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.

EMF2 gene maintains vegetative development by repressing flowering program,and plays an important role in the process of flower organ development.In order to offer a reference for the researches on epigenetic regulation of Camellia oleifera characters,the full-length cDNA of EMF2 gene in C.oleifera(Huaxin) was cloned and analyzed by 5′RACE and 3′RACE techniques.The results show that the full length of the gene is 2 210 bp,the ORF box included 1 866 bp and encodes 621 amino acids,and it is named Co-EMF2.The results of multiple sequence alignment show that Co-EMF2 has the typical conserved sequences of EMF2 and relevant characters,and it has high homology with EMF2 in other plant species.the protein is a labile and hydrophobic nuclear protein,and its lsoelectric point is 6.17.

A cDNA library and an EST library of Camellia oleifera were constructed.A 14-3-3 protein gene was cloned from Camellia oleifera.This gene is 1 156 bp in length,including 57 bp of 5'untranslated region and 301 bp of 3'untranslated region.It has an open reading flame of 777 bp,encoding a protein of 259 amino acid residues with protein molecular weight of 29.466 ku and isoelectric point of 4.78.No signal peptide sequence was found,which shows it's a non-secreted protein.This gene is named Co-14-3-3a.The speculated secondary structure of Co-14-3-3a consists of nine helices and an extended-beta located between αC and αD.

The full-length cDNA of a new S-RNase (S 34-RNase) (GenBank accession No.DQ414813) was successfully cloned from pistils of Chinese pear by RT-PCR and RACE methods. The S 34-RNase shows a complete CDS of 687 nucleotides for putative 229 amino acids. The theoretical three-dimension structure of S 34-RNase was predicted by homology modeling method and its structural characteristic was analyzed. Aligning of S 34-RNase with other 39 S-RNases of pears revealed the question about unifying naming system.

PAL(phenylalanine ammonia lyase) is one of the key enzymes in plant secondary metabolism.The full-length cDNA of PAL was identified from the cDNA library and EST library of Camellia oleifera by the method of molecular cloning in this paper.The results showed that the cDNA of C.oleifera PAL contained 2 118 bp,encoding 706 amino acids,and had a high similarity in the evolution of a high degree of homology with other species of PAL;C.oleifera PAL encoded protein sequence contained the same deamination site and catalytic active site with protein PAL of rice and corn.PAL phylogenetic tree analyses revealed that the C.oleifera PAL had closer relationship with PALs from Shrub plants than from other plants.

In order to realize rapid propagation in Chirita langshanica,taking tender leaves as experimental materials,tissue culture techniques in Chirita langshanica were studied under different culture conditions.The results indicated that MS supplemented with BA 1.0 mg·L-1 and NAA 0.5 mg·L-1 was the optimal medium for inducing callus,MS supplemented with BA 0.5 mg·L-1 and NAA 0.3 mg·L-1 was the best medium for cluster buds differentiation,and MS supplemented with NAA 0.3 mg·L-1 and quartz sand was the optimal medium for inducing root.Hardening seedlings should be carried out under 80% shading-net,and the survival rate of seedlings replanted was up to 80% after 25 days.

以构建的油茶EST文库为基础,采用电子克隆技术,分离克隆一个脱水素基因的全长cDNA序列,该基因的cDNA全长1406bp,含一个600bp的CDS,编码200aa的小分子蛋白。推测该基因编码蛋白的分子质量为21ku,等电点7,暂命名为CoDHNI。同源性分析发现该基因的编码蛋白具有2个Y