及转基因油菜的获得

通过构建RNAi载体抑制油菜脂肪酸延长酶基因（fae1）表达，获得了油菜脂肪酸延长酶基因（fae1）功能缺失突变体。经酶切及序列测定证实，成功构建完成油菜脂肪酸延长酶基因（fae1）反向重复结构RNAi载体；通过农杆菌介导的油菜转化，经RCR及印染检测，获29个油菜转基因再生株系。     

以油菜脂肪酸延长酶基因fae1为靶标RNAi载体的构建

通过基因工程手段抑制脂肪酸延长酶基因fae1的表达,以阻止芥酸合成,培育不受高芥酸窜粉影响的低芥酸品种.利用GenBank中的fae1基因序列AF490462为模板设计引物和多聚酶链式反应(PCR)从白菜、甘蓝和甘蓝型油菜的12个不同品种中扩增出长1007bp的fae1基因片段,通过对fae1基因片段DNA序列的测定和序列比较,找到长度为428bp高度保守区作为RNA干涉(RNAi)的靶标区.根据RNA干涉(RNAi)双向表达载体设计原则,将428bp fae1基因片段以正反两个方向插入双向表达载体pMCG161中,两个片段用一个wax基因的内含子连接,植物筛选标记基因采用bar基因,从而构建以油菜fae1基因为靶标的RNAi载体.     

油菜PEPase基因的克隆及其对应RNAi载体的构建

磷酸烯醇式丙酮酸羧化酶(PEPase)是控制油菜蛋白质/油脂含量比例的一个关键酶.采用RT-PCR方法从甘蓝型油菜中双4号中扩增出PEPase基因cDNA片段,与已发表的PEPase基因(登录号为D13987)序列同源性为98%.根据RNA 干扰(RNAi)目标序列的选取原则选取两段长度约为400bp的DNA序列,分别克隆到RNAi载体pHBM1301中,构建了油菜中对应于PEPase基因的RNAi载体pHBM1301-PEP1和pHBM1301-PEP2.     

草莓NCED基因正反义表达载体和RNAi载体的构建

根据草莓（Fragaria ananassa Duch）NCED基因序列（GenBank： HQ399498），克隆NCED基因开放阅读框，将该片段插入植物表达载体pBI 121的CaMV 35S 启动子和NOS 终止子之间，构建了正义表达载体pBI 121NCED。克隆NCED基因正义、反义片段和作为内含子的gusA基因片段，以植物表达载体pBI 121为基础，以pCAMBIA2301作为中间载体，通过多次酶切和连接，成功构建了草莓NCED基因RNAi表达载体pBI 121NCEDRNAi和反义表达载体pBI 121NCEDF。经PCR、限制性内切酶酶切和测序鉴定后，成功将pBI 121NCED、pBI 121NCEDF和pBI 121NCEDRNA 3个重组表达质粒导入农杆菌EHA105中。研究结果为进一步研究草莓NCED基因的功能奠定了基础。     

RNAi干扰油菜Δ12-脂肪酸脱饱和酶基因的表达效果

为进一步研究RNAi介导的Δ12-脂肪酸脱饱和酶（FAD2）干扰基因对油菜内源FAD2基因表达的影响,以油菜肌动蛋白(β-Actin)基因为内参照基因,提取转基因油菜幼嫩种子总RNA,通过半定量RT-PCR检测内源FAD2基因的相对表达量。结果表明,T1,T3代转基因种子中FAD2基因的相对表达量与对照相比明显降低。对T3代种子的油酸含量的分析表明：转基因油菜种子中油酸的含量比野生型增加了13.90到32.20个百分点,直接说明RNAi干扰体下调了FAD2基因的表达。因此,种子内源表达的FAD2基因被RNAi干扰体有效沉默,并且产生了能够稳定遗传两代的表型变化。     

