水稻叶绿体16S启动子克隆改造、载体构建及转化研究

利用PCR方法从水稻叶绿体基因组DNA中分离16S启动子,并在其下游加入rbcL基因SD序列,以增强该启动子的翻译能力;序列分析表明,除加入的SD序列外,扩增片段与水稻(Oryza sativa)叶绿体基因组DNA序列16S启动子相应区域同源性为100％。将16S启动子与bar基因和gfp基因的融合基因连接,以psbA基因的3′序列为终止子,并以烟草叶绿体trnH—psbA和trnK为同源片段构建了烟草叶绿体表达载体pRl6S。用基因枪转化烟草,转化植株经Southern、Northern检测及后代遗传学分析,发现：16S启动子具有启动活性,融合基因已在烟草叶绿体中稳定整合并遵循母系遗传规律。     

水稻叶绿体转化载体pBS的构建及融合基因功能的初步鉴定

利用PCR方法分别从载体pchN-CAT-Bar和Psk51中分离得到编码膦丝菌素乙酰转移酶基因bar和绿色荧光蛋白基因sgfp,并在bar基因前段连接T7 leader sequence/5′-UTR和T7 10 N-terminus的8个氨基酸序列.运用融合PCR技术,得到bar和sgfp的融合基因片段BS.以水稻叶绿体基因组中的trnI和trnA为同源重组片段,来自烟草叶绿体16S rRNA的Prrn为启动子,psbA基因的TpsbA为终止子,融合片段作为双效筛选标记,构建水稻叶绿体转化载体pBS.大肠杆菌原核表达检测到明显荧光,基因枪轰击TP309愈伤,筛选抗性愈伤.     

甘薯叶绿体表达载体构建及其融合基因在叶绿体中的瞬间表达

利用叶绿体基因组进化中高度保守的特点,根据烟草叶绿体基因组全序列设计引物,从甘薯(Ipomoea batatas)叶绿体基因组中克隆了2个相邻的功能基因rbcL(GenBank登录号为AY942199)和accD(GenBank登录号为AY942200),并以此作为定点整合外源基因的同源重组片段.以来自叶绿体基因组的强启动子Prrn和RpsbA-pro分别驱动选择标记基因aadA及phaC-gfp融合基因,构建成表达盒prrn-aadA-TpsbA-ter与RpsbA-pro-phaC-gfp-RpsbA-ter,然后将这2个表达盒串联在一起克隆进甘薯叶绿体同源片段中,获得甘薯叶绿体定点整合表达载体pSC-GFP.酶切分析证明,所构建的载体符合预期设计;采用该载体对甘薯叶片进行基因枪转化,结果显示,phaC-gfp融合基因可在叶绿体特异启动子和终止子的调控下在甘薯幼嫩叶片中瞬间表达,证明构建的载体pSC-GFP可用于甘薯叶绿体转化.     

小麦叶绿体基因组定点整合表达载体的构建

从小麦中分别克隆了包括rbcL基因3′端部分和完整的psaI、ycf4基因的DNA片段.利用克隆到的DNA片段作为同源重组片段、烟草叶绿体16S rRNA基因的启动子Prrn和PsbA基因的终止子PsbA3′控制筛选标记基因aadA和报告基因gfp的转录,构建了小麦叶绿体基因组定点整合表达载体pRAGY. 用该载体转化大肠杆菌,在激光扫描共聚焦显微镜下,检测到了gfp基因成功表达的产物被激发出的强烈的绿色荧光.     

拟南芥生长素受体基因TIR1启动子的初步分析

以野生型拟南芥（Arabidopsis Columbia）基因组DNA为模板,通过PCR扩增得到拟南芥生长素受体基因T1R1启动子的5个不同长度的系列缺失片段,将这些片断分别克隆到PGM—T载体上。序列分析表明,该启动子系列缺失片段的大小分别为2008bp、1524bp、939bp、532bp和321bp。与已报道的序列完全相同。将不同长度的启动子克隆片断分别与GUS基因融合,构建成表达载体后,在烟草叶片中作遗传转化。分析结果显示：不同长度的启动子片段已整合到烟草基因组中并有GUS酶活性存在,且不同长度启动子片段的表达活性有较明显差异。     

甘薯叶绿体rbcL基因的克隆与序列分析

根据烟草、水稻和菠菜叶绿体的全基因组序列设计引物,以甘薯的叶绿体基因组DNA为模板,PCR扩增包舍甘暑叶绿体rbcL完整基因(GenBank登录号为AY942199)在内的一段序列.序列分析表明:此片段的全长为1 627 bp,包括1 443bp的编码区序列在内.推测编码480个氨基酸,同时构建了此片段的限制性酶切图谱.相似性比较显示,此基因编码区序列与烟草、菠菜、小麦、水稻、玉米、矮牵牛、紫花苜蓿、拟南芥、莨菪、葡萄以及甜菜的rbcL基因核苷酸的同源性为85%～98%,氨基酸的同源性为92%～95%.     

