝子的获得

采用农杆菌介导的遗传转化方法,将来自枯草杆菌的果聚糖蔗糖转移酶基因(Sac B) 导入美丽胡枝子,以提高胡枝子抵御干旱胁迫和盐胁迫的能力。以美丽胡枝子子叶节为外植体,通过与含有植物双元表达载体pKP的农杆菌LBA4404 共培养,将Sac B 基因导入美丽胡枝子基因组。经卡那霉素筛选后,共获得62 株卡那霉素抗性植株。经PCR特异性扩增和PCR-Southern杂交,证明有5株再生植株基因组DNA 中整合了Sac B 基因。通过RT-PCR分析,结果表明SacB 基因均获得表达。经过200 mmol/L NaCl和5% PEG模拟胁迫,发现转基因植株美丽胡枝子中,可溶性糖含量在任何时候均高于未转化植株,并比对照拥有更高的抗干旱胁迫和盐胁迫能力。     

‘云香’水仙NtWRKYY2基因的克隆及功能分析

以‘云香’水仙为材料,采用PCR技术分离中国水仙WRKY转录因子家族成员NtWRKYY2(GenBank登录号为KX056496),其开放阅读框(ORF)长度为867bp,编码289个氨基酸。氨基酸序列比对及系统进化树分析显示,NtWRKYY2编码蛋白含1个WRKY结构域和C2H2锌指结构(Csx4Cx23HxH),属于第Ⅱd类WRKY转录因子。实时荧光定量PCR(qRT-PCR)分析显示,NtWRKYY2在‘云香’水仙应对盐胁迫中显著上调表达。利用InFusion克隆技术成功构建过表达载体pMDC140-NtWRKYY2,并采用农杆菌介导叶盘法转化烟草,获得11株卡那霉素抗性植株,转化子进一步PCR检测结果显示,其中有8株目的基因已成功导入烟草基因组中,转化率为72%。盐胁迫处理和叶绿素荧光参数分析显示,盐胁迫处理后NtWRKYY2过表达的转基因烟草萎蔫和黄化程度小于野生型植株,Fv/Fm值下降幅度小于野生型植株。研究表明,NtWRKYY2过表达的转基因烟草具有抵抗盐胁迫的能力。该研究为水仙抗盐转基因育种提供备选目的基因。     

根癌农杆菌介导AtNHX1基因转化番茄的研究

构建AtNHX1基因植物表达载体,通过农杆菌介导法将其转入番茄。探讨了外植体类型、农杆菌菌株和不同筛选标记对芽诱导分化的影响。对抗性植株进行PCR检测,获得15株转基因植株。对转基因番茄T1代进行80mmol/LNaHCO胁迫处理,转基因植株的相对生长量高于对照植株,显示AtNHX1基因的导入提高了番茄对碱性盐的耐受性。     

‘神马’菊花DREB1A基因的分离及同源遗传转化

逆境诱导转录因子DREB1A基因在植物中的超量表达能够提高植株对低温、干旱、盐渍、高温等非生物胁迫的耐性。从菊花中克隆得到DgDREB1A基因,构建了转基因表达载体pBIG-DREB1A,并转入农杆菌LBA4404中;在建立了菊花高效再生体系的基础上,优化受体再生体系,通过根癌农杆菌介导将DgDREB1A基因转入切花菊‘神马’品种。PCR结果显示,在获得的38株卡那霉素抗性植株中有8株为阳性,表明外源基因已整合到转化植株基因组中,转化效率为3‰。本研究为培育综合抗逆性良好的菊花新品种奠定工作基础。     

菠菜甜菜碱醛脱氢酶基因在烟草中的表达

质粒pLS9含有1．5kb的编码菠菜甜菜碱醛脱氢酶(BADH)基因。经限制酶切后克隆到植物表达载体的35S启动子和PolyA终止子之间。经农杆菌介导转化烟草,获得90多株抗卡那霉素再生植株。经PCR检测证明60％以上再生植株含有BADH基因。转基因植株经Western blot,BADH酶活性测定,BADH酶活性特异性染色法检查和耐盐性分析,证明菠菜BADH基因在烟草正常表达。在叶绿体和胞液中均有BADH酶存在。转基因植株能耐较高浓度盐。     

