cDNA cloning and functional analysis of 4-coumarate --CoA: ligase (4CL) gene in Chinese white aspen

cDNA encoding 4-coumarate: CoA ligase (4CL) was isolated from the secondary developing xylem of Chinese white aspen (Populus tomentosa) by RT-PCR for the first time, which was 1619 bp in length. The coding sequence and putative amino acid sequence showed 97.53% and 97.00% identity to that of Pt4CL1 in quaking aspen (P. tremuloids), respectively. Molecular analysis indicated that 4CL was encoded by multiple genes in P.tomentosa, its mRNA was highly accumulated in xylem and the expression of 4CL revealed a biphasic pattern in one growing season, almost in phase with the expression of other related enzymes in lignin biosynthesis. Transgenic research showed that expression of antisense 4CL cDNA led to the decreasing of lignin content in transgenic tobaccos, among which the average reduction was 10.3% and the highest could be up to 18.9%. These data suggested that 4CL gene was a potential gene used in altering lignin biosynthesis by biotechnology for producing new materials of papermaking.     

GRP1.8融合4CL1基因调控杨树木质素生物合成

将形成层定位启动子GRP1.8与毛白杨的4-香豆酸:辅酶A连接酶基因融合,叶盘法转化毛白杨741,southern杂交检测表明融合基因已经整合到转基因杨树的基因组中.通过实时定量PCR技术测定mRNA的表达量,GC-MS分析4CL底物香豆酸的含量和定量硫酸木质素含量,分析了4CL基因的过量表达对转基因杨树生理生长的影响.实验结果表明,整合了正义4CL1的转基因植株在茎中的酶活性比野生植株增加了30%,木质素含量增加了40%.     

Introduction of antisense waxy gene into main parent lines of indica hybrid rice

Amylose content in rice endosperm is one of thekey determinants of rice eating and cooking quality, and thepoor quality of indica hybrid rice is closely related to thehigh amylose level in rice grains. In order to improve thegrain quality of the indica hybrid rice by genetic engineering,an antisense fragment of rice waxy gene, driven by theintroduced into three major parent lines of indica hybrid rice,all contain a high amylose level in the grains, via Agrobacte-rium, and more than 100 hygromycin-resistant plants wereregenerated. The analysis of PCR amplification and South-ern blots indicated that the T-DNA containing the antisensewaxy gene had been integrated into the genome of transgenicrice plants. Most of the primary transgenic rice plants grewnormally, and the mature seeds from these transgenic plantswere performed for analysis of the amylose content. Theresults showed that the amylose content in the endosperm ofsome grains was reduced and the lowest reached 7.02% inone homozygous transgenic line, 72.4% lower than that ofthe wild type. The influence of the altered amylose contenton the gelatinization temperature and gel consistency wasalso observed in several homozygous transgenic rice plants.     

毛白杨4CL基因启动子的克隆及初步功能分析

4CL(4-coumarate:CoA ligase,4-香豆酸:辅酶A连接酶)在植物木质素合成途径中催化羟基香豆酸生成羟基肉桂酰CoA,主要在木质部中表达,对植物木质素生物合成具重要调控作用.为研究4CL基因启动子在转基因植物中的表达特性,探索其在植物基因工程研究中的潜在应用价值,利用PCR方法从毛白杨基因组DNA中扩增得到了4CL启动子片段.序列分析表明与美洲山杨(P.tremuloids)的4CL启动子同源性为95%.采用生物信息学方法对该序列进行分析.与GUS基因融合构建双元表达载体,转化烟草的瞬时表达检测可见明显GUS活性.     