油菜叶绿体基因组同源重组片段的克隆及phb基因定点整合载体的构建

利用叶绿体基因组在进化过程中高度保守的特点,根据烟草、水稻和玉米叶绿体基因组全序列资料,设计合成引物,PCR扩增并克隆了油菜叶绿体两个重要的功能基因rbcL和atpB（GenBank登录号分别为AF267640和AF267641）,并以此作为定点整合外源基因的同源重组片段。以来自叶绿体的强启动子PpsbA和Prrn等驱动PHB合成途径中3个关键酶基因phbA、phbB和phbC,分别构建表达盒,并将它们按照其在原始菌株中的自然转录顺序phbC-phbA-phbB相串联,最后连同选择标记基因aadA表达盒一起,克隆到油菜叶绿体同源片段中,构建成phb基因定点整合载体pRCABZ和pRCABF。酶切及Southern杂交结果证明所构建的转化载体符合预期设计。叶绿体转化及后续工作目前正在进行之中。     

油菜叶绿体基因组同源重组片段的克隆及phb基因定点整合载体的构建

利用叶绿体基因组在进化过程中高度保守的特点,根据烟草、水稻和玉米叶绿体基因组全序列资料,设计合成引物,PCR扩增并克隆了油菜叶绿体两个重要的功能基因rbcL和atpB(GenBank登录号分别为AF267640和AF267641）,并以此作为定点整合源基因的同源重组片段。以来自叶绿体的强启动子PpsbA和Prrn等驱动PHB合成途径中3个关键酶基因phbA、phbB和phbC,分别构建表达盒,并将它们按照其在原始菌株中的自然转录顺序phbC-phbA-phbB相串联,最后连同选择标记基因aadA表达盒一起,克隆到油菜叶绿体同源片段中,构建成pgb基因定点整合载体pRCABZ和pRCABF.酶切及Southem杂交结果证明所构建的转化载体符合预期设计。叶绿体转化及后续工作目前正在进行之中。     

丙肝病毒融合抗原基因NS3-C定点整合入衣藻叶绿体基因组的研究

用ＰＣＲ 方法从丙型肝炎病毒（ＨＣＶ） ｃＤＮＡ 文库中克隆了两段ＤＮＡ 片段,即ＨＣＶ 基因组非结构ＮＳ３区抗原基因（约０．７ ｋｂ）和核心抗原Ｃ区抗原基因（约０．６ ｋｂ）的ｃＤＮＡ 片段。在两段ｃＤＮＡ 间加入连接肽Ｓｅｒ－ Ｐｒｏ－ Ｇｌｙ－ Ｓｅｒ 的密码子序列,构建成融合抗原基因ＮＳ３－ Ｃ。将该融合基因与衣藻叶绿体基因ａｔｐＡ 的启动子和ｒｂｃＬ 基因的３′末端连接,得到丙肝病毒融合抗原基因ＮＳ３－ Ｃ表达盒,再将该表达盒与选择标记基因ａａｄＡ 表达盒和衣藻叶绿体基因组同源片段连接,构建成衣藻叶绿体转化载体ｐＳＳ６。基因枪法转化衣藻叶绿体,经壮观霉素筛选获得转化再生的单藻落,对转基因衣藻的ＰＣＲ 和Ｓｏｕｔｈｅｒｎ 杂交分析表明,融合抗原基因ＮＳ３－ Ｃ已整合到衣藻叶绿体基因组中。     

Bt基因甜菜叶绿体转化载体构建及毒蛋白表达

利用植物叶绿体基因组在进化中高度保守的特点,根据烟草、菠菜、水稻叶绿体基因组全序列资料设计合成引物,PCR扩增并克隆了甜菜叶绿体两个重要功能基因rbcL和atpB(GenBank登录号分别为DQ067450和DQ067451),并以其作为定点整合外源基因的同源重组片段,构建了Bt基因CryIAc甜菜叶绿体定点转化载体pSKARBt,酶切鉴定表明:所构建载体符合预期设计。对克隆菌菌体总蛋白进行了生物杀虫试验,结果表明:Bt基因CryIAc能够在叶绿体特异性启动子及终止子的调控下表达,并对二龄末甘蓝夜蛾有很强的毒杀作用。该载体构建对培育甜菜高抗虫品种具有重要应用价值。叶绿体转化及后续工作正在进行中。     

