β-伴大豆球蛋白α′-亚基基因ihp-RNAi表达载体的构建

【目的】构建β-伴大豆球蛋白α′-亚基基因具有功能性间隔序列的发夹结构（Intron-hairpin RNAi,ihp-RNA）的RNA干扰(ihp-RNAi)表达载体并转化大豆,为通过RNA干扰技术改良大豆的营养品质奠定基础。【方法】以大豆总RNA反转录获得的cDNA为模板,通过PCR扩增克隆了β-伴大豆球蛋白α′-亚基基因的核心保守序列(400 bp),并将该片段的反义和正义片段插入到重组植物表达载体p3301P的种子特异性启动子7αp下游,将功能性间隔序列intron-SSR插入反义片段与正义片段之间,构建α′-亚基基因ihp-RNAi安全型表达载体p3301-PFNZ-α′-BADH,并进行PCR及双酶切鉴定。利用农杆菌介导法将带有p3301-PFNZ-α′-BADH 的菌株转化“吉农27”大豆植株,对转基因植株进行PCR、Southern杂交检测,并对转基因植株α′-亚基基因的表达量进行RT-PCR。【结果】成功构建了β-伴大豆球蛋白α′-亚基基因ihp-RNAi表达载体p3301-PFNZ-α′-BADH,利用农杆菌介导法转化大豆得到7株阳性转化植株;Southern杂交结果显示,外源基因以1～2个拷贝整合于大豆基因组中;RT-PCR检测表明,β-伴大豆球蛋白α′-亚基基因的表达被明显抑制。【结论】成功构建了β-伴大豆球蛋白α′-亚基基因ihp-RNAi表达载体,获得了α′-亚基基因被明显抑制的转基因大豆植株,为应用基因工程技术进行大豆品质改良奠定了基础。     

LYC-b基因反义表达对番茄果实番茄红素含量的影响

【目的】研究番茄红素β-环化酶(LYC-b)基因反义表达对番茄果实中番茄红素含量的影响,为番茄品质育种提供新方法。【方法】以番茄品种"TTI1117A"和"灵光3号"为材料,构建了分别由CaMV35S启动子驱动的GUS标记基因和反义LYC-b5′基因双价植物表达载体,通过农杆菌GV3101介导,将基因整合到番茄基因组中,采用GUS、PCR以及RT-PCR、Northern杂交进行检测,并测定了转基因植株果实中的番茄红素含量。【结果】由GUS、PCR检测结果可知,筛选出1株"TTI1117A"和4株"灵光3号"转基因植株;RT-PCR、Northern杂交检测结果表明,目的基因在RNA水平上已经表达;对番茄果实中番茄红素含量的测定结果表明,转基因植株果实中番茄红素含量均高于未转基因植株。【结论】LYC-b基因的反义表达具有提高番茄果实中番茄红素含量的作用。     

番茄红素环化酶基因片段克隆及反义表达载体构建

根据已知的番茄红素环化酶基因,即13环化酶基因和s环化酶基因的保守序列设计特异性引物。提取番茄叶片的RNA,经RT—PCR反应,扩增出723bp和569bp的目的片段,将它们分别连接到pEASY—T1 Cloning Vector上,测序鉴定其正确性。克隆到载体上的13环化酶基因用BamHI和SmaI双酶切,将基因反向插入PROK2表达载体上,构建PROK2-LYCb反义表达载体。同样连接到克隆载体上的s环化酶基因用BamHI和XbaI双酶切,将基因反向插入PROK2表达载体上,构建PROK2-LYCe反义表达载体。     

