毛白杨CCoAOMT cDNA片段的克隆与转基因杨木质素含量的调控

用RT_PCR方法从毛白杨 (PopulustomentosaCarr.)中克隆了CCoAOMT基因的cDNA片段 ,并对其序列进行了分析。Northern杂交表明该基因在毛白杨正在木质化的次生木质部高水平表达 ,且与木质化进程同步。构建了该基因反义表达载体 ,根癌土壤杆菌 (Agrobacteriumtumefaciens (SmithetTownsend)Conn)介导转化杂交杨 (欧洲山杨×银白杨 ,P .tremula×P .alba)。采用PCR、PCR_Southern及Southern杂交对获得的转基因植株进行分子检测 ,并测定移栽 5～ 6个月的转基因植株的Klason木质素含量 ,其中一个转基因株系的Klason木质素含量比未转基因对照杨树下降 17.9%。结果表明利用反义RNA技术对杨树CCoAOMT基因的表达可以降低转基因植株的木质素含量 ,达到改善其造纸性能的目的     

烟草花叶病毒装配起始位点反义RNA表达型中间载体的构建

利用基因工程方法培育抗病毒植物新品种的途径之一,是在植株中建立一个产生病毒基因组功能片段的反义RNA的系统。本工作设计并合成了一段烟草花叶病毒(TMV)装配起始位点反义RNA的基因,再以pBR 325为基本质粒,构建了包含带有花椰菜花叶病毒(CaMV)35S启动子和PolyA信号的反义RNA表达单元,以及为筛选转基因植株所必须的NPT-Ⅱ表达单元的中间载体,为以后经土壤农杆菌而获得转基因烟草植株打下了基础。     

葡萄蔗糖转运蛋白在烟草中的反义表达及其对转基因烟草的影响

将葡萄蔗糖转运蛋白VvSUC27cDNA以反义方向插入到含有CaMV35s启动子的真核表达载体pBI121载体中,然后转化到烟草(Nicotianatobacumcv.Samsun)植株中.转反义VvSUC27cDNA的烟草植株在含有20g/L蔗糖的培养基上能够正常生长发育,但是通过切片观察,发现其根部发育较弱,且叶片叶绿体含量增加.可溶性糖测定发现,转基因烟草根部蔗糖含量只有野生型烟草的51%.14C蔗糖吸收实验发现转基因烟草转运外界蔗糖的能力大大降低.     

外源基因在烟草绿色组织中的高效表达研究

研究外源基因在受体绿色组织中的特异表达情况,分别构建由棉花PsbP启动子驱动的GUS基因及CP4 epsps 基因的植物表达载体pBI121-P、pBI121-PE.农杆菌介导法转化到烟草中,获得20株转pBI121栽体、40株转pBI121-P栽体和32株转pBI121-PE载体的烟草阳性植株.组织化学染色分析表明,GhPsbP启动子驱动的GUS基因只在转基因烟草的叶片和茎中表达,在根中不表达;Real-time PCR分析表明,PsbP启动子驱动CP4 epsps基因在叶和茎中的表达量分别是根中的15倍和10倍;草甘膦抗性试验证明,PsbP启动子驱动的CP4 epsps基因在烟草叶及茎的绿色组织中的表达量足以忍受1%浓度草甘膦的毒害.证明GhPsbp启动子可以有效地驱动外源基因在烟草的绿色组织中高效特异的表达.     

甜味蛋白thaumatin基因转入烟草的研究

利用基因工程技术,将分别克隆在两个不同载体上的甜味蛋白thaumatin cDNA基因片段连接成一个完整的cDNA基因,并将该基因克隆进pBI121,构建成表达载体pBI121-tha。通过冻融法导入农杆菌,农杆菌介导叶盘法转入烟草,经过组培,得到转基因的植株。提取转基因烟草总DNA,经PCR,PCR—Southern和Southern杂交证实,甜味蛋白基因已整合到烟草基因组中。RT—PCR结果证明,thaumatin基因已在转基因烟草中转录成mRNA,但SDS—PAGE和甜味尝试都表明thaumatin基因在转基因烟草中没有表达出甜味蛋白。     