Studies on Insect Resistance of Bt Transplastomic Plants and the Phenotype of Their Progenies

Insecticidal protein gene CrylA (c) from Bacillus thuringiensis (Bt toxin gene) was placed under the control of psbA5'- and 3'- regulatory regions of rice (Oryza sativa L. ) chloroplast to construct Bt expression cassette, which was ligated with selectable marker aadA cassette and homology regions of tobacco ( Nicotiana tabacum L. ) chloroplast genome to generate transformation vector pTRS8. Leaves of tobacco plant cv. NC89 were transformed with particle bombardment method, plastid transformants were selected by their resistance to 500 mg/L of spectinomycin. Some transplastomic plants were toxic to the third-instar larvae of Helicoverpa zea, and the growth of the survived insects was remarkably inhibited. Genetic and molecular analyses of T1 and T2 progenies of plants with highly efficient insect resistance showed that Bt toxin gene had been inherited in progenies, and spectinomycin resistance was inherited maternally.     

Processing of a chimeric protein in chloroplasts is different in transgenic maize and tobacco plants

Transgenic maize (Zea mays L.) and tobacco (Nicotiana tabacum Petit Havana SR1) plants have been generated, which overproduce a mitochondrial Nicotiana plumbaginifolia manganese superoxide dismutase (MnSOD) in chloroplasts. For this, the mature MnSOD-coding sequence was fused to a chloroplast transit peptide from a Pisum sativum ribulose-1,5-bisphosphate carboxylase (Rubisco) gene and expression of the chimeric gene was driven by the cauliflower mosaic virus (CaMV) 35S promoter. The transgenic MnSOD gene product was correctly targeted to the chloroplasts both in maize and tobacco. However, despite the use of the CaMV 35S promoter, the MnSOD was predominantly localized in the chloroplasts of the bundle sheath cells of maize. Furthermore, the transit peptide was cleaved off at a different position in maize and tobacco.     

Chloroplast transformation in plants: polyethylene glycol (PEG) treatment of protoplasts is an alternative to biolistic delivery systems

Nicotiana plumbaginifolia protoplasts were directly transformed by PEG treatment with a cloned 16S rRNA gene isolated from a double antibiotic-resistant Nicotiana tabacum plastid mutant. Putative plastid transformants were selected in cell culture by their spectinomycin resistance and identified by their unselected streptomycin resistance. Alternatively, cell lines were selected in the presence of both antibiotics. The cell line (and its regenerated plants) selected solely for spectinomycin resistance demonstrated an extensive segregation of streptomycin resistance in subsequent tests, while the double-selected line showed stable resistance for both antibiotics. The resistance markers were inherited maternally. In the putative plastid transformants the origin of the resistance mutations was identified by the absence of an Aat ll site, missing in the donor N. tabacum plastid gene (spectinomycin resistance site) but present in that of wild-type N. plumbaginifolia , and a sequence analysis of the particular nucleotide changes in both resistance sites. Restriction enzyme analysis of total plastid DNA (ptDNA), and the recloning and full sequencing of the fragment introduced, investigated in one of the plastid transformants, showed no DNA rearrangements accompanied with the integration process. Sequence analysis indicated a targeted, homologous integration of the DNA fragment introduced but an unexpectedly complete homology of the parental ptDNA sequences in this region prevented the location of borders. Although the frequency of plastid transformant colonies (2 × 10⁻⁵) should still be improved, this method for stable chloroplast DNA transformation is comparable with or more efficient than the particle bombardment techniques.     

Promoter Function of a Rice Repetitive DNA Sequence RRD3 in Transgenic Plants

A 820 bp rice (Oryza sativa L.) repetitive DNA sequence, the RRD3, was cloned by annealing kinetics. From the sequence analysis, there are several conserved promoter motifs in the sequence such as TATA-box, CAAT-box, etc. In order to detect the promoter function of RRD3, RRD3 was inserted into Ti plasmid pBI121 to replace the CaMV 35S promoter DNA fragment. Both transgenic tobacco (Nicotiana tabacum L.) G28 and rice callus showed the β-glucuronidase (GUS) activity by histochemical assays, the GUS activities of the transgenic tobacco were primarily localized at or around the vascular tissue in leaf and stem. These results indicated that RRD3 can exercise promoter function. The core sequence of promoter of RRD3 will be located.     