AtNHX1基因对草木樨状黄芪的转化和耐盐性表达研究

应用RT-PCR技术从100mmol/LNaCl胁迫处理的拟南芥幼中克隆得到编码液泡膜Na /H 逆向转运蛋白的AtNHX1基因cDNA 编码ORF.并在该ORF上游分别插入CaMV 35启动子和TMV RNA5'UTR的Ω片段,而在下游插入NOS polyA构建真核表达盒,进而将该表达盒插入双元植物表达栽体pNT质粒的T-DNA区构建了携带AtNHX1 基因的植物表达载体质粒pNT-AtNHX1.将pNT-AtNHX1 导入农杆菌LBA4404,用农杆菌介导法将AtNHX1 基因导入豆科牧草草木樨状黄芪中,共获得103株Kan抗性再生植株.通过对农杆菌茵液浓度、侵染时间和乙酰丁香酮浓度等影响转化效率的因素进行优化,初步建立了稳定的草木樨状黄芪农杆菌转化体系.经过PCR检测、Southern杂交和RT-PCR检测表明,AtNHX1 基因已被成功整合到草木樨状黄芪基因组中,并且能够正常转录.野生型和转基因株系诱发的愈伤组织进行耐盐生长实验,结果显示相同盐胁迫条件下,转基因愈伤组织的相对生长率显著高于野生型愈伤组织.施加梯度NaCl胁迫后,植株叶片K ,Na 含量和叶片相对电导率测定结果显示,转基因植物叶片比野生型积累更多的Na 和K ,维持较高的K /Na ;转基因株系叶片相对电导率显著低于野生型.上述结果表明,AtNHX1 基因的导入和表达在提高草木樨状黄芪耐盐性的同时减轻了盐胁迫对植物细胞膜的伤害.关键词: AtNHX1 草木樨状黄芪农杆菌遗传转化耐盐性.     

羽衣甘蓝胁迫应答基因BoRS1转化烟草的初步研究

将克隆于羽衣甘蓝的胁迫应答基因BoRS1连入中间载体p35S-2300：：gus：：noster相应位点,成功地构建了含BoRS1基因的植物双元表达载体p35S-2300：：BoRS1：：noster,并通过农杆菌介导法对烟草进行了遗传转化。PCR检测结果表明目的基因BoRS1已成功地导入并整合到烟草基因组中。RT-PCR分析显示,在不同的转基因烟草植株中BoRS1表达量存在差异。转BoRS1烟草的耐干性和甘露醇胁迫研究表明,BoRS1基因的表达对提高植物抗干旱胁迫能力有一定的作用。     

反义磷脂酶Dγ(Anti-PLDγ)基因转化美洲黑杨G2的研究

通过农杆菌介导法将反义磷脂酶DT(Anti-PLDγ)基因转入美洲黑杨G2中以提高其耐盐性。本研究建立了美洲黑杨G2组培再生体系,确立了美洲黑杨G2叶片分化最适宜培养基和生根培养基,进行了卡那霉素(选择性抗生素)敏感性试验,改良了传统的农杆菌介导转化法。经诱导不定芽及生根阶段卡那霉素(选择性抗生素)连续筛选,获得了21株卡那霉素抗性植株,抗性植株经PCR及PCR-Southern杂交检测,有13株均呈阳性,证明Anti-PLDγ基因成功整合到美洲黑杨G2基因组中。耐盐性实验表明,4株转基因植株抗NaCl能力比对照有不同程度提高。     

转HS1prol基因番茄植株的获得

目的为了减少根结线虫对番茄的危害,研究并获得转抗线虫基因HS1prol番茄植株.方法在鉴定表达载体之后,采用CaCl2法制作农杆菌EHA105感受态细胞,然后用冻融法将HS1prol基因转入农杆菌中.通过农杆菌介导法将HS1prol基因导入无菌番茄外植体中,获得抗根结线虫转化再生植株.用卡那霉素筛选到再生植株后,提取抗性芽的基因组,利用设计好的引物进行PCR鉴定.结果与结论目的基因已整合到番茄基因组中,获得了转HS1prol基因番茄植株.     