荠菜CRABS CLAW的克隆与拟南芥遗传转化

根据GenBank收录的CRC基因cDNA序列设计引物,以短角果荠菜(Capsella bursa-pastoris)为材料,通过RT-PCR扩增出与拟南芥CRC基因同源的全长cDNA,进行测序、比对及同源性分析.结果显示,克隆的该基因cDNA序列与报道的拟南芥CRC序列一致性达到93%,可以推断其为荠菜CRC基因的cDNA(cbCRC).以Ti质粒pWM101为载体,构建了由CaMV35S启动子调控的cbCRC基因植物表达载体pWM101-cbCRC, 采用根癌农杆菌滴注柱头法转化拟南芥,获得了转cbCRC基因的拟南芥植株.转基因拟南芥心皮果荚形态大小发生了一定的变化,说明荠菜cbCRC基因在拟南芥中的表达对拟南芥心皮形态和大小都产生了一定影响,但其并没有使拟南芥表现出荠菜短角果的形态.     

UGPase和反义4CL基因对转基因烟草纤维素和木质素合成的调控

用根癌农杆菌介导法将源于紫穗槐的尿苷二磷酸葡萄糖焦磷酸化酶（UGPase）基因、反义4-香豆酸辅酶A连接酶（4CL）基因以及两者的双价基因分别转移至烟草中。PCR和Southern杂交检测证实外源基因已整合到转基因烟草基因组中。测定全纤维素和Klason木质素含量的结果显示,增强UGPase基因的表达可提高转基因植株的纤维素含量,但对木质素含量没有影响;抑制4CL基因的表达可显著降低转基因植株的木质素含量,但对纤维素含量没有影响;转移双价基因的转基因植株中纤维素含量增加而木质素含量降低。     

油茶CoSAD基因载体的构建、鉴定及功能分析

为揭示油茶( Camellia oleifera Abel)硬脂酰-ACP脱饱和酶( SAD)基因(即CoSAD基因)的功能,构建了该基因的原核表达载体pET28b-CoSAD、植物表达载体pBI121-CoSAD和RNA干扰载体pBI121-CoSAD RNAi,并采用PCR扩增及双酶切方法对3类载体进行鉴定;在此基础上,对原核表达载体中的CoSAD基因进行诱导表达分析,并对pBI121-CoSAD转化的拟南芥〔Arabidopsis thaliana ( Linn.) Heynh.〕sad突变体植株和pBI121-CoSAD RNAi转化的拟南芥野生型植株进行转基因鉴定和主要脂肪酸成分含量分析。 PCR扩增和双酶切结果显示：从 pET28b-CoSAD、pBI121-CoSAD和pBI121-CoSAD RNAi 载体的阳性克隆中均可获得目的条带,表明这3类载体均构建成功;用1 mmol·L-1 IPTG分别诱导0.5、1.0、2.0、3.0、4.0和5.0 h,CoSAD基因均能够在pET28b-CoSAD转化的大肠杆菌BL21感受态细胞中正常表达,能够获得与预测结果相符的相对分子质量约47000的特异目的蛋白条带,且蛋白活性随诱导时间的延长而升高。从pBI121-CoSAD转化的拟南芥突变体植株和pBI121-CoSAD RNAi转化的拟南芥野生型植株中也均可扩增出目的条带。 GC-MS分析结果显示：与拟南芥野生型植株相比,其突变体植株的硬脂酸和棕榈酸含量较高、油酸和棕榈油酸含量较低;但突变体植株经pBI121-CoSAD转化后,硬脂酸和棕榈酸含量降低而油酸和棕榈油酸含量提高;野生型植株经过pBI121-CoSAD RNAi转化后,硬脂酸和棕榈酸含量提高、油酸和棕榈油酸含量降低,表明pBI121-CoSAD转化能够促进拟南芥sad突变体植株体内饱和脂肪酸向不饱和脂肪酸转化,而pBI121-CoSAD RNAi转化对拟南芥SAD基因的表达有明显的抑制作用,这2种重组质粒均可影响拟南芥植株的脂肪酸含量。研究结果表明：油茶CoSAD基因具有调控饱和脂肪酸(硬脂酸和棕榈酸)向不饱和脂肪酸(油酸和棕榈油酸)转化的功能,对茶油的脂肪酸组成具有关键的调控作用。     

