农杆菌介导的橡胶草遗传转化体系的建立

以橡胶草为试验材料,利用农杆菌介导法进行遗传转化研究。结果表明：50 mg/L的卡那霉素(Kan)对橡胶草的抗性芽具有很好地筛选效果,生根筛选时,Kan的适宜质量浓度为35 mg/L;最佳抑菌抗生素为羧苄青霉素（cb）,适宜质量浓度为500 mg/L,获得35株抗性试管苗,通过GUS染色及分子生物学检测,最终获得6株转基因植株,转化率为17.1%。可见,获得的橡胶草转基因植株,为橡胶草后续的遗传转化研究奠定了基础。     

纤枝短月藓BeLEA2基因的克隆及表达分析

为探究纤枝短月藓LEA2基因的结构和表达特征,该研究以纤枝短月藓为材料,首次利用PCR克隆技术得到纤枝短月藓BeLEA2基因序列,并对该基因进行分析。结果表明:(1)该基因序列中含有2个外显子和1个内含子,其开放阅读框(ORF)为456 bp,编码151个氨基酸,预测其相对分子质量为16515.96 Da。(2)将纤枝短月藓与其他植物LEA2基因氨基酸序列进行比对,构建系统进化树,结果显示纤枝短月藓与小立碗藓的亲缘关系最近。(3)利用HiTail-PCR技术克隆获得1072 bp的BeLEA2启动子序列,用PlantCARE在线工具对该启动子的顺式作用元件进行预测,结果表明该启动子除了含有核心启动子元件TATA-box和CAAT-box外,还含有ABRE、MYB、MYC、MYB结合位点(MBS)等其他顺式元件。(4)实时荧光定量PCR分析表明,BeLEA2基因在纤枝短月藓不同发育时期和不同组织中都有表达,且对脱水胁迫有响应。以上结果为进一步探究LEA2基因在苔藓植物中的功能及作用机制奠定了基础。     

The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene

Positive selection of transgenic plants is essential during plant transformation. Thus, strong promoters are often used in selectable marker genes to ensure successful selection. Many plant transformation vectors, including pPZP family vectors, use the 35S promoter as a regulatory sequence for their selectable marker genes. We found that the 35S promoter used in a selectable marker gene affected the expression pattern of a transgene, possibly leading to a misinterpretation of the result obtained from transgenic plants. It is likely that the 35S enhancer sequence in the 35S promoter is responsible for the interference, as in the activation tagging screen. This affected expression mostly disappeared in transgenic plants generated using vectors without the 35S sequences within their T-DNA region. Therefore, we suggest that caution should be used in selecting a plant transformation vector and in the interpretation of the results obtained from transgenic approaches using vectors carrying the 35S promoter sequences within their T-DNA regions.     

小蓬NeMT2基因的克隆及其植物表达载体的构建

该研究利用RACE ( Rapid amplification of cDNA ends)技术从小蓬中成功分离编码金属硫蛋白( Metal-lothionein,MT)的cDNA序列,命名为NeMT2,在GenBank中登录号为KT835290。该基因全长590 bp,开放阅读框为237 bp,编码78个氨基酸,编码的氨基酸序列中含有14个半胱氨酸残基( Cys,C),呈C-C,C-X-C,C-X-X-C排列,集中分布在肽链的N端和C端,基因编码蛋白的分子量为7.6036 kD,等电点为4.71。系统发育分析表明,小蓬金属硫蛋白NeMT2与藜科的海蓬子( AEF01492)和盐穗木( AHI62953)同源性最高,其次是甜菜( XP 010667708.1)。生物信息学分析表明,金属硫蛋白NeMT2无信号肽结构,属于非跨膜亲水性蛋白;疏水性分析表明,NeMT2蛋白的35~45个氨基酸之间有较强的疏水性,其中第41位Asp具最强的疏水性(1.444);结构预测分析该蛋白质二级结构的主要元件是无规则卷曲。通过RT-PCR对NeMT2基因的表达分析发现, NeMT2基因在铜矿区和非铜矿区的小蓬叶片中均有表达,但该基因在铜矿区小蓬叶片的表达量明显高于非铜矿区。将小蓬NeMT2基因定向克隆到植物表达载体pCAMBIA1300的35S 启动子下游,构建该基因的植物超表达载体pCAMBIA1300＋NeMT2。该研究结果为进一步研究该基因的功能和小蓬响应重金属胁迫的分子机制提供了一定基础。     