转HSl^prol基因番茄植株的获得

目的：为了减少根结线虫对番茄的危害,研究并获得转抗线虫基因HSl^prol。番茄植株。方法：在鉴定表达载体之后,采用CaCl2法制作农杆菌EHAl05感受态细胞,然后用冻融法将HSl^prol基因转入农杆菌中。通过农杆菌介导法将HSl^prol基因导入无菌番茄外植体中,获得抗根结线虫转化再生植株。用卡那霉素筛选到再生植株后,提取抗性芽的基因组,利用设计好的引物进行PCR鉴定。结果与结论：目的基因已整合到番茄基因组中,获得了转HSl^prol基因番茄植株。     

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。     

转基因烟草在PEG6000模拟干旱胁迫条件下的生理响应

该研究以转高山离子芥的CbPLDα、CbPLDβ基因烟草为材料,研究了渗透调节物质和保护酶系对PEG6000溶液模拟干旱胁迫的响应机制.结果表明:渗透调节物质脯氨酸、可溶性糖、可溶性蛋白分别以各自不同的响应方式在干旱胁迫下增强转基因烟草的抗旱性,且在所有浓度PEG6000模拟的干旱胁迫下,转基因烟草的脯氨酸、可溶性糖、可溶性蛋白的含量始终显著高于野生型烟草(P<0.05).说明干旱胁迫下两种转基因烟草的渗透调节能力要强于野生型烟草.保护酶系中,超氧化物歧化酶(SOD)和过氧化物酶(POD)在减轻干旱胁迫下转基因烟草膜脂过氧化伤害中起到协同互补作用,而过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)在干旱胁迫下转基因烟草清除过氧化氢机制中发挥主要作用,说明保护酶系在抵制干旱胁迫和保护转基因烟草免受干旱伤害方面具有重要的生物学功能,这从生理角度揭示了高山离子芥CbPLDα、CbPLDβ响应干旱的生理生态机理.综上,高山离子芥CbPLDα、CbPLDβ基因参与了干旱胁迫下烟草的膜稳定性调节、渗透调节物质的积累和抗氧化酶系的调控.该研究结果为提高植物抗旱性研究及应用提供了新的基因资源,对于加强PLD功能研究、补充植物抗干旱理论及抗低温干旱育种种质资源的开发利用具有重要意义.     

Overexpression of monodehydroascorbate reductase in transgenic tobacco confers enhanced tolerance to ozone,salt and polyethylene glycol stresses

Ascorbate (AsA) is a major antioxidant and free-radical scavenger in plants. Monodehydroascorbate reductase (MDAR; EC 1.6.5.4) is crucial for AsA regeneration and essential for maintaining a reduced pool of AsA. To examine whether an overexpressed level of MDAR could minimize the deleterious effects of environmental stresses, we developed transgenic tobacco plants overexpressing Arabidopsis thaliana MDAR gene (AtMDAR1) in the cytosol. Incorporation of the transgene in the genome of tobacco plants was confirmed by PCR and Southern-blot analysis and its expression was confirmed by Northern- and Western-blot analyses. These transgenic plants exhibited up to 2.1-fold higher MDAR activity and 2.2-fold higher level of reduced AsA compared to non-transformed control plants. The transgenic plants showed enhanced stress tolerance in term of significantly higher net photosynthesis rates under ozone, salt and polyethylene glycol (PEG) stresses and greater PSII effective quantum yield under ozone and salt stresses. Furthermore, these transgenic plants exhibited significantly lower hydrogen peroxide level when tested under salt stress. These results demonstrate that an overexpressed level of MDAR properly confers enhanced tolerance against ozone, salt and PEG 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.     

谷子SiRLK35基因克隆及功能分析

为探讨谷子(Setaria italica L.)耐旱抗逆机制,解析类受体蛋白激酶(receptor like protein kinase, RLKs)基因功能,进而为培育谷子抗逆新品种提供依据,本文以干旱处理的谷子“豫谷1号”为材料,通过iTRAQ技术筛选到1个干旱响应的类受体蛋白激酶基因,命名为SiRLK35。以谷子RNA反转录的单链cDNA为模板,经PCR扩增获取SiRLK35基因全长序列。应用qRT-PCR方法,对SiRLK35在NaCl、PEG、ABA、GA、MeJA等不同处理下的表达模式进行分析。进一步构建基因原核表达载体pET28a-SiRLK35,结合斑点法对SiRLK35的抗盐能力进行初步评价。同时构建过表达载体pCAMBIA1301P-SiRLK35转化水稻,并对转基因植株抗盐能力进行检测。结果显示：胁迫及激素处理均可不同程度诱导SiRLK35基因的表达;斑点法研究结果显示,在相同NaCl浓度的LB平板上,含有SiRLK35基因的原核表达载体的大肠杆菌菌株生长状态较阴性对照好,SiRLK35具有一定的抗盐能力;获得的转SiRLK35基因水稻植株对盐胁迫的耐受性高于对照。SiRLK35基因对不同胁迫均可以产生响应,但对盐胁迫的响应较为明显,推测该基因可能在谷子的抗盐及抗逆过程中发挥作用。     