铁蛋白基因表达对烟草耐低铁能力的影响

铁是植物生长发育的必需元素。由于土壤中的三价铁离子不能被植物直接利用, 使一些植物经常表现出缺铁症状。为探讨利用铁蛋白基因提高植物耐低铁胁迫的作用, 利用农杆菌介导法将大豆铁蛋白基因SoyFer1和内源反义铁蛋白基因NtFer2的cDNA分别导入烟草基因组, 采集转基因烟草种子。对T1转基因烟草的卡那霉素抗性分析表明, 整合到烟草基因组的外源基因多为单拷贝基因, 也有少数为多拷贝基因。对具有卡那霉素抗性的转基因植株进行PCR检测和Northern杂交分析表明, 外源基因已整合到烟草基因组中, 并且得到了正确表达。将转基因株系移栽到铁离子浓度不同的培养基中生长2个月后进行比较表明, 转大豆铁蛋白基因烟草株系的生长量明显高于非转基因烟草株系, 而转内源反义铁蛋白基因烟草株系的生长量则明显低于非转基因烟草株系。转大豆铁蛋白基因和转内源反义铁蛋白基因烟草株系的叶绿素含量、丙二醛(MDA)含量和过氧化物酶(POD)活性等生理性状也发生了明显变化, 表现为转大豆铁蛋白基因株系的叶绿素含量明显增加, POD活性明显增强, MDA含量明显降低; 而转内源反义铁蛋白基因株系的叶绿素含量、POD活性和MDA含量等则表现为与转大豆铁蛋白基因株系的相反。铁蛋白过量表达提高了烟草耐低铁能力, 而铁蛋白抑制表达则降低了烟草耐低铁能力。     

铁蛋白基因表达对烟草耐低铁能力的影响

铁是植物生长发育的必需元素。由于土壤中的三价铁离子不能被植物直接利用。使一些植物经常表现出缺铁症状。为探讨利用铁蛋白基因提高植物耐低铁胁迫的作用,利用农杆菌介导法将大豆铁蛋白基因SoyFer1和内源反义铁蛋白基因NtFer2的cDNA分别导人烟草基因组,采集转基因烟草种子。对T1转基因烟草的卡那霉素抗性分析表明,整合到烟草基因组的外源基因多为单拷贝基因,也有少数为多拷贝基因。对具有卡那霉素抗性的转基因植株进行PCR检测和Northern杂交分析表明,外源基因已整合到烟草基因组中,并且得到了正确表达。将转基因株系移栽到铁离子浓度不同的培养基中生长2个月后进行比较表明,转大豆铁蛋白基因烟草株系的生长量明显高于非转基因烟草株系,而转内源反义铁蛋白基因烟草株系的生长量则明显低于非转基因烟草株系。转大豆铁蛋白基因和转内源反义铁蛋白基因烟草株系的叶绿素含量、丙二醛（MDA）含量和过氧化物酶（POD）活性等生理性状也发生了明显变化,表现为转大豆铁蛋白基因株系的叶绿素含量明显增加,POD活性明显增强,MDA含量明显降低：而转内源反义铁蛋白基因株系的叶绿素含量、POD活性和MDA含量等则表现为与转大豆铁蛋白基因株系的相反。铁蛋白过量表达提高了烟草耐低铁能力,而铁蛋白抑制表达则降低了烟草耐低铁能力。     

甜味蛋白thaumatin基因转入烟草的研究

利用基因工程技术 ,将分别克隆在两个不同载体上的甜味蛋白 thaum atin c DNA基因片段连接成一个完整的 c DNA基因 ,并将该基因克隆进 p BI12 1,构建成表达载体 p BI12 1- tha.通过冻融法导入农杆菌 ,农杆菌介导叶盘法转入烟草 ,经过组培 ,得到转基因的植株 .提取转基因烟草总 DNA,经 PCR,PCR- Southern和 Southern杂交证实 ,甜味蛋白基因已整合到烟草基因组中 .RT- PCR结果证明 ,thaumatin基因已在转基因烟草中转录成 m RNA,但SDS- PAGE和甜味尝试都表明 thaumatin基因在转基因烟草中没有表达出甜味蛋白     