大豆7S球蛋白(α+β)-亚基缺失型种质的α-与β-亚基基因的测序与分析

 【目的】明确大豆7S球蛋白（α+β）-亚基双缺失特性是否是由于α-与β-亚基基因的缺失或变异引起的。【方法】聚丙烯酰胺凝胶电泳(SDS-PAGE)分析和α-与β-亚基基因的PCR扩增、克隆、测序和序列比较。【结果】该种质α-亚基基因特异性引物的PCR扩增结果与对照基本一致,β-亚基基因的则与对照不同。α-亚基基因扩增片段与对照的同源性较高,为90.9%, 二者的区别主要存在于扩增片段的5′-端;β-亚基基因间的同源性较低,仅为53.1%。另外,在该材料β-亚基基因扩增片段的两端,检测到一对短的反向重复序列。【结论】该种质α-与β-亚基同时缺失的特性不是由α-与β-亚基基因的缺失引起的;α-与β-亚基基因的扩增序列与对照存在一定的差异,基因序列间的差异是否为导致α-与β-亚基缺失的直接原因,尚需进一步研究确认。     

杜氏盐藻番茄红素ε-环化酶基因DsLYCE表达载体构建及其在烟草中瞬时表达分析

[目的]ε-环化酶(lycopeneε-cyclase,LYCE)可将番茄红素环化,并与番茄红素β-环化酶共同作用合成α-胡萝卜素,参与植物体内类胡萝卜素的合成途径。富含β-胡萝卜素的杜氏盐藻(Dunaliella Salina)LYCE功能还未解析。[方法]本文利用RT-PCR分离杜氏盐藻DsLYCE基因ORF,并克隆到植物表达载体pCAMBIA1303、构建DsLYCE组成型表达载体pCAMBIA1303-DsLYCE。通过农杆菌介导渗入法在烟草叶组织中瞬时表达DsLYCE,鉴定DsLYCE功能。[结果]烟叶生理生化分析显示,与野生型和空载体对照相比,DsLYCE瞬时表达的烟叶组织中类胡萝卜素积累显著提高(P0.05),含量增加了24%。相似地,转DsLYCE基因的烟草叶片中叶绿素a和叶绿素b含量也明显高于野生型空载对照烟草。这表明杜氏盐藻DsLYCE基因异源表达不仅能促进宿主细胞类胡萝卜素的合成与积累,而且还可以改善宿主细胞的光合作用。[结论]本研究为深入解析杜氏盐藻DsLYCE参与类胡萝卜素生物合成的分子机制以及应用于其他植物的色素代谢遗传改良提供科学参考。     

反义Trx-S基因对小麦子粒淀粉酶活性的影响

研究了反义Trx-S基因对小麦Trx-h基因表达抑制情况，对两个转反义Trx-S基因品系(OOTY5和OOT89)的T3代进行了PCR检测，证明反义Trx-S基因已经遗传到转基因品系T3代植株当中、对不同成熟时期的转基因品系种子的α-淀粉酶和β-淀粉酶活性测定表明，转基因种子在不同成熟时期的α-淀粉酶和β-淀粉酶活性有较大的变化，但与对照相比均有不同程度的降低，其中α-淀粉酶活性最低值出现在花后33～36d，平均降低幅度达到61％；而β-淀粉酶活性最低值出现在花后22d、方差分析表明不同成熟时期的差异均达到显著或极显著水平，表明反义Trx-S基因对小麦Trx-h基因的表达具有明显抑制作用。     

普通烟草β-胡萝卜素羟化酶2基因克隆与生物信息学分析

β-胡萝卜素羟化酶(beta-carotene hydroxylase,BCH)是植物类胡萝卜素合成代谢中的关键限速酶,为获得烟草β-胡萝卜素羟化酶2(NtBCH2)基因序列,采用同源克隆法从烟草中分离NtBCH2的基因组DNA序列,基因登录号为JX101477。结果表明:1)同源克隆法从烟草中克隆出NtBCH2基因,NtBCH2基因组DNA全长2131bp,cDNA为1 083bp,含7个外显子和6个内含子。2)NtBCH2蛋白有309个氨基酸,分子量为34.79kD,具有7个糖基化位点,11个磷酸化位点,4个跨膜域。3)系统发育树与BLAST分析表明,NtBCH2是番茄的SlBCH和辣椒CaBCH2的同源基因;GENEVESTIGATOR数据库芯片结果表明,NtBCH2在幼嫩的茎和子叶中表达量最高。     