Mapping of rRNA genes in an inverted repeat in Nicotiana tabacum chloroplast DNA.

Nicotiana tabacum chloroplast DNA contains two copies each of 16S and 23S rRNA genes. These genes are located in an inverted order as determined from restriction fragment mapping and Southern hybridization to restriction fragments. The position of these genes on the N. tabacum chloroplast DNA molecule has been established relative to a complete map of SalI and SMaI restriction enzyme cleavage sites.     

Characterization of FaRB7, a near root-specific gene from strawberry (Fragariaxananassa Duch.) and promoter activity analysis in homologous and heterologous hosts

Many insect and fungal pathogens posing agronomically important threats specifically target the roots in strawberry. The use of a root-specific promoter to confer expression of resistance genes in a targeted manner has the potential appreciably to benefit the genetic improvement of commercial strawberry varieties. A novel gene, FaRB7, was isolated from strawberry (Fragariaxananassa Duch.) and found to contain motifs characteristic of tonoplast intrinsic proteins (TIPs). Phylogenetic analysis revealed that FaRB7 represents an RB7-type TIP. In strawberry, this gene is expressed predominantly in roots, with very low expression in petioles. A 2.843 kb region representing the FaRB7 gene upstream regulatory sequence was isolated and found to share a number of sequence motifs with the promoter of the Nicotiana tabacum TobRB7 root-specific RB7-type TIP. When cloned upstream of the gusA reporter gene and introduced into strawberry plants, the FaRB7 promoter was shown to direct strong, near root-specific expression with expression patterns very similar to that of the endogenous gene. Furthermore, the FaRB7 promoter was found to confer constitutive expression, comparable to that produced by the cauliflower mosaic virus (CaMV) 35S RNA promoter, in tobacco. Thus, the FaRB7 promoter may be used to achieve near-root-specific transgene expression in strawberry and also represents an alternative to the CaMV 35S promoter for producing constitutive foreign gene expression in heterologous hosts. The FaRB7 full-length genomic sequence and 5' upstream regulatory region have been submitted to the EMBL/GenBank database under accession number DQ178022.     

Expression of 2S Seed Storage Protein Gene of Brassica juncea in Transgenic Tobacco Plants under Constitutive and Seed-specific Promoters

The coding and upstream promoter regions of Brassica juncea 2S seed storage protein gene were amplified by polymerase chain reaction. The plant expression cassettes containing 2S seed storage protein gene under the control of either a constitutive Caulimovirus 35S promoter or a seed specific 2S protein promoter and the polyadenylation signal of a pea rbcS gene were used for Agrobacterium — mediated transformation of tobacco (Nicotiana tabacum cv Petit Havana). Integration of 2S gene was confirmed by Southern blot and PCR analysis of plant genomic DNA. Expression of introduced 2S protein gene was monitored by slot blot analysis of total RNA using 2S protein sequence as hybridization probe and also by immunodot blot analysis using specific antiserum of 2S protein. Expression was either constitutive with CaMV 35S promoter or highly seed-specific with Brassica 2S promoter.     

Molecular characterization of fatty acid alpha-hydroperoxide-forming enzyme (alpha-oxygenase) in rice plants

Genes encoding an alpha-oxygenase, in Nicotiana tabacum and Arabidopsis thaliana, have been recently isolated. However, the reaction mechanism of the enzyme has not so far been elucidated. In this study, a cDNA encoding the fatty acid alpha-oxygenase gene in rice plants was isolated. The deduced amino acid sequence showed high similarity (63.6%) to that of N. tabacum. The gene was cloned into an expression vector system, pQE-30, and expressed in Escherichia coli as a host cell. Palmitic acid as a substrate was incubated with the lysate of the cells, and the products were analysed by HPLC. A compound formed predominantly by the recombinant enzyme was shown to be n-pentadecanal. By incubating the mixture at 0 degrees C, 2-hydroperoxypalmitic acid was detected as a primary product and little formation of n-pentadecanal was detected. Furthermore, uptake of molecular oxygen was observed with an oxygen electrode. This indicated that the gene in rice plants encodes the alpha-oxygenase.