小麦淀粉合酶基因III片段的克隆及反义和RNA干扰载体的构建

采用RT-PCR方法从小麦品种豫教2号的发育籽粒中克隆出淀粉合酶III基因（starch synthase III, SSIII）部分cDNA片段(509bp) (GenBank No. EF466009),同源性比较结果显示,它与GenBank 上已报道的SSIII基因有高度同源性。以pWM101质粒为基础,构建了由35S启动子调控的SSIII基因的反义表达载体pWM101SSIII;另外,还以pFGC5941质粒为基础,构建了SSIII基因的RNAi干扰载体pFGC5941SSIII,这些载体的构建为研究此基因的功能打下了很好的基础。     

Essential role of caffeoyl coenzyme A O-methyltransferase in lignin biosynthesis in woody poplar plants

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) has recently been shown to participate in lignin biosynthesis in herbacious tobacco plants. Here, we demonstrate that CCoAOMT is essential in lignin biosynthesis in woody poplar (Populus tremula x Populus alba) plants. In poplar stems, CCoAOMT was found to be expressed in all lignifying cells including vessel elements and fibers as well as in xylem ray parenchyma cells. Repression of CCoAOMT expression by the antisense approach in transgenic poplar plants caused a significant decrease in total lignin content as detected by both Klason lignin assay and Fourier-transform infrared spectroscopy. The reduction in lignin content was the result of a decrease in both guaiacyl and syringyl lignins as determined by in-source pyrolysis mass spectrometry. Fourier-transform infrared spectroscopy indicated that the reduction in lignin content resulted in a less condensed and less cross-linked lignin structure in wood. Repression of CCoAOMT expression also led to coloration of wood and an elevation of wall-bound p-hydroxybenzoic acid. Taken together, these results indicate that CCoAOMT plays a dominant role in the methylation of the 3-hydroxyl group of caffeoyl CoA, and the CCoAOMT-mediated methylation reaction is essential to channel substrates for 5-methoxylation of hydroxycinnamates. They also suggest that antisense repression of CCoAOMT is an efficient means for genetic engineering of trees with low lignin content.     

Cloning of cDNA Encoding CCoAOMT from Populus tomentosa and Down-regulation of Lignin Content in Transgenic Plant Expressing Antisense Gene (in English)

cDNA encoding caffeoyl CoA O-methyltransferase (CCoAOMT) from Chinese white poplar ( Populus tomentosa Carr.) was cloned by RT-PCR and sequenced. Northern analysis displayed that the CCoAOMT was expressed specifically in the developing secondary xylem and its expression was coincident with lignification. The antisense CCoAOMT cDNA was transformed into P. tremula×P. alba mediated by Agrobacterium tumefaciens (Smith et Townsend) Conn. Transgenic plants were identified with PCR, PCR-Southern and Southern analysis. Lignin content in 5- to 6-month-old transgenic plants was measured. One of the transgenic lines had significant reduction of 17.9% in Klason lignin content as compared with that of untransformed poplar. The results demonstrate that antisense repression of CCoAOMT is an efficient way to reduce lignin content for improving pulping property in engineered trees.     

Cloning of cDNA Encoding COMT from Chinese White Poplar (Populus tomentosa), Sequence Analysis and Specific Expression

cDNA encoding caffeoyl CoA O-methyltransferase (CCoAOMT) from Chinese white poplar ( Populus tomentosa Carr.) was cloned by RT-PCR and sequenced. Northern analysis displayed that the CCoAOMT was expressed specifically in the developing secondary xylem and its expression was coincident with lignification. The antisense CCoAOMT cDNA was transformed into P. tremula×P. alba mediated by Agrobacterium tumefaciens (Smith et Townsend) Conn. Transgenic plants were identified with PCR, PCR-Southern and Southern analysis. Lignin content in 5- to 6-month-old transgenic plants was measured. One of the transgenic lines had significant reduction of 17.9% in Klason lignin content as compared with that of untransformed poplar. The results demonstrate that antisense repression of CCoAOMT is an efficient way to reduce lignin content for improving pulping property in engineered trees.     