转基因大麦茎秆木质素含量及相关特性

以不同转基因大麦(T1,T2)及非转基因对照(WT,NT1)为供试对象,采用比色法及组织化学定位的显微分析并结合傅里叶红外光谱分析(FT-IR),研究了其茎秆木质素含量、代谢关键酶活性、茎秆解剖结构及FT-IR特征.结果表明:不同基因型大麦茎秆木质素含量、薄壁组织及维管束的数目、细胞壁中愈创木基木质素(G)、紫丁香基木质素(S)及A(S)/A(G)(吸收峰面积比)均未见显著改变.与野生型大麦比较,转基因大麦T1的木质素代谢关键酶苯丙氨酸解氨酶(PAL)活性的显著下调(降幅达66%)、过氧化物酶(POD)活性的显著上调(增幅达76%)、茎秆厚壁组织明显变薄及对-羟基苯基木质素(H)比例的显著减小...     

Introduction of antisensewaxy gene into main parent lines ofindica hybrid rice

Amylose content in rice endosperm is one of the key determinants of rice eating and cooking quality, and the poor quality ofindica hybrid rice is closely related to the high amylose level in rice grains. In order to improve the grain quality of theindica hybrid rice by genetic engineering, an antisense fragment of ricewaxy gene, driven by the 5′-franking sequences of the ricewaxy gene, was successfully introduced into three major parent lines ofindica hybrid rice, all contain a high amylose level in the grains, viaAgrobacterium, and more than 100 hygromycinresistant plants were regenerated. The analysis of PCR amplification and Southern blots indicated that the T-DNA containing the antisensewaxy gene had been integrated into the genome of transgenic rice plants. Most of the primary transgenic rice plants grew normally, and the mature seeds from these transgenic plants were performed for analysis of the amylose content. The results showed that the amylose content in the endosperm of some grains was reduced and the lowest reached 7.02% in one homozygous transgenic line, 72.4% lower than that of the wild type. The influence of the altered amylose content on the gelatinization temperature and gel consistency was also observed in several homozygous transgenic rice plants. The two authors contributed equally to this work.     

镉胁迫对转PprI基因油菜生理生化的影响

利用露天盆栽试验研究了不同浓度Cd胁迫下转PprI基因油菜和非转基因野生型油菜对Cd的吸收特性以及生理响应。结果表明：随着Cd胁迫浓度的升高,转基因和非转基因野生型油菜对Cd的吸收均显著增加,且二者地下部分的吸收都大于地上部分;在0.5 mg/kg Cd处理下,转基因和非转基因油菜对Cd的吸收差异不显著,但1和2 mg/kg处理下转基因油菜对Cd的吸收显著低于非转基因野生型油菜。两种油菜对Cd的富集系数和转运系数均随处理浓度的升高而降低;随着Cd浓度的增加,超氧化物歧化酶（SOD）活性呈现先上升后下降的趋势,且在相同处理浓度下,转基因油菜的SOD活性均高于非转基因野生型油菜;丙二醛（MDA）含量呈上升趋势,且其在转基因油菜中的含量均低于非转基因野生型油菜;叶绿素a和叶绿素b的含量随Cd浓度的升高而下降。因此PprI基因的导入,显著降低了油菜对Cd的吸收,提高了其可食部分安全性。     

Introducing antisense <Emphasis Type="Italic">waxy</Emphasis> gene into rice seeds reduces grain amylose contents using a safe transgenic technique

Rice (Oryza sativa L.) eating quality is one of themost important traits. Amylose content (AC) in rice en-dosperm is a major index affecting rice eating quality[1,2].It has a negative correlation with gel consistency of rice[3].Based on amylose content, r…     

甘蓝型油菜3个AP3重复基因植物反义表达载体的构建及遗传转化

利用pFGC5941构建了甘蓝型油菜3个AP3重复基因的植物反义表达载体pFGC5941-BnAP3-2、pFGC5941-BnAP3-3和 pFGC5941-BnAP3-4.采用快速冻融法,将载体导入农杆菌EHA105,转化甘蓝型油菜下胚轴.在5mg/L PPT的MS培养基中筛选,获得反义BnAP3-2基因的抗性再生植株31株,反义BnAP3-3基因的抗性再生植株20株,反义BnAP3-4基因的抗性再生植株42株.PCR鉴定结果显示,获得BnAP3-2反义的基因植株12株,BnAP3-3反义的转基因植     

