中间锦鸡儿CiDR1的克隆及干旱胁迫下的表达分析

[目的]研究并了解中间锦鸡儿CiDR1基因功能及其对干旱胁迫的响应,为抗性育种提供候选基因。[方法]通过RACE技术从中间锦鸡儿中克隆CiDR1基因的c DNA全长,利用生物信息学分析软件对其基因结构及功能进行分析和预测。再通过qRT-PCR技术对干旱胁迫后的幼苗中的CiDR1表达模式进行研究。[结果]从中间锦鸡儿中克隆到CiDR1基因的c DNA全长共计4 297 bp,Gen Bank登录号为KP277100。生物信息学分析表明,预测的CiDR1蛋白序列中含有1 243个氨基酸残基,具有抗病基因特征结构域TIR、NB-ARC、LRR等,其等电点为6.35,不稳定指数为42.91,不具备信号肽,为非分泌蛋白,定位于细胞质中。定量PCR检测发现,CiDR1基因在幼年期的茎中表达量较低,在成年期的叶片中表达量较高;在干旱胁迫处理后的幼苗中,CiDR1表达水平有明显下降,表明该基因的表达受干旱抑制,可能与中间锦鸡儿适应干旱相关。[结论]中间锦鸡儿在干旱胁迫后其根、茎和叶中CiDR1的表达均明显下降,表明CiDR1的表达受干旱抑制,可能与中间锦鸡儿适应干旱相关,进一步研究发现CiDR1在根、茎、叶中的表达水平可能受发育阶段调控。     

Isolation and sequence analysis of a Dof protein gene (PtDof1) in Populus

Both cDNA and DNA clones of PtDof1 (GenBank Accession No. FJ402844 and FJ402845) were isolated from plants grown in tissue culture of Populus tomentosa. The DNA sequence is 1597 bp including two exons and one intron. The cDNA is 969 bp in length with a 765 bp open reading frame which is capable of encoding 255 amino acids. The deduced amino acids sequence of the PtDof1 protein shares 65%, 56% and 55% identity with Vitis vinifera (CAO48618), Nicotiana tabacum (CAA08755) and Glycine max (ABI16022) Dof protein by blast analysis in GenBank. Phylogenic analysis suggests PtDof1 gene could belong to the Dof gene family. PtDof1 protein contains an unusual conserved single zinc finger with the pattern of C-X2-C-X21-C-X2-C, which may play a functional role in tissue-specific expression and possibly the auxin response of endogenous plant genes.     

哈茨木霉天冬氨酸蛋白酶基因SA76的3＇RACE扩增和序列分析

由EST获得全长cDNA对于结构基因组学和功能基因组学都是至关重要的,cDNA末端快速扩增技术RACE是该领域中的重要研究方法.利用BD SMART RACE技术扩增编码分泌天冬氨酸蛋白酶SA76基因的3＇末端,将其与哈茨木霉cDNA文库中的SA76基因的EST序列进行序列拼接,获得2019bp的全长cDNA序列,其开放读码框长1593bp,5＇非编码区266bp,3＇非编码区201bp,编码530个氨基酸,有信号肽.哈茨木霉天冬氨酸蛋白酶基因与玉蜀黍赤霉、粗糙脉孢菌、球毛壳菌天冬氨酸蛋白酶基因的同源性分别为53%, 37%, 36%.利用BD SMART RACE技术首次从哈茨木霉中克隆天冬氨酸蛋白酶基因,为验证SA76基因的功能奠定基础,为进一步研究蛋白酶的作用机制及生物防治功能提供依据.     