白及小分子热激蛋白BsHsp17.3基因的克隆与表达分析

为了探索人工栽培白及的适宜条件,该研究以湖北省十堰市野生白及为对象,采用同源克隆和3'RACE技术,从白及(Bletilla striata)中获得与热激蛋白合成有关的BsHsp17.3基因,并分析BsHsp17.3基因对不同胁迫的响应。结果表明:BsHsp17.3基因开放阅读框长度为453 bp,编码150个氨基酸;蛋白的分子量为17.42 kD,等电点为6.33。进化树分析表明BsHSP17.3蛋白与同为兰科的铁皮石斛进化关系较近,同在一分支上。半定量RT-PCR分析显示BsHsp17.3基因在白及根、叶、鳞茎及花组织中的表达具有特异性,且BsHsp17.3基因在叶中的表达量较高,在鳞茎及花中不表达。实时荧光定量PCR检测显示BsHsp17.3对非生物胁迫高温、低温具有明显应答反应,20%PEG模拟干旱胁迫不诱导该基因表达,推测该基因在白及防止倒苗过程中可能发挥一定作用。     

Stable expression of the GUS reporter gene in chrysanthemum depends on binary plasmid T-DNA

Three chrysanthemum (Dendranthema grandiflora) cultivars were cocultivated with 2 Agrobacterium tumefaciens strains in combination with 4 pBIN19 derived binary plasmids, all carrying the Nosnptll selection gene and 35Sgus(intron) reporter gene. All binary plasmids transferred DNA to chrysanthemum explants but only pMOG410 gave good stable expression of GUS. This plasmid differs from the other plasmids in 2 aspects: 1) It carries a restored nptll gene and 2) the selection gene is positioned at the left border side of the reporter gene. Cocultivation with AGLO(pMOG410) yielded up to 13 GUS positive shoots per 100 explants. The presence of the gus and nptll gene in recovered shoots was confirmed by PCR and Southern blot analysis.     

The effect of additional virulence genes on transformation efficiency,transgene integration and expression in rice plants using the pGreen/pSoup dual binary vector system

We assessed the effect of four different virulence (vir) gene combinations on plant transformation efficiency and transgene behaviour in rice using the pGreen/pSoup dual binary vector system. Transformation experiments were conducted using a pGreen vector containing the bar and gusA expression units with, or without, the virG542, virGN54D, virGwt or the virG/B/C genes added to the backbone. Additonal vir gene(s) significantly altered plant transformation efficiency and the integration of vector backbone sequences. However, no differences in transgene copy number, percentage of expressing lines and expression levels could be detected. Addition of virGwt was the most beneficial, doubling the overall performance of the pGreen/pSoup vector system based on transformation frequency, absence of backbone sequence integration and expression of unselected transgenes. In 39 of the plant lines, the additional vir genes were integrated into the rice genome. The contribution of super dual binary pGreen/pSoup vectors to the development of efficient rice transformation systems and to the production of plants free of selectable marker genes are discussed.     

pGFPGUSPlus, a new binary vector for gene expression studies and optimising transformation systems in plants

A binary vector containing two reporter gene cassettes has been developed. This vector is ideal for optimising new plant transformation systems. Following optimisation, one of the reporter genes can be replaced with a gene of interest; the second can be used as a marker to confirm transgenic lines, and to estimate locus number and determine zygosity. This allows simple, efficient and economical screening for homozygous single-insert lines and azygous controls.     

The nifH, nifM and nifN genes of Azotobacter vinelandii: Characterisation by Tn5 mutagenesis and isolation from pLAFR1 gene banks

Summary Tn5 was introduced into Azotobacter vinelandii on a suicide vector, pGS9. Three Nif^- mutants were found to carry Tn5 in nifH (MV6), in nifN (MV22), and in or near nifM (MV21), from the results of hybridisation experiments. For MV21 and MV22 this was also shown by complementation with the nif genes of Klebsiella pneumoniae on pRD1. MV6 failed to synthesis the nifH, D and K gene products. MV6 and MV22 fixed nitrogen in the absence of supplied molybdenum while mutant MV21 did not, suggesting that the nifM gene product may be required for the alternative nitrogenase system synthesised in azotobacteria under conditions of molybdenum deprivation. Reconstitution experiments with mutant extracts showed that MV22 (nifN ^-) lacked the FeMo cofactor and that MV21 (NifM^-) synthesised inactive Fe protein. These biochemical phenotypes are identical to those of the K. pneumoniae nifN and nifM mutants, respectively, demonstrating that these genes have the same function in both K. pneumoniae and A. vinelandii. Complementation of the A. vinelandii mutants with pLAFR1 gene banks of A. vinelandii or a. chroococcum yielded three cosmids of interest. pLV10 complemented UW91, a nifH mutant, and corrected the defect in MV6 after recombination with the mutant genome. It also carried nifD (but not nifK) and about 18 kb of DNA upstream from nifH. pLV1 from the A. vinelandii gene bank complemented both MV21 and MV22 as did pLC11, isolated from the A. chroococcum gene bank. Both pLV1 and pLC11 carried part of the nif cluster downstream of nifHDK which also includes nifEN and nifMVS on about 22 kb of DNA.     