毛白杨阿魏酸-5-羟基化酶对烟草木质素的影响

为了研究阿魏酸-5-羟基化酶(F5H)在木质素合成途径中的调控作用,采用CTAB法提取毛白杨RNA,反转录得到cDNA并克隆出毛白杨F5H基因,将其以正义、反义、干涉的基因形式,使用农杆菌侵染法导入烟草基因组中.将得到的转基因植株茎段进行解剖分析,并测定其木质素细胞壁化学组成.转基因植株的木质素、纤维素总量和木质部解剖分析结果与野生型相比均为未表现出明显差异,植株正常生长.F5H的过量表达和基因沉默均不影响植株木质化和生长,可以说明它是木质素合成途径可供选择的一个很好的调控点.     

拟南芥AtSEC14基因反义RNA植株的获得及抗病性检测

为确定拟南芥抗灰霉病相关基因AtSEC14的功能,本试验构建了AtSEC14基因的反义RNA载体;通过农杆菌介导的遗传转化方法,将其转化拟南芥野生型Col-0中;利用潮霉素抗性筛选和PCR检测,获得了阳性转基因植株。利用半定量RT-PCR技术,在转基因株系中未检测到AtSEC14基因的表达,说明该基因反义RNA载体的转入能特异影响AtSEC14基因的表达,表明试验所获得的转基因植株是AtSEC14基因的反义RNA转基因植株。对所获得的反义RNA转基因植株进行抗病性鉴定,发现反义RNA转基因植株对灰葡萄孢的敏感性增强,表明AtSEC14基因在拟南芥抗灰葡萄孢过程中起正调控的作用。     

中国牦牛β-珠蛋白基因的克隆和序列分析

根据黄牛β-珠蛋白基因的序列设计引物,扩增了中国牦牛的β-珠蛋白基因,并对其进行了克隆测序和氨基酸序列比较分析。结果显示,其与黄牛β-珠蛋白基因的同源性很高,达到97%以上;检测到中国牦牛β-珠蛋白基因的2个等位基因7β3A sn和1β35A sn,其中67号牦牛个体中检测到2个等位基因7β3A sn和1β35A sn,64号牦牛个体中检测到等位基因1β35A sn,1078号牦牛个体中检测到等位基因7β3A sn;等位基因1β35A sn是中国牦牛特有的一个等位基因,推测该等位基因编码的第135位氨基酸天冬酰胺,可能会降低牦牛血红蛋白的氧亲和力。     

小麦花粉电穿孔转化因素优化和转基因植株获得及鉴定

[目的]通过优化电穿孔转化技术主要参数,得到候选转基因植株,探索普通小麦(Triticum aestivum L.)电穿孔遗传转化体系.[方法]以冬小麦品种济麦19为受体,GUS为外源基因,将扬花期成熟花粉以最适电场强度和操作温度进行电穿孔处理后,人工授粉济麦19,最终收获种子;利用PCR技术、Southern技术、化学染色方法对T1、T2植株进行鉴定.[结果]电穿孔转化以电场强度6 kV·cm-1、冰上处理花粉,花粉密度为5×106个/mL,以及去雄后第5天授粉为最佳参数;Southern杂交检测结果表明,T2阳性植株的2个株系检测到杂交信号,同时其幼根、叶片等组织经GUS表达活性的组织化学染色检测,呈现蓝色反应.[结论]利用小麦花粉电穿孔转化法可以成功将外源基因整合到小麦基因组中并稳定遗传至T2,且外源基因GUS能够得到表达.     