平榛ChWRKY28基因克隆及表达模式分析

[目的]研究平榛ChWRKY28基因序列特征及其在不同非生物胁迫下的表达规律.[方法]以平榛为试材,采用RACE-PCR方法进行基因克隆;利用实时荧光定量PCR方法检测基因在不同组织及不同非生物胁迫下的表达模式.[结果]表明:克隆得到的WRKY基因,全长1 342 bp,基因内部包含1个长963 bp的完整开放阅读框,编码320个氨基酸残基,命名为ChWRKY28.构建的系统发育树表明:该序列与拟南芥AtWRKY28及杨树PtrWRKY93的关系最近,相似性分别为49%和60%.基因表达分析表明:ChWRKY28在雄花序、雌花芽及茎中均有表达,但在茎部(皮)中的表达量高于雄花序和雌花芽中的表达量,具有组织表达特异性;低温、干旱及盐胁迫均能诱导ChWRKY28基因的表达,但受诱导程度存在差异.亚细胞定位分析结果表明:ChWRKY28蛋白分布在细胞核内,是一个核蛋白.[结论]推测ChWRKY28基因可能参与植物响应非生物胁迫的信号转导过程.     

Development of transgenic hybrid sweetgum (<Emphasis Type="Italic">Liquidambar styraciflua</Emphasis> × <Emphasis Type="Italic">L. formosana</Emphasis>) expressing γ-glutamylcysteine synthetase or mercuric reductase for phytoremediation of mercury pollution

Using Agrobacterium-mediated gene transfer, we generated transgenic hybrid sweetgum (Liquidambar styraciflua × L. formosana) overexpressing two types of genes to enhance plant remediation of mercury-contaminated soil and water: bacterial γ-glutamylcysteine synthetase gene (ECS), the first and most important enzyme in phytochelatin synthesis, or various genes encoding a mercuric ion reductase (merA9, merA18, merA77). Hybrid sweetgum proembryogenic masses (PEMs) constitutively overexpressing ECS were able to grow in the presence of 50 μM HgCl₂, which inhibited wild-type PEMs, but plantlets regenerated from the PEMs had abnormal form and did not survive for more than a few weeks following germination. In contrast, mature somatic embryos generated from PEMs constitutively overexpressing merA9 and merA18 converted to normal plantlets on germination medium containing 25 μM HgCl₂, while control embryos were killed on 25 μM Hg(II)-medium. Transgenic merA plantlets displayed enhanced resistance to Hg(II) and released Hg(0) two to three times more efficiently than the wild-type plantlets.     

杜仲1-脱氧-D-木酮糖-5-磷酸还原异构酶基因cDNA全长克隆与序列分析

以杜仲叶片cDNA为模板,采用反转录RCR及RACE技术分离出DXR基因cDNA全长。序列分析结果表明该基因序列全长1 814 bp,共编码478个氨基酸,推导的蛋白质分子量为51.71 kD,理论等电点5.79,命名为EuDXR。推导的EuDXR蛋白质序列具有植物DXR酶的5个典型基序,并预测出26个潜在的功能位点。系统进化树分析表明EuDXR蛋白与玉米、水稻亲缘关系最近,其次为橡胶、拟南芥、烟草。     

Over-expression of Tryptophan Decarboxylase Gene in Poplar and its Possible Role in Resistance Against Malacosoma disstria

Amines and their derivatives are known to influence insect behavior involved in feeding and reproduction. In order to examine the feasibility of improving the resistance of poplar to insect pests by the introduction of a plant-derived amine-generating transgene, explants from the hybrid poplar clone ‘INRA 717 1B4’ (P. tremula ×P. albo) were transformed with a Camptotheca acuminata tryptophan decarboxylase (TDC, EC 4.1.1.28) cDNA driven by the CaMV 35S promoter. The enzyme TDC catalyzes the decarboxylation of tryptophan to tryptamine, which, in addition to being a bioactive amine itself, is known to act as a precursor of various other indole derivatives. Putative transgenic lines were confirmed by PCR for the TDC1 gene sequence and by the expression analysis of the transgene mRNA and encoded protein. No visible phenotypic changes were associated with ectopic TDC1 expression. Chemical and radiotracer analyses of the transgenic plants revealed tryptamine accumulation as high as 4 mM in leaf tissue, and suggested that the tryptamine produced by ectopically expressed TDC was not further metabolized. Insect bioassays with the TDC transgenic plants showed that the tryptamine accumulation was consistently associated with adverse effects on feeding potential and physiology of Malacosoma disstria (forest tent caterpillar).     

