[目的]为揭示油茶基因的生物学功能及品种遗传改良提供参考.[方法]以优良油茶品种'横冲89'为研究对象,采集叶、花芽、雄蕊、雌蕊、子房、成熟种子,提取总RNA,其中,以花芽、雄蕊、雌蕊、子房总RNA的混合样构建花的转录组.用磁珠分离富集mRNA,利用Illumina RNA-seq高通量测序技术构建油茶叶、花和成熟种子的转录组,利用NCBI、SwissProt、Nr蛋白数据库与GO、KEGG、KOG数据库对这3个转录组中的Unigene进行注释和分类,分析这些转录组中参与糖代谢、能量代谢、脂质代谢途径的Unigene,并进行KEGG通路的富集分析,比较光合作用、脂肪酸合成途径中基因的转录表达.[结果]构建了油茶叶、花、种子转录组文库,在这些转录组中包括6.652×107条净读长序列,82964个Unigene,185190个转录本.其中,Unigene和转录本的平均长度分别为671、966 bp.这些Unigenes与NCBI、SwissProt数据库基因的相似性约为46.51％,与Nr蛋白数据的相似性为53.49％;对82994条组装Unigene进行GO分类,其中24313条Unigene与KEGG 31个代谢途径相匹配,35773条Unigene与KOG 26个代谢途径匹配.此外,在上述代谢途径中发现光合作用关键酶Rubisco、脂肪酸合成关键酶ACCase存在同源基因,并且发现叶绿素a/b结合蛋白4、脂肪酸合成酶、类脂肪酸合成酶异构体、乙醇脱氢酶等基因在叶、花、种子中差异表达.[结论]油茶叶、花、种子转录组中的Unigene参与糖代谢、能量代谢、油脂代谢途径,在光合作用及油脂合成过程中差异表达并发挥重要作用.

乙酰辅酶A羧化酶(Acetyl-CoA carboxylase,ACCase)是催化脂肪酸合成的关键和限速步骤.该文介绍了ACCase的分类及结构特征,阐述了其在脂肪合成代谢中的作用和在除草剂中的应用,分析了它的表达调控及反馈机理,揭示了ACCase基因的克隆及表达鉴定,并展望了ACCase在植物育种中的应用前景.

从油茶(Camellia oleifera Abel.)叶片中分离克隆了Rubisco rbcL和rbcS基因的cDNA序列,全长分别为1522和615 bp.其中,前者的编码区为1425 bp,编码475个氨基酸;后者的编码区为615 bp,编码176个氨基酸,分别命名为Co-rbcL和Co-rbcS.利用荧光定量PCR研究其在油茶品种'湘林14'、'湘林1'和'横冲89'中的表达,结果显示,油产量较高的品种,这2个基因的转录表达量也较高,3个品种的净光合速率呈现类似于上述基因的表达模式.Pearson分析表明:Co-rbcL与3个品种的油产量和种子产量显著相关(P<0.001);而Co-rbcS与油产量和种子产量具有相关性.另外,净光合速率与产油量、种子产量具有相关性(P<0.001;P<0.01).因此,这2个基因具有作为早期高油产量油茶品种选择分子标记的潜力,结合净光合速率测定,这种早期鉴定将会缩短育种时间并提高育种效率.

The heteromeric acetyl coenzyme A carboxylase (ACCase) in plant seeds catalyzes the formation of malonyl-CoA from acetyl-CoA, which is the rate-limiting step in de novo fatty acid biosynthesis. In this study, we cloned biotin carboxylase (BC) and beta-subunits of carboxyltransferase (beta-CT) genes of ACCase subunits from Camellia oleifera, namely Co-accC and Co-accD, respectively. The full-length Co-accC (GeneBank accession no.FJ965288) was 1901 bp encoded 530 aa, and the coding regions of Co-accD was 1530 bp encoded 510 aa (accession no. FJ965289), which was in the 2574-bp fragments of rbcL, rbcL-accDintergenic spacer and accD. The comparison of genomic and cDNA sequences showed that they were all intronless. Structural analyses showed that their putative amino acid sequences shared high identity with those of other oil-plants, and that they possessed the ATP-binding site, biotin carboxylation site in the Co-accC protein, and zinc finger motif CX2CX15CX2C in the Co-accD protein. Moreover, transcript expressions of the two genes were carried out in cultivars as well as in tissues and fruit development stages of 'Huashuo', the former showed that their expression levels in cultivars were almost correlated with oil contents of matured seed kernels, the higher expression levels, the higher oil content to some extent, suggesting that they could potentially be as molecular markers for selection of higher oil production cultivars. The latter revealed their expression rules in lipid synthesis and accumulation. Taken together, information from our study indicates that they may be significant in the fatty acid and lipid biosynthesis of seeds, thus be valuable for oil yield of C. oleifera.