滇龙胆GrWRKY5基因的克隆和植物表达载体构建

WRKY蛋白是目前被广泛研究的一类DNA特异结合转录因子,在植物次生代谢物生物合成、植物生长和发育及衰老等生理过程以及生物、非生物防御反应中起重要的调控作用。该研究从滇龙胆幼叶中克隆萜类合成的关键转录因子基因Gr WRKY5,并利用生物信息学方法对基因功能进行预测,构建植物过表达载体。结果表明:根据三年生滇龙胆转录组Gr WRKY5基因序列,设计特异性引物,通过RT-PCR扩增Gr WRKY5 ORF序列,并进行TA克隆、测序和序列分析;构建入门载体p ENTRTM2B-Gr WRKY5,经LR反应后构建植物过表达载体。Gr WRKY5 ORF全长591 bp,编码196个氨基酸,Gen Bank登录号为KF922375,其中第167与170之间的色氨酸(W)、精氨酸(R)、赖氨酸(K)、酪氨酸(Y)组成WRKY蛋白所特有的"WRKY"结构。序列分析表明Gr WRKY5是WRKY超家族的成员。经生物信息学在线软件分析发现,Gr WRKY5的等电点为6.29,脂肪族指数为61.37,不稳定指数为57.80。总平均疏水性为-0.708,为亲水蛋白;含有20种氨基酸,其中天冬氨酸(Asp)和脯氨酸(Pro)含量最高,为8.1%;半胱氨酸(Cys)含量最低,仅为1.0%。氨基酸序列系统发育分析表明,Gr WRKY5与拟南芥中WRKY家族中遗传距离最接近的是WRKY27,属于Ⅱe类成员;与Cr WRKY22和Vv WRKY22蛋白的亲缘关系较近;与Jc WRKY47和Tc WRKY27亲缘关系较远;BLASTp结果显示,Gr WRKY5与欧洲油菜Bn WRKY27-1的同源性最高(为69%);与拟南芥Aa WRKY22的一致性最低(仅为31%)。以Gateway入门载体p ENTR2B和目的载体p K2GW7为基础,成功构建了植物过表达载体p K2-35S-Gr WRKY5,该载体的成功构建为该基因在拟南芥、滇龙胆等植物中的遗传转化奠定了基础,同时为WRKY基因功能的深入研究提供依据。     

Fluorescent labeling of (Na + K)-ATPase by 5-iodoacetamidofluorescein

5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na⁺ + K⁺)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K⁺-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na⁺, K⁺, Mg²⁺, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na⁺, and Mg²⁺, and decreased by K⁺. These fluorescence changes likely reflect ligand-induced stabilization of the E₁ or E₂ states of the enzyme.     

The switching gene swi6 affects recombination and gene expression in the mating-type region of Schizosaccharomyces pombe

Summary The products of 11 switching (swi) genes are required for efficient mating-type (MT) switching in homothallic (h ⁹⁰) strains of Schizosaccharomyces pombe. The MT region of h ⁹⁰ comprises three cassette genes: the expression site mat1: 1 and two silent loci, mat2: 2 and mat3: 3. Besides reducing MT switching, the swi6 mutation leads to deletions in the MT region caused by intrachromosomal cross-overs between two paired cassettes. These deletions only arise if DNA double-strand breaks are present at mat1: 1, which initiate MT switching. Furthermore, swi6 allows meiotic recombination in the K region, a region of 16 kb between mat2: 2 and mat3: 3; in wild-type strains no recombination occurs in K. swi6 also allows the simultaneous expression of two different cassettes in the same haploid cell. Thus swi6 may have an influence on the general chromatin structure in the MT region.