A comparison of genetic diversity and differentiation in five Chinese pines using cpSSR and AFLP markers

Gene diversity and genetic differentiation in five Chinese pines, Pinus henryi, P. tabulaeformis, P. yunnanensis, P. taiwanensis and P. massoniana, were compared using amplified fragment length polymorphism (AFLP) and simple chloroplast sequence repeat (cpSSR). High genetic differentiation and median gene diversity with cpSSR markers were found both at population and species level, while median differentiation and higher gene diversity in AFLP data. Measures of subdivision that consider similarity between haplotypes offered better information on the geographic structure of plants than the standard subdivisions. Among several methods analyzed in AFLPs, the square root method provided downwardly biased estimates of the genetic parameters, while the Lynch and Milligan method over-estimated genetic diversity due to a small sample size. The Bayesian statistic was the most accurate and popular method for these dominant species and its value of species differentiation (θ ^B = 0.1035) was close to the parameter given by analysis of molecular variance (AMOVA). __________ Translated from Acta Botanica Boreali-Occidentalia Sinica, 2007, 27(12): 2385–2392 [译自: 西北植物学报]     

Isolation and sequence analysis of the promoter and an allelic sequence of the iron-sulfur protein subunit gene from the white-rot fungusPleurotus ostreatus

We have isolated a structural gene ofsdil, which encodes the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (EC 1.3.99.1), from a white-rot basidiomycete,Pleurotus ostreatus. Here we report isolation of the promoter region ofsdil and an allelic sequence encoding the second-type cDNA fragment isolated in the former experiments. The nucleotide sequence analysis of the promoter region revealed the existence of putative CAAT and TATA boxes, which permits us to develop an expression system in this species. The Southern blot analysis and the restriction fragment length polymorphism assay using monokaryotic strains demonstrated that no family genes tosdil exist in the haploid genome ofP. ostreatus. Moreover, a genetic analysis to detect a linkage between thesdil genotypes and flutolanil resistance in the mutantP. ostreatus strains was also developed.     

Nutrient cycle of planted forest of Pinus tabulaeformis in the Miyun Reservoir Watershed, Beijing

We studied the nutrient cycle of a planted forest of Pinus tabulaeformis in the Miyun Reservoir Watershed, Beijing. Results show that the total biomass of P. tabulaeformis stands at age 29 in the experimental area is 92627 kg/hm², and the total nutrient store is 695.17 kg/hm² including nitrogen (N), phosphorus (P), kalium (K), calium (Ca) and magnesium (Mg). The sequence of their contents in different organs was given as follows: needle>branch> trunk>root. The annual amount of 85.37 kg/hm² of five nutrient elements were assimilated by P. tabulaeformis, about 0.34% of the total store in soil, and 3.30% of available nutrient store in soil depth from 0 to 30 cm. The nutrient annual retention is 35.92 kg/hm², annual returning 49.46 kg/hm², the rain input 26.04 kg/hm² to the five nutrient elements. The parameter absorption coefficient, utilization coefficient, cycle coefficient and turnover period were cited to describe the nutrient elements cycle characteristic of the planted forest ecosystem of P. tabulaeformis. The absorption coefficient is the ratio of plant nutrient element content to soil nutrient element content, and its sequence of five nutrient elements was given as follows: N>P>K>Ca>Mg. Utilization coefficient is the ratio of the nutrient element annual uptake amount to the nutrient element storage in standing crops, and its sequence of five nutrient elements was: Mg>K> P>N>Ca. The big utilization coefficient means more nutrients stored in the plant. The cycle coefficient is the ratio of the nutrient element annual return amount to the nutrient element annual uptake amount, its sequence: Ca>N>P>K>Mg. Turnover period is the ratio of the nutrient storage in the crops to the annual returning, its sequence: Mg>K>P>N>Ca. __________ Translated from Journal of Beijing Forestry University, 2008, 30(3): 51–56 [译自: 北京林业大学学报]     

Prokaryotic expression analysis of an NBS-type PtDRG01 gene isolated from Populus tomentosa Carr.