根癌农杆菌介导转化获得耐逆性增强的高羊茅转基因植株

 为改良草坪型高羊茅（Festuca arundinacea Schreb.）的耐逆性，以成熟种子来源的胚性愈伤组织为受体材料，通过农杆菌介导法将拟南芥（Arabidopsis thaliana）耐逆相关CBF1基因导入4个供试品种的基因组，经GUS染色、PCR检测和Southern杂交分析验证，获得了112株转基因植株，转化频率为0.92% ~2.87%，不同品种间存在差异。试验表明，在高盐与高渗胁迫下，转基因植株具有显著生长优势，存活率极显著高于非转化对照植株，经低温、高温、干旱和高盐等逆境胁迫处理后的叶片相对电导率平均较对照植株低25% ~ 30%，证明转基因植株的耐逆性有所增强。考察发现，非胁迫条件下CBF1基因的组成型超表达使转基因植株的生长受到抑制。     

农杆菌介导rolC基因转化烟草植株的研究

通过根癌农杆菌介导手段，将来自发根农杆菌的rolC基因转化到烟草（Nicotiana tabacum)植株的基因组，并借助GUS组织化学法，PCR扩增法和Southen杂交进行了鉴定证实。 rolC基因改变转基因烟草植株某些性状的特点，如植株高变矮，花冠变小，雄蕊 变短等，还发现rolC基因具有延迟转基因烟草植株花期的作用。     

Production of Transgenic Tall Fescue Plants with Enhanced Stress Tolerances by Agrobacterium tumefaciens-Mediated Transformation

In order to improve stress tolerances of turf-type tall fescue （Festuca arundinacea Schreb.）, Agrobacterium tumefaciens strain EHA105 carrying plasmid pCMD containing stress tolerance-related CBF1 gene from Arabidopsis thaliana was used to transform mature seeds-derived embryogenic calli of four cultivars. A total of 112 transgenic plants were regenerated from 32 independent lines and verified by histochemical detection of GUS activity, PCR assay and Southern hybridization analysis. The transformation frequency ranged from 0.92 to 2.87% with apparent differences among the cultivars. Stress tolerances of transgenic plants were enhanced, which was shown by the facts that transgenic plants had distinct growth superiority and significantly higher survival rate than non-transformed ones under high salinity and high osmosis stresses, and that relative electronic conductivity of in vitro leaves treated with low and high temoeratures, dehvdration and high salinity stresses was 25-30% lower in transgenic plants than in control plants.In addition,it was observed that growth of transgenic plants was inhibited due to constitutive overexpression of CBF1 gene under normal environmental conditions.     

Molecular Detection and Disease Resistance Identification of Transgenic Wheat with pti5-vp16 Gene

The immature embryos of wheat cultivars Liaochun10, Tiechun1 and Fengqiang3 were bombarded with gold particles coated with pti5-vp16 by gene gun and disease resistant regenerated plants were attained. In order to confirm that the plants are genuine transformed ones, a series of molecular tests were conducted as follows. Firstly, transient GUS expression test on embryos two days after bombardment was done. There were many obvious blue spots produced on the surface of bombarded embryos after GUS staining, in which the maximum reached to 85 blue spots per embryo. Secondly, PCR test was performed with DNA from the regenerated plants obtained after double selection with ppt. 6 plants were found PCR test positive. At last, further verification analysis using dot hybridization and southern blotting was carried out on those PCR positive plants and the strong hybridization signals appeared as expected. All the above tests were uniformly indicated that the disease resistant regenerated plants were true transgenic plants. When inoculated with Blumeria graminis, transgenic wheat plants of PCR positive results were mostly resistant(R) after 7 days, and resis-tant, moderate resistant(MR), moderate susceptible(MS) at 14 days respectively. The disease severity of them was distinctively lighter than that of control.     

RolB基因对三倍体毛白杨的转化研究

为了提高三倍体毛白杨的生根能力,把rolB基因转入三倍体毛白杨中,获得20个转化植株,在无激素培养基上,转化植株的生根率达到100%,而未转化植株的生根率只有15%~20%。PCR扩增证明目的基因已经插入到植物的基因组中。对部分rolB基因转化植株进行了GUS组织化学染色检测,转化植株叶片中有GUS活性的表达。     