Cloning and expressional analyses of a cinnamoyl CoA reductase cDNA from rice seedlings

Cinnamoyl CoA reductase (CCR: EC 1.2.1.44),the entry-point enzyme of the llgnin specific biosynthetic pathway, catalyzes the conversion of cinnamoyl CoA esters to their corresponding dnnamaldehydes. Multiple sequence alignment showed that the deduced polypeptide shared 70% similarity and 30% sequence identity at the amino acid level with defined CCR genes from other plant species and they all contain the common signature sequences thought to be the catalytic site as well as the putative NADP binding domain.Using a conserved OsCCR cDNA fragment as the probe for library screening, we isolated the genomic DNA that covered the whole coding region of OsCCR with total length of 3045bp including 4 introns and 5 exons. The open reading frame for our OsCCR gene coBtAin~ 337 amino adds. Northern blot indicated that OsCCR was expressed in different organs with the highest level found in stems. In situ hybridization results showed that OsCCR mRNA was localized mainly along the vascular bundles in stems and leaves, and also in lateral roots that was differentiating from the tiilering node. We conclude that the vascular-localized expression of OsCCR gene may suggest its possible involvement in llgnin biosynthesis. Cloning and characterization of OsCCR will help to clarify how llgniflcations in plants are regulated and will provide a physical basis for creating genetically engineered rice plants with optimal lignin contents.     

转基因小黑杨根际土壤微生物群落特征研究

【目的】根际土壤细菌及真菌群落组成不仅受植物种类及植物生长时期等影响,外源基因的导入也可改变根际微生物群落组成。通过基因工程技术获得的转betA或转TaLEA基因小黑杨(populus simonii x p.nigra)能够提高甜菜碱或晚期胚胎富集蛋白的含量,进而增强转基因植物的抗旱耐盐性,PsnWRKY70是胁迫应答信号转导网络中的负调控因子,WRKY70干扰表达小黑杨的耐盐性显著高于对照株系。在明确了转betA、转TaLEA、转WRKY70小黑杨抗旱耐盐性的同时,开展转基因小黑杨根际土壤细菌、真菌群落组成分析,为其环境生态安全性评价提供参考。【方法】以2年生转TaLEA、betA、WRKY70基因小黑杨及对照野生型(WT)小黑杨根际土壤为试材,利用Illumina-Miseq高通量测序平台对根际土壤微生物进行16S rRNA和ITS测序分析,对根际土壤细菌和真菌群落丰富度和多样性变化、结构差异性、群落组成进行分析,了解转基因活动对小黑杨根际土壤微生物组成的影响。【结果】Alpha多样性分析显示,针对根际细菌群落组成,转betA小黑杨的Simpson指数显著低于WT小黑杨,而转WR...     

转基因抗虫棉花木质素含量及其生物合成关键酶活性研究

通过盆栽试验,以不同转基因抗虫棉花及其亲本对照为供试对象,研究木质素含量及其生物合成关键酶活性.结果表明:与非转基因棉花比较,在蕾期及铃期,转基因抗虫棉花Z30,CCRI41,SGK321叶片、茎秆组织中的木质素含量及叶片内的苯丙氨酸解氨酶和过氧化物酶活性发生了一些变化,但其显著性改变仅特异性地发生在某些品种、某些组织或某些时期内.在蕾期,转Bt基因棉花Z30茎秆组织中的木质素含量比其常规棉对照Z16显著下降,幅度达34.96%;转CpTI-Bt棉花CCRI41茎秆组织和SGK321叶片组织中的木质素含量比其常规棉对照显著下降,幅度分别为27.15%,13.22%.所有供试转基因抗虫棉花的木质...     