In order to investigate the protein features of an NBS gene (PtDRG01, EF157840) isolated from Populus tomentosa Carr., the full-length open reading frame was fused into a prokaryotic expression vector pGEX-KG. PCR analysis and double endonuclease digestion showed that the recombinant vector was successfully constructed and transferred into an expression host E. coli strain XA₉₀. It was indicated by SDS-PAGE analysis that IPTG treatment successfully induced the expression of a fusion protein of about 79 kD, which was consistent with the predicted value. In addition, the prokaryotic expression system was also optimized. The result suggests that 1 mmol/L IPTG treatment for 4 h at 37°C was most effective, and the product was predominately soluble and not extra-cellular secreting. Moreover, the fusion protein was purified with an affinity chromatography column using Glutathione Sepharose 4B. This work will lay a foundation for further studies on biological functions of the PtDRG01 gene. __________ Translated from Acta Botanica Boreali-Occidentalia Sinica, 2008, 28 (5): 0882–0888 [틫自: 컷놱횲컯톧놨]     

Cloning and sequence analysis of a glucose-6-phosphate dehydrogenase gene <Emphasis Type="Italic">PsG6PDH</Emphasis> from freezing-tolerant <Emphasis Type="Italic">Populus suaveolens</Emphasis>

A 1207 bp cDNA fragment (PsG6PDH) was amplified by RT-PCR from cold-induced total RNA of the freezing-tolerant P. Suaveolens, using primers based on the highly conserved region of published plant glucose-6-phosphate dehydrogenase (G6PDH) genes. The sequence analysis showed that PsG6PDH coding region had 1 101 bp and encoded 367 predicted amino acid residues. Moreover, the nucleotide sequence of PsG6PDH showed 83%, 82%, 79%, 79% and 78% identity, and the derived amino acid sequence shared 44.2%, 44.7%, 42.0%, 40.5% and 43.9% identity with those of the Solanum tuberosum, Nicotiana tabacum, Triticum aestivum, Oryza sativa and Arabidopsis thaliana, respectively. The results show that PsG6PDH is a new member of G6PDH gene family and belongs to the cytosolic G6PDH gene. This is the first report on cloning of the G6PDH gene from woody plants.     

山鸡椒1-脱氧木酮糖-5-磷酸还原异构酶 DXR基因的克隆和SNP分析

在转录组测序结果分析基础上,以山鸡椒cDNA 为模板,克隆得到山鸡椒1-脱氧木酮糖-5-磷酸还原异构酶DXR 基因cDNA 全长,以山鸡椒基因组DNA 为模板,设计引物、扩增拼接后获得山鸡椒DXR 基因全长,命名为LcDXR。序列分析表明,LcDXR cDNA 全长为1 501 bp,5'非编码区长34 bp,3'非编码区长53 bp,开放阅读框长1 413 bp,预测编码含有470个氨基酸残基的蛋白质,等电点为6.62,分子量为51.12 kD。LcDXR 基因全长为12 601 bp,其中外显子12 个,内含子11 个。对来自10个种源的LcDXR 基因编码区单核苷酸变异位点进行分析表明:在cDNA 区间内共发现10 个SNP(single nucleotide polymorphism)位点,其中有4 个单核苷酸变异导致了所编码的氨基酸的改变,为了分析氨基酸突变导致的蛋白质精细结构的变化,利用Swiss-PDB Viewer 模拟4 个突变位点氨基酸残基的替换。其中江西安远(AY)的突变Lys119Thr 引起了氢键的变化,推测可能对酶的活性产生影响。研究结果为深入研究山鸡椒脱氧木酮糖5-磷酸还原异构酶的活性和功能奠定了基础,同时为山鸡椒遗传育种提供理论依据。     