农杆菌介导水稻幼胚转化获转基因植株

本研究以根癌农杆菌ＬＢＡ４４０４（ｐＴＯＫ２３３）为供试菌株,以水稻幼胚为供试材料,对影响农杆菌介导基因的转化效率的因素进行了研究,确立了农杆菌介导的水稻高效转化体系。采用所确立的转化体系转化了Ｒａｄｏｎ、中国９１、０２４２８ ３个水稻品种（系）幼胚,结果表明,ｇｕｓ Ａ基因的瞬时表达率均在７０％左右,最高为７６％;ｇｕｓ Ａ基因稳定表达率均在２０％以上,平均为２３％。转基因植株的ＧＵＳ活性检测及Ｓｏｕｔｈｅｒｎ杂交分析表明,外源基因事于水稻基因组中,进行了有效地表达,且能稳定地遗传给后代,外源基因在后代遗传符合孟德尔遗传规律。     

Xa21基因导入水稻广亲和恢复系SWR20的研究

以成熟胚愈伤组织为转化受体 ,经农杆菌介导法将显性广谱白叶枯病抗性基因 Xa2 1导入高感白叶枯病的水稻广亲和恢复系 SWR2 0中 ,共获得 4 3个独立的转基因株系 ,1 0 0余株转基因水稻植株。经 GUS活性、潮霉素抗性检测及PCR分析表明 ,外源基因已整合到转基因水稻基因组中 ;白叶枯病原菌接种试验表明 ,大部分 T0 代转基因水稻植株的抗病性有明显提高 ,多数植株表现抗或高抗 ,个别为感病。经对农杆菌介导的水稻转化技术作进一步改进后 ,GUS瞬间表达率可高达 92 .5% ,稳定转化频率达到 2 7.6%     

Enhanced Stem Nematode Resistance of Transgenic Sweetpotato Plants Expressing Oryzacystatin-I Gene

Enhanced stem nematode resistance of transgenic sweetpotato (cv. Lizixiang) was achieved using Oryzacyslatin-I (OCI)gene with Agrobacterium tumefaciens-mediated transformation. A. Tumefaciens strain EHA 105 harbors a binary vector pCAMBIA1301 with OCI gene, gusA gene and hptⅡ gene. Selection culture was conducted using 25 mg L-1 hygromycin. A total of 1715 plants were produced from the inoculated 1 450 cell aggregates of Lizixiang via somatic embryogenesis. GUS assay and PCR analysis of the putative transgenic plants randomly sampled showed that 90.54% of them were transgenic plants. Transgenic plants exhibited significantly enhanced resistance to stem nematodes compared to the untransformed control plants by the field evaluation with stem nematodes. Stable integration of the OCI gene into the genome of resistant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated OCI gene ranged from 1 to 4. Transgene overexpression in stem nematode-resistant plants was demonstrated by quantitative real-time PCR analysis. This study provides a way for improving stem nematode resistance in sweetpotato.     

Performance of autoregulatory Senescence-inhibition Gene in Rice

The Performance of autoregulatory senescence - inhibition gene PSAG12 - IPT in rice has been investigated in the study. 422 transgenic plants from 134 independent resistant calli were ohmined from 4 rice varieties through Agrobacterium -mediated transformation. Among them, 233 were positive PSAG12 - IPT transgenic plants identified by GUS histochemical assay and PCR analysis.Southern analysis shorted the transgene was randomly integrated into rice genome, of which 42.29% was single copy. Investigations on photmynthesis function and agronomic characters R1 generation shorted that chlorophy Ⅱ content and photmynthesis rate of flag leaves in transgenic plants, were 41.23% and 50.24% higher than the control wild-type rice, respectively. The growth duration and plant height of the transgenic plants were similar to the control. Variations of other characters were dependent on the varieties. For the variety Millin with significant aging phenomenon in China, its total grains per hill, its seed setting rate and 1000-grain wdght were increased by 40.44%, 8.05% and 8.32% respectively. The results indicated that after leaf senesomce of varieties liable to age was delayed, the seed setting rate and the filling degree of seeds were improved, which finally resulted in significantly increased seed yield and biomass per hill. The new variety Wuyujing 2 without serious aging problem, was also increased in the panicles per hill, the totslgrains per hill, the seed yidd per hill and biomass in different degrees.