康乃馨ACC氧化酶cDNA的克隆及其反义植物表达载体的构建

以康乃馨（Dianthus caryophyllus L.)花瓣为材料,用改进的异硫氰酸胍一步法提取总RNA,根据已报道的康乃馨ACC氧化酶（1-aminocyclopropane-1-carboxylic acid oxidase,CO)基因的序列设计产合成一对引物,通过RT-PCR方法获得一约1.2kb特异片段,把该片段连接pGEM^(R)-Teasy vector上进行测序,其全长共1156bp,编码区915bp。共编码304个氨基酸残基,序列分析结果表明该序列与GenBankL35152中的康乃馨ACC氧化酶基因的cDNA序列完全相符,推断该基因在康乃馨种内可能是完全或高度保守的,随 后将此片段反向插入植物表达载体pBI121的35S启动子和NOS终止子之间,构建了一反义植物表达载体pBO;又把花特异表达启动子PchsA插入pBI121的HindⅢ Xbal位点构建中间载体pGHB,再把康乃馨ACC氧化酶基因反向插入中间载体pCHB的XbaI Satl位点构建成另一反义植物表达载体pCBO。     

双价抗虫基因植物表达载体的构建

将蝎毒基因BmKITS和几丁质酶基因chitinase2个抗虫基因采用不同的启动子ubi或35S,连到2个高效的植物表达载体pWM101和pBI101中,2个重组表达质粒分别经过限制性酶切分析和PCR鉴定,实验结果表明2个含有双价抗虫基因的植物重组表达质粒均已构建成功．     

番茄转录调控因子基因Pti5植物表达载体的构建及其转化烟草的研究 Construction of a Plant Expression Vector with Tomato Transcription Factor Gene Pti5 and Studies on Transgenic Tobaccos

本实验构建了含有CaMV35S启动子控制下的Pti5-VP16基因的植物双元表达载体pBI121UCH1。通过根癌农杆菌叶盘转化法,将Pti5-VP16基因导入烟草SRI中,经卡那霉素筛选,获得了抗性植株。经PCR和Southern印迹分析,表明抗性植株中整合了Pti5-VP16基因,经抗病性鉴定转基因烟草植株的抗病性明显提高。 Abstract:The plasmid pBI121UCH1 carrying Pti5-VP16 gene under the control of the cauliflower mosaic virus 35s promoter was constructed.Leaf segments of tobacco SRI were infected by Agrobacterium tumefaciens EHA105 with plasmid pBI121UCH1,from which kanamycin resistant plants were obtained.PCR and Southern analysis proved that the Pti5-VP16 gene was integrated into the genomes of the tobacco plants.The disease resistance assay showed that the disease resistance was enhanced in the transgenic tobacco plants.     

Dual methylation pathways in lignin biosynthesis

Caffeoyl-coenzyme A (CoA) O-methyltransferase (CCoAOMT) has been proposed to be involved in an alternative methylation pathway of lignin biosynthesis. However, no direct evidence has been available to confirm that CCoAOMT is essential for lignin biosynthesis. To understand further the methylation steps in lignin biosynthesis, we used an antisense approach to alter O-methyltransferase (OMT) gene expression and investigated the consequences of this alteration. We generated transgenic tobacco plants with a substantial reduction in CCoAOMT as well as plants with a simultaneous reduction in both CCoAOMT and caffeic acid O-methyltransferase (CAOMT). Lignin analysis showed that the reduction in CCoAOMT alone resulted in a dramatic decrease in lignin content. The reduction in CCoAOMT also led to a dramatic alteration in lignin composition. Both guaiacyl lignin and syringyl lignin were reduced in the transgenic plants. However, guaiacyl lignin was preferentially reduced, which resulted in an increase in the S/G (syringl/guaiacyl) ratio. We have also analyzed lignin content and composition in transgenic plants having a simultaneous reduction in both CCoAOMT and CAOMT. The reduction in both OMTs resulted in a further decrease in total lignin content. This is in sharp contrast to the effect that resulted from the reduction in CAOMT alone, which only decreased the syringl lignin unit without a reduction in overall lignin content. These results unequivocally demonstrate that methylation reactions in lignin biosynthesis are catalyzed by both CCoAOMT and CAOMT.