植物4CL基因家族结构功能与表达特性研究进展

4-香豆酸：辅酶A连接酶（4CL）是植物苯丙烷衍生物,如类黄酮和木质素等,生物合成途径的一种关键酶,在大多数维管植物中4CL基因以基因家族的形式出现.4CL基因家族成员在植物组织中存在差异表达,并参与不同的苯丙烷类衍生物的生物合成.从4CL酶的基因结构、基因家族组成与表达特性等方面详细论述了当前最新研究进展,这将有助于更好地理解植物4CL基因参与苯丙烷类衍生物途径的合成与代谢机制.     

Characteristics of photosynthesis in rice plants transformed with an antisense Rubisco activase gene

Transgenic rice plants with an antisense gene inserted via Agrobacterium tumefaciens were used to explore the impact of the reduction of Rubisco activase (RCA) on Rubisco and photosynthesis. In this study, transformants containing 15% to 35% wild type Rubisco activase were selected, which could survive in ambient CO2 concentration but grew slowly compared with wild type controls. Gas exchange measurements indicated that the rate of photosynthesis decreased sig-nificantly, while stomatal conductance and transpiration rate did not change; and that the intercellular CO2 concentration even increased. Rubisco determination showed that these plants had approximately twice as much Rubisco as the wild types,although they showed 70% lower rate of photosynthesis, whichRubsico activase and/or the reduction in carbamylation.was likely an acclimation response to the reduction in Rubsico activase and/or the reduction in carbamylation.     

小麦根尖细胞分化过程中木质素合成及其相关酶的活性变化

小麦（Triticumaestivum）根尖细胞分化过程中，苯丙氨酸解氨酶[PAL．EC4．3．1．5]和对香豆酸辅酶A联结酶[4CL，EC6．2．1．12]的活性从分生区至成熟区逐渐增加．PAL活性在分生区细胞中为0.4CL活性近似为0．在成熟区细胞中PAL和4CL活性均增加到最大．木质素的含量变化与PAL和4CL活性存在着平行性，同样在分生区细胞中木质素含量很低，到伸长区稍许增加，至成熟区增加最多．PAL和4CL是木质素合成的开始步骤和关键酶．木质素主要在管状分子和细胞壁次生加厚时沉积，是细胞分化的重要标志之一．     

Secretive expression of Aspergillus fumigatus phytase in tobacco improves phosphorus nutrition in plant

To generate transgenic plants capable of utilizing exogenous phytate,an Aspersgillus fumigatus phytase gene(fphyA) was constitutively expressed in tobacco and recombinant enzyme was secreted from plant roots into the rhizosphere using the signal sequence from tobacco calretieulin.After 40 days of plant growth in hydroponic media,phytase activities in leaves,stems,roots and growth media of transgenic plants were 8.6-fold,7.4-fold,12.6-fold and 14.3-fold higher than those of wild-type plants.Signifi-cant improvements in plant growth and phosphoms(P)utilization were observed in the transgenic plants.When phytate was supplied as the sole P source.45-day-old transgenic tobaccos accumulated 1.0-fold and 0.5-fold more shoot and root biomass than wild-type tobaccos.with a concomitant of 1.7-fold increase in total P concentration.These results indicate that secretive expression of the A.fumigatus phytase improves acquisition and use of P from phytate in plants.     

人参亲环素基因表达载体构建及其在拟南芥中的抗盐活性分析

摘 要 为研究人参亲还素基因的抗盐活性,为该基因在人参抗逆育种方面的应用提供参考,通过植物转基因技术和外施不同浓度NaCl的方法获得阳性拟南芥植株,研究了不同植株类型不同盐浓度下的种子萌发率、植株生存率、植株主根长、植株分支数等相关指标。结果表明：在盐胁迫作用下转基因拟南芥种子萌发率高于野生型拟南芥;在盐胁迫作用下转基因拟南芥植株生存率显著高于野生型拟南芥;在盐胁迫作用下转基因拟南芥植株主根长大于野生型拟南芥;在盐胁迫作用下转基因拟南芥植株分支数与野生型拟南芥植株分支数没有明显差异。可见人参亲还素基因提高了转基因拟南芥抵御高盐胁迫的能力。