Based on the conducted cDNA library of Camellia oleifera,two full-length cDNA genes were obtained,and were 953 bp and 1 024 bp,respectively,designated as CoCaM1 and CoCaM2 (GertBank access numbers:EU856536 and FJ649316).They shared high similarity at nucleotide level in the coding regions(CDS).They both contained the 447-bp CDS with 25 nucleotide substitutions,and encoded an identical polypeptide of 149 amino acids(predicted relative molecular weight of 16.83 kDa).The protein is a hydrophilic acidic protein,and comprised of 19 amino acids with pI(theoretical isoelectric point) of 4.10.The characteristic is consistent with the hypothesis "Multigenes possess an identical amino acid sequence".It possesses four EF-hand domains,cysteine and other enzymebinding sites,etc.The protein has some identical degree in hydrophilicity,flexible regions and antigenicity,and thus shows high flexibility.The blast and phylogenetic analysis indicated that the amino acid sequence of C oleifera CaM displayed highly homologous compared with those of other higher plants.The higher expression levels of CoCaM1 were observed in the stage from flower bud to ovary formation.The expression levels of CoCaM2 were much higher in fruit formation and oil accumulation,while in leaves and mature seeds were lower.This shows that they may play different roles in the development of flower buds,fruit formation and oil synthesis and accumulation.

通过将机械精量穴水直播、机械精量穴旱直播和人工撒播三种播种方式进行对比,对甬优4949生育进程和经济效益进行分析.结果表明,机械穴水直播比人工撒播单产增加15.2%,而机械穴旱直播比人工撒播单产增加6.51%.

In order to reveal sequence features and structural function of the UDP-glucuronate decarboxylase gene in Camellia oleifera,and based for the further research,the specific primers were designed according to the UDP-glucuronate decarboxylase gene sequence from Camellia oleifera seed EST library.Using RT-PCR technology,the gene full-length cDNA clones were obtained.Then the sequence was analyzed by bioinformatic software.The results showed that:the ORF box included 1032 bp and encoded 344 amino acids.The sequence had typical UXS family motif sequence.The gene had closest phylogenetic relationship with GhUXS3 gene of Island cotton and localized in the cytoplasm with strong hydrophilicity.The gene was named CoUXS1 and the GenBank accession number was JN017094.

Calmodulins, members of the EF-hand family of Ca 2+ -binding proteins, play significant regulatory roles in the processes of plant cell proliferation, growth, development, photosynthesis and stress resistance. Here, the two full-length complementary DNA (cDNA) clones were isolated from the constructed Camellia oleifera cDNA library. They are 953 base pair (bp) and 1024 bp in the length, respectively, and designated as CoCaM1 and CoCaM2 (GenBank access numbers EU856536 and FJ649316). They contain the complete protein-coding region of 447 bp encoded an identical polypeptide of 149 amino acids and various lengths of untranslated segments. The result supported the hypothesis “Multigenes possess an identical amino acid sequence”. The structure analyzed results indicated the putative CoCaM protein possessed four EFhand domains, hydrophobic residues and crucial residues, in which the amphipathic helices might possess lipid affinity via binding and altering the associated plasma or organelle membranes. In addition, the protein had one to seven substitutions, and displayed more than 89% identity at the amino acid level in comparison to other species CaMs. Finally, the expression of the 2 cDNA genes in matured seeds of 6 C. oleifera varieties was analyzed by real-time fluorescence quantitative PCR, and the result showed that there were different expression levels. In conclusion, the structure properties together with the observed expression levels indicate that they may play different roles in the processes of development, lipid biosynthesis and stress responses of C. oleifera seeds.

Cyclophilins are folding enzymes belonging to the class of peptidyl-prolyl cis-trans isomerases(PPIases) that catalyze the cis-trans isomerization of peptidyl-prolyl bonds in proteins.The full-length cyclophilin gene clone was isolated,cloned,identified,and expressed using the cDNA library conducted by the nearly matured Camellia oleifera seeds.Its size was 975 bp containing an open reading frame(ORF) of 621 bp,encoded a 22.24-kDa protein of 207 amino acid residues,and was named co-cyp.The secondary structure of the protein mainly contains extended strands acompanying with alpha helixs,beta turns,and numerous random coils.It contains a 26-amino acid singal sequence in the N-terminal domain,and has a transmembrane region and fine flexibility.It also possesses N-glycosylation sites,protein-kinase C phosphorylation sites,Casein kinaseII phosphorylation sites,Tyrosine kinase phosphorylation site,N-myristoylation sites,Cyclophilin-type peptidyl-prolyl cis-trans isomerase signature YKGSKFHRIIPSFMLQGG,seven amino acid residues KSGKPLH of plant cyclophilins,et al.It showed that the protein belonged to CyPB,and was a globular secretory protein.It shares overall identities of 80%~94% with those of some higher plants.On the basis of the full-length cDNA,the approximately 21 kDa protein was obtained via the constructed recombinant pET30-CyP in E.Coli strain BL21(DE3) induced by IPTG under the optimum expressed conditions.This study may be of very importance for the pathway of lipid biosynthesis and stress response of Camellia oleifera.

With the near-matured seeds of 'Xianglin No.1' of Camellia oleifera as material to extract total RNA,the whole length cDNA of BC gene that is a subunit of ACCase from Camellia oleifera was obtained by means of the techniques such as degenerate PCR,RACE and the overlap extension PCR.The full-length of cDNA sequence of BC gene is 1 901 bp,and has a 1 599 bp ORF coding 533 amino acids.The pI value of BC protein is 6.88,the molecular mass of the protein is 58 509.3 u,the protein bears two trans-membrane domain,has no signal peptide,and is a unstable protein.There are several pro-sites in BC,such as N-glycosylation site,Protein kinase C phosphorylation site,ATP-grasp fold profile and so on.In the three-dimensional model of BC,we found 12 alpha helixes and a cupped area in the central of this protein.The gene was submitted to the GeneBank and named co-bc.

Cyclophilins (CyPs) are ubiquitous proteins and involved in protein folding and stress response due to peptidyl-prolyl cis-trans isomerase (PPIase) activity. In this study, a 975-base pair (bp) full-length complementary DNA (cDNA) gene, Camellia oleifera CyP (CoCyP, GenBank access no. FJ377540), was isolated from the conducted cDNA library of C. oleifera seeds. This cDNA encodes a polypeptide of 207 amino acids, which has an endoplasmic-reticulum-localized signal sequence at its N-terminus, but not the typical signal sequence at its C-terminus. Moreover, its putative amino acid sequence shares highest identity with those of Arabidopsis thaliana CyPB (84%) and Triticum aestivum CyPB (81%), and possesses a highly conserved central core domain including cyclosporin A (CsA)-binding sites, the tetrapeptide Ala-Ala-Pro-Ala-binding sites, residues required for PPIase catalysis and an insertion of seven amino acids of unknown function. Collectively, it is suggested that the CoCyP protein belongs to CyPB and has PPIase activity. An approximately 21 kDa protein was expressed via the recombinant pET-30b(+)/CoCyP in Escherichia coli. In this study, we tentatively put forward to the hypothesis that the gene expressing during the peak of seed lipid biosynthesis, might correlate with the caused harmful products in the lipid biosynthesis process, and protect cells against reactive oxygen species (ROS) damage; thus it may be crucial during lipid biosynthesis and stress responsiveness.

The genomic DNA extracted from tender leaves of 23 elite clones of Camellia oleifera(Native species for edible oil in China)was used to perform ISSR(inter-simple sequence repeat)analysis.Of the 60 primers screened,10 pieces of DNA fingerprint profiles containing 102 bands with 70 distinct polymorphic bands(68.6%)were obtained,and each primer produced 3 to 10 bands with the band size ranging from 200 bp to 2 000 bp.Result indicated that Shannon's information index was 0.479 3,and the mean observed number of alleles and the mean effective number of alleles were 2.000 0 and 1.518 5,respectively.The genetic distance varied from 0.154 2 to 0.693 1.There exists abundant polymorphism among these elite clones of C.oleifera.The UPGMA analysis result shows that the 23 elite clones of C.oleifera could be classified into 4 groups,namely,18 elite clones(including Xianglin 156,Xianglin 210,Xianglin 4,etc)belong to the first group,Jinruan 2 and Xianglin 97 to the second group,Xianglin 34 and Xianglin 11 to the third group,Xianglin 81 to the fourth group when the genetic distance is 0.392.On the basis of these analyses,the relationship between the clustering result and the agricultural characters of C.oleifera was discussed,which provides some references for the identification of elite clones,phylogenetic analyses and the utilization of germplasm resources.

In order to determine the molecular mechanism of forming high quality tea-oil, the EST library technology was used to find out the main expressed genes controlling the fatty acid biosynthesis in camellia oleifera seeds. A high quality full-length cDNA library was constructed successfully using the nearly matured seeds of elite cultivar Xianglin-1 planted widely in China,then more than two thousand clones were randomly sequenced from the 5' orientation, which was used to construct a high representational EST library. After the bioinformatic analysis of this EST library, 15 main genes controlling the biosynthesis of unsaturated fatty acids in camellia oleifera seeds were obtained.

South Dongting Lake is a Ramsar Site of International Importance and is rich in the World Natural and Cultural Heritages, PF but it has been ignored for a long period. We studied its outstanding world natural and cultural heritage features and the results showed that South Dongting Lake is well qualified for the standards of Convention Concerning the Protection of the World Cultural and Natural Heritage (CCPWCNH), and proposed firstly the application for World Heritage Lists as soon as possible. We established the ecological countermeasures of eco-tourism and conservation strategy, which is of great theoretical and practical significance in promoting the world heritage protection and sustainability.

With the destruction of natural environment by human beings, scenic resources are no longer inexhaustible in supply and use. Human beings begin to lay the scenic resources on the same important strategic status as other natural resources, while landscape resources assessment is the prerequisite of their sustainable exploitation and conservation. This paper illustrated the psychological mechanisms of aesthetic and its approaches, compared with the methodologies of traditional and modem landscape aesthetic research, discussed the characteristics of important aesthetic theories (Platonism, Kant paradigm, Empathizing theory, Gestalt paradigm, Marxism aesthetics theory, and Appleton theory) and the landscape assessment theories of 4 paradigms (expert, psychological, cognitive, and empirical) and 2 groups (landscape environment science and landscape architecture culture), and summarized the important practices and successful examples at home and abroad. It was demonstrated that the historical development of landscape assessment had the feature of a contest between expert- and perception-based approaches, with the expert approach dominated in landscape management, while the perception-based approach dominated in landscape research. Both of these approaches generallty accepted that landscape quality was derived from the interaction between the biophysical features of landscape and the percepultual (judgmental) processes of human viewer. In the future, landscape quality assessment will evolve toward a shaky marriage, both expert- and perceptual approaches will be applied in parallel and merged in the final landscape management decision-making process in some but unspecified way, landscape information and complex geo-temporal dynamics representation central to scenic ecosystem management will present major challenges to the traditional landscape aesthetic assessment, and modem science and technology will continue to help meet these challenges. The main trends of landscape resources aesthetic assessment in this century will be the amalgamation and incorporation of diverse paradigms, the cross-disciplinary intersection and quantitative assessment, the emphasis of environment information and landscape ecological value recognition, the universal application of modern science and technology and of the dynamic and intellectual approaches of landscape information, the reality of landscape simulation and its scene technology of human perception, and the more attention to history and landscape culture.

Dongting Lake is a famous freshwater lake wetland in China,and is also only a lake in the world,which consists of three Ramsar Sites of international importance.As Dongting Lake's most important component,South Dongting Lake Wetland has particular ecological values in world wetland involvement.This paper analyzed the formation reason,involvement regularity,studied the ecosystem structure characteristics of South Dongting Lake Wetland,evaluated its important functions such as the animal habitat,plants gene pool,ecological improvement,wetland production,base of the research education,tourism holidays.In addition,the authors pointed out the existing problems in protection and exploitation of South Dongting Lake Wetland,namely the lake area function declination,attenuation of the wild biology species,low efficiency of wetland resource utilization,serious environment pollution.At last,we proposed the sustainable development approach of South Dongting Lake,which is enlarging lake area,stressing wetland resource protection,enhancing construction of the nature reserve,establishing high efficiency eco-agriculture,and controlling pollution.

This paper summarized the latest 10 years research advances in urban green space at abroad, including urban green space effects, green space aesthetics, vertical greening, plants landscaping, landscape ecology, information technology application, urban forestry, vegetation cultivation, and green space planning and design. The development trend of urban green space research in the future was pointed out.

Dongting Lake is a traditional landscape and famous scenery in China and also only a lake in the world which consists of three Ramsar Sites of international importance. As Dongting Lake’s most important component, South Dongting Lake Wetland plays a key role of the Yangtze River reaches civilization, China civilization and even the global civilization. It is rich in diversity of characteristic wetland landscape culture resources. Basing on detailed field investigation from 1999 to 2005, we analyzed systematically the structures and special value of the following landscape cultures:substance culture like the rice culture, ship culture, architecture culture, producing culture, engineering culture;spirit culture such as Duanwu Festival, customs & arts, fishery’s taboos, food culture, fighting against flooding, plants culture; regulation culture such as Nuo culture, Chu culture, historical site and remains, wetland historical relics. We studied further scientifically the wetland culture's origin and position, and expounded further the contents of the 15-type wetland landscape cultures. At last, we concluded their total essential features of the wetland culture and water culture, including Ramsar Site, Xiang-chu culture, wetland culture, outstanding water culture, world cultural heritage, excellent integrality and authenticity.