甘蔗锌指蛋白基因ShSAP1的RNAi载体构建及对甘蔗的转化

植物中具有A20/AN1锌指结构域的蛋白参与了逆境应答,为研究甘蔗A20/AN1型锌指蛋白基因ShSAP1的功能及在甘蔗抗逆育种方面的价值,本研究通过构建ShSAP1基因的RNAi载体并进行甘蔗的遗传转化,以期通过反向遗传学进行ShSAP1的功能分析。将ShSAP1锌指编码区片段分别以正向和反向插入到中间载体pTCK303内含子的两侧,构建中间载体pTCK-iShSAP1,而后把干扰片段连入pCAMBIA-GUS中,获得RNAi表达载体pCAMBIA-iShSAP1,将该载体转导根癌农杆菌EHA105,通过农杆菌介导法侵染甘蔗胚性愈伤组织,并对部分转化幼苗进行了初步的PCR鉴定。经过限制性内切酶分析和测序验证,RNAi载体pCAMBIA-iShSAP1构建成功;转化幼苗经PPT抗性筛选后,获得了58株抗性植株,对抗性幼苗进行了Bar基因的PCR检测,得到6株PCR阳性植株。本研究完成了ShSAP1的RNAi载体构建及对甘蔗的遗传转化,为ShSAP1的功能研究打下了一定的基础。     

RNAi研究及在植物中的应用

RNAi（RNA interference）技术可通过降解靶基因的mRNA进行基因干涉，是研究多种生物基因功能的有效手段。概述了RNAi的作用机制、特点及其植物RNAi载体的构建。同时介绍了RNAi在植物的基因功能、抗病性、雄性不育等方面的应用。     

香蕉Maasr1基因RNAi植物表达载体的构建及鉴定

以香蕉Maasr1基因为目的基因,载体pBS为中间载体,pBBB、pBI121为表达载体,分别构建含有Maasr1基因的RNAi植物表达载体DBBB-S-I-A和含有Maasr1基因正义片段的植物表达载体pBl121-S,并通过农杆菌介导的方式对构建的该RNAi载体沉默效果进行鉴定.结果表明:(1)利用中间载体pBS和植物表达载体pBBB成功构建了香蕉Maasr1基因的RNAi植物表达载体pBBB-S-I-A;(2)利用载体pBI121成功构建了1个含有Maasr1基因正义片段的植物表达载体pBI121-S:(3)构建的RNAi载体pBBB-S-I-A可以抑制Maasr1基因的表达,具有较好的沉默效果.     

大豆脂肪酸脱氢酶GmFAD3C-1基因的克隆及功能分析

研究通过对大豆脂肪酸脱氢酶GmFAD3家族中4个关键酶基因序列进行比对,与对照JN18相比,大豆低亚麻酸突变体MT72中GmFAD3C-1基因在起始密码子后+966bp处存在一个碱基位点的缺失（G→?）,产生移码突变,导致氨基酸序列上产生较大变化（该突变基因命名为gmfad3c-1）。构建GmFAD3C-1基因超表达及CRISPR/Cas9编辑载体,利用花粉管通道法获得T₂转化植株。脂肪酸脱氢酶酶活测定结果显示,超表达植株T₂籽粒中,酶活与对照相比上升47.62%~78.85%,编辑载体T₂籽粒中,酶活与对照相比下降25.67%~47.11%。脂肪酸相对含量测定的结果进一步表明,GmFAD3C-1基因的表达与植株亚麻酸含量密切相关。     

Inhibition of growth ofErwinia carotovora in vitro by phenolics

Summary The phenolic acids caffeic, cinnamic, ferulic, salicylic, sinapic, and vanillic, together with scopoletin and coniferyl alcohol, at concentrations of ≤1 mg/ml, variously inhibited growth ofErwinia carotovora in Nutrient Broth buffered to pH 6 by 0.2 M phosphate buffer. Chlorogenic acid at 1 mg/ml did not inhibit growth but it did so in agar diffusion bioassays at high concentrations (3–10 mg/well) probably by lowering of the pH of the agar.     

A New Polyunsaturated Brominated Fatty Acid from a Haliclona Sponge

A new polyunsaturated brominated fatty acid possessing acetylenic bonds 1 was isolated from the Indonesian sponge Haliclona sp. The structure of compound 1 was elucidated by analyzing its spectral data. It showed moderate cytotoxicity against cultured cells.     