荔波连蕊茶GA2ox1基因的克隆及表达分析

[目的]GA2氧化酶是赤霉素生物合成代谢过程中关键酶之一,GA2ox家族基因常被用于植物矮化基因工程育种。[方法]根据植物GA2氧化酶基因编码区的保守序列设计引物,以山茶属荔波连蕊茶嫩枝为材料,提取总RNA,进行RT-PCR。采用RACE技术扩增获得1 371 bp的GA2ox基因全长cDNA序列,命名为ClGA2ox1(GenBank登录号KJ502290)。[结果]序列分析表明,ClGA2ox1放阅读框(ORF)为1 002 bp,编码333个氨基酸,5'非编码区59 bp,3'非编码区310 bp。预测的蛋白质分子量为37.31 kD,等电点为5.92,具有GA2ox超基因家族的保守结构域和特有的氨基酸残基。ClGA2ox1蛋白与GenBank中收录的其它植物GA2ox蛋白氨基酸的相似性达到80%。构建系统进化树,结果显示山茶GA2氧化酶与烟草GA2ox蛋白的亲缘关系最为密切。实时定量PCR结果显示,该基因在荔波边蕊茶不同器官及发育不同时期的均有表达,表达量有所不同:ClGA2ox1基因在2年生茎段中的表达量最高,在嫩枝和根中也有较高的表达,而在新抽生的嫩叶中最低。[结论]试验结果为进一步明确ClGA2ox1基因的功能特征及揭示其参与调控植物生长的分子机制奠定基础。     

白桦果胶甲酯酶抑制剂(PMEI)基因克隆及序列分析

从白桦(Betula platyphylla Suk.)cDNA文库中获得1个果胶甲酯酶抑制剂(PMEI)基因全长cDNA序列,该序列去除polyA后长946 bp,其ORF全长624 bp,编码208个氨基酸,编码蛋白的分子量22 486.4D,理论等电点为10.09,保守区分析确定其含有PMEI蛋白保守序列,属于植物PMEI超家族。该结果为研究白桦细胞壁代谢的分子调控奠定了基础。     

思茅松1-脱氧-D-木酮糖-5-磷酸合酶(DXS)基因的克隆及功能分析

1-脱氧-D-木酮糖-5-磷酸合酶(DXS)是甲基-D-赤藓醇-4-磷酸(MEP)途径中的第一个酶,也是限速酶。本文根据思茅松(Pinus kesiya var. langbianensis(A. Chev.) Gaussen)树皮转录组数据分析结果,获得思茅松DXS基因片段,然后根据获得的基因片段设计特异引物,运用RT-PCR和RACE技术从思茅松树皮中克隆得到完整的DXS基因(PkDXS1)。PkDXS1基因的cDNA全长序列2888 bp,含有1个2223 bp的开放阅读框(ORF),编码740个氨基酸,该基因推断的蛋白与赤松(Pinus densiflora Siebold & Zucc)DXS蛋白的相似性为99%,与欧洲云杉(Picea abies(L.) H. Karst.)DXS的相似性为97%;经氨基酸序列比对,推断思茅松DXS具有高等植物DXS酶特有的叶绿体转运肽,二磷酸硫胺结合位点和转酮醇酶结构域。半定量RT-PCR检测表明树皮的创伤促进DXS基因的表达。     

Construction and analysis of a subtracted cDNA library of Betula platyphylla female inflorescence

Female inflorescence of Betula platyphylla was sampled at an interval of each two days to analyze the background of gene expression in floral phase. On the basis of SMART strategy, the driver cDNA was obtained from total RNA of the last sample and the tester cDNA was from that of the others by RT-PCR which were subsequently used to construct a subtracted cDNA library. The result of the ESTs (expression sequence tags) blastX showed that the genes in the subtracted cDNA library could be mainly clustered into 5 groups related to metabolism, transportation and signal transduction, cell cycle, stress response, and regulation. The relationship between gene expression and development was also discussed.