Increase of glycinebetaine synthesis improves drought tolerance in cotton

The tolerance to drought stress of the homozygous transgenic cotton (Gossypium hirsutum L.) plants with enhanced glycinebetaine (GB) accumulation was investigated at three development stages. Among the five transgenic lines investigated, lines 1, 3, 4, and 5 accumulated significantly higher levels of GB than the wild-type (WT) plants either before or after drought stress, and the transgenic plants were more tolerant to drought stress than the wild-type counterparts from young seedlings to flowering plants. Under drought stress conditions, transgenic lines 1, 3, 4, and 5 had higher relative water content, increased photosynthesis, better osmotic adjustment (OA), a lower percentage of ion leakage, and less lipid membrane peroxidation than WT plants. The GB levels in transgenic plants were positively correlated with drought tolerance under water stress. The results suggested that GB may not only protect the integrity of the cell membrane from drought stress damage, but also be involved in OA in transgenic cotton plants. Most importantly, the seedcotton yield of transgenic line 4 was significantly greater than that of WT plants after drought stress, which is of great value in cotton production.     

Genetic modification of Gossypium arboreum universal stress protein (GUSP1) improves drought tolerance in transgenic cotton (Gossypium hirsutum)

Cotton crop suffers shortage of irrigation water at reproductive stage which reduces the yield and fibre quality. Universal stress proteins belong to Pfam00582 which enables several plants to cope with multiple stresses via ATP binding. GUSP1 (Gossypium arboreum USP) is one of such proteins; its amino acids were mutated after in silico simulations including homology modeling and molecular docking analysis. Transgenic cotton plants were developed through Agrobacterium mediated genetic transformation by using mutated pmGP1 and non mutated pGP1 constructs under CaMV35S promoter. PCR and semi-quantitative PCR analyses confirmed the amplification and expression of transgene in transgenic plants. It was revealed that leaf relative water content, total chlorophyll content, CO₂ assimilation as net photosynthesis, stomatal conductance, total soluble sugars and proline content was significantly increased at P ≤ 0.0001 and P ≤ 0.001 in both the pmGP1 and pGP1 transgenic plants as compared to non transgenic control plants. Moreover, relative membrane permeability and the transpiration rate were reduced significantly at P ≤ 0.0001 and P ≤ 0.001 respectively in transgenic plants under drought stress. Furthermore, the T1 transgenic seedlings containing pmGP1 mutated construct showed longer roots under desiccation stress imposed by 5% PEG. Transgene inheritance into the T1 progeny plants was confirmed by amplification through PCR and integration through Southern blot. Hence, our results pave the way to utilize the mutagenized known genes for increasing endurance of plants under drought stress. This will help to increase our understanding of drought tolerance/ sensitivity in cotton plants at the molecular level.Supplementary Information The online version contains supplementary material available at 10.1007/s12298-021-01048-5.     

Transgenic tomato plants overexpressing chloroplastic monodehydroascorbate reductase are resistant to salt- and PEG-induced osmotic stress

RNA gel hybridization showed that the expression of monodehydroascorbate reductase (MDHAR) in the wild type (WT) tomato was decreased firstly and then increased under salt- and polyethylene glycol (PEG)-induced osmotic stress, and the maximum level was observed after treatment for 12 h. WT, sense transgenic and antisense transgenic tomato plants were used to analyze the antioxidative ability to cope with osmotic stresses. After salt stress, the fresh mass (FM) and height of sense transgenic lines were greater than those of antisense lines and WT plants. Under salt and PEG treatments, sense transgenic plants showed a lower level of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), a higher net photosynthetic rate (P _N), and the maximal photochemical efficiency of PSII (F_v/F_m) compared with WT and antisense transgenic plants. Moreover, sense lines maintained higher ascorbate peroxidase (APX) activity than WT and antisense plants under salt- and PEG-induced osmotic stress. These results indicate that chloroplastic MDHAR plays an important role in alleviating photoinhibition of PSII by elevating ascorbate (AsA) level under salt- and PEG-induced osmotic stress.