Fatty Acid Composition of Three Rice Varieties Following Storage

The petroleum ether extractable lipids (PEE-L) and aqueous propan-1-ol extractable lipids (PWE-L) of three varieties of rice were determined gravimetrically and characterised by fatty acid profiles. The content of PEE-L (22·5–28·2 mg g⁻¹) was higher than that of PWE-L (7·4–11·5 mg g⁻¹) in brown rice with the situation reversed in milled rice (3·0–4·5 mg g⁻¹vs. 7·2–8·7 mg g⁻¹). The ratio of unsaturated fatty acid to saturated fatty acid was about two times higher in PEE-L than that in PWE-L for both brown and milled rice reflecting the selective complexation of saturated fatty acids. Rice storage at 37 °C resulted in some minor but statistically significant changes in the fatty acid profile. In the case of brown rice, the only notable changes were a reduction in the amounts of oleic and linoleic acids in the aqueous propan-1-ol extractable fatty acid fraction (PWE-FA) following storage at the higher temperature. Milled rice of all three varieties showed a decrease in linoleic acid content of PEE-L following storage at 37 °C for 4 and 7 months compared to storage at 4 °C. There was no change in fatty acid contents of PWE-L of milled rice when stored at 4 and 37 °C for 4 and 7 months. This implies that the PWE-L (or bound lipids) were more stable than PEE-L (or free lipids) during storage.     

化学杀雄剂1号对棉花花药氨基酸的影响

利用化学杀雄剂 1号及其改良配方对棉花植株进行杀雄处理 ,研究结果表明 ,使用化学杀雄剂 1号后 ,花药 16种蛋白质氨基酸中 Met,Ile,L eu,His,Tyr等 5种氨基酸的含量不受杀雄剂 1号的影响 ,而 Pro,Asp,Glu,Phe,L ys,Val,Thr,Ser,Gly,Arg和 Ala等 11种氨基酸含量受到不同程度的影响。化学杀雄剂 1号中添加脯氨酸以及添加混合氨基酸 (脯氨酸和谷氨酸 )的两种改良配方使花药 Pro,Glu,Phe,L ys,Thr,Gly含量恢复到接近对照水平 ,而对其它氨基酸含量则没有显著影响 ;其杀雄效率与单独喷施化学杀雄剂 1号无显著性差异 ,而毒副作用明显减轻。杀雄剂 1号引起的氨基酸代谢失调可能是造成雄性不育的主要原因之一。     

三种浸种剂对Na2SO4胁迫下小麦种子萌发期耐盐性的影响

为了解黄腐酸、褪黑素、水杨酸三种浸种剂对硫酸盐胁迫下小麦种子萌发期耐盐性的调节效应,分别采用不同浓度的黄腐酸(0.5、1.0和1.5 g·L^-1)、褪黑素(0.01、0.05和0.10 mmol·L^-1)、水杨酸(0.1、0.5和1.0 mmol·L^-1)对小麦进行浸种处理,随后与未处理的小麦种子一并采用100 mmol·L^-1 Na₂SO₄溶液培养,测定各处理下小麦种子萌发期的生长生理指标,并通过主成分分析综合评价其耐盐性。结果表明,0.5～1.5 g·L^-1的黄腐酸、0.01～0.10 mmol·L^-1褪黑素、0.1～0.5 mmol·L^-1水杨酸浸种均提升了小麦根系活力、体内抗氧化酶活性,降低了小麦体内超氧阴离子自由基■产生速率及丙二醛(MDA)含量,缓解了盐分对小麦种子萌发的胁迫程度,增强了小麦种子萌发期的耐盐性,促进了小麦的萌发生长。其中,1.5 g·L^-1的...     

大叶紫薇叶中corosolic acid的分离和测定

采用2次制备TLC分离法,从大叶紫薇叶(LagerstroemiaspeciousL.)中提取了corosolicacid纯品。通过IR,MS,1HNMR和13CNMR谱图鉴定了corosolicacid的结构,用高效液相色谱法(HPLC)测得corosolicacid的纯度为98.8%,并且采用HPLC法对大叶紫薇叶中corosolicacid含量进行了测定,测定方法准确性高、重现性好。     

Variability in dehydrodiferulic acid composition of durum wheat (Triticum durum Desf.) and distribution in milling fractions

The dehydrodiferulic acid content of different common and durum wheat grains and milling fractions was determined by an HPLC procedure. The 8-O-4′, 5–8′ benzofuran, 5–8′ and 5-5′ dehydrodimers were identified in all samples studied and occurred in decreasing relative amounts, respectively. Durum wheats were twice as concentrated in dimers as common wheats. An important genetic variation for dehydrodiferulic acid content was shown within durum wheat grains, whereas the agronomic conditions had no effect. There was 10 to 20 times more dehydrodiferulic acids in external layers (aleurone, bran) than in the starchy endosperm of durum wheat grains. The content and composition in dimers of the inner endosperm did not vary according to genotypes and cultivation conditions. The ratio of dehydrodimers to monomers of ferulic acid which represented an index of dimerisation, was 1·6 times higher in the external layers of the grain than in the endosperm. No relation was found, however, between the degree of ferulic acid dimerisation and the milling behaviour of durum wheat grains.     

Identification of KCS gene family and functional analysis of FAE-like genes from Malania oleifera

KCS(3-ketoacyl-CoA synthase)is the key enzyme catalyzing the first step of very long chain fatty acid(VLCFA)biosynthesis.Studies showed that different KCSs possessed different substrate preference.Malania oleifera are abundance of VLCFAs in its mature seeds,especially the nervonic acid,which is essential for human health.In this study,we identified and characterized 18 KCS genes in M.oleifera genome.Phylogenetic analysis showed that these KCS genes were classified into four subfamilies,including two FAE-like,six KCS-like,eight FDH-like and two CER6.We concentrated on the functional role of two FAE-like genes,Maole003085.T1 and Maole004215.T1 which encoded predicted amino acid residues of 516 and 518 in protein,respectively.Multiple sequence alignment showed that their two proteins contained the known and conserved active sites among FAE-like subfamily.Upon heterologous expression in wild type yeast(Saccharomyces cerevisiae)INVSc1,we found that Maole004215.T1 could produce four new fatty acids including C22:0/C22:1 and C24:0/C24:1,but Maole003085.T1 only produced C22:1.Besides,upon heterologous expression in mutant yeast BY4741-△elo3,we found the Maole003085.T1 could produce C24:0 and C26:0,while the Maole004215.T1 could catalyze the formation of fatty acids C24:0,C26:0 and C28:0.These results showed Maole003085.T1 and Maole004215.T1 had fatty acid elongation activity in yeast,and possessed different substrate preference in the production of different VLCFAs.Interestingly,we found Maole004215.T1 could produce nervonic acid in yeast,which provides molecular basis on the genetic improvement and genic engineering for producing nervonic acid resources by using biotechnological methods.     

香蕉叶片微管免疫荧光染色方法的建立与应用

以香蕉幼叶为材料,通过优化间接荧光免疫染色流程,建立了香蕉微管显微观察方法.结果如下:在微管稳定缓冲溶液中加入37g/L的多聚甲醛,10 mL/L的Triton x-100,10 mL/L的甘油以及10 mL/L的DMSO作为固定液,固定60 min,可以保持微管骨架的原有形态;水解细胞壁30 min,可使抗体有效通过细胞壁.进一步用镰刀菌酸处理香蕉叶片后发现:微管骨架出现解聚和断裂,呈现出典型的抗性响应特征,暗示微管骨架参与了香蕉和枯萎病病原镰刀菌的互作.     

Generation of Internal-Image Functional Aptamers of Okadaic Acid via Magnetic-Bead SELEX

Okadaic acid (OA) is produced by Dinophysis and Prorocentrum dinoflagellates and primarily accumulates in bivalves, and this toxin has harmful effects on consumers and operators. In this work, we first report the use of aptamers as novel non-toxic probes capable of binding to a monoclonal antibody against OA (OA-mAb). Aptamers that mimic the OA toxin with high affinity and selectivity were generated by the magnetic bead-assisted systematic evolution of ligands by exponential enrichment (SELEX) strategy. After 12 selection rounds, cloning, sequencing and enzyme-linked immunosorbent assay (ELISA) analysis, four candidate aptamers (O24, O31, O39, O40) were selected that showed high affinity and specificity for OA-mAb. The affinity constants of O24, O31, O39 and O40 were 8.3 × 10⁸ M⁻¹, 1.47 × 10⁹ M⁻¹, 1.23 × 10⁹ M⁻¹ and 1.05 × 10⁹ M⁻¹, respectively. Indirect competitive ELISA was employed to determine the internal-image function of the aptamers. The results reveal that O31 has a similar competitive function as free OA toxin, whereas the other three aptamers did not bear the necessary internal-image function. Based on the derivation of the curvilinear equation for OA/O31, the equation that defined the relationship between the OA toxin content and O31 was Y = 2.185X − 1.78. The IC₅₀ of O31 was 3.39 ng·mL⁻¹, which was close to the value predicted by the OA ELISA (IC₅₀ = 4.4 ng·mL⁻¹); the IC₁₀ was 0.33 ng·mL⁻¹. The above data provides strong evidence that internal-image functional aptamers could be applicable as novel probes in a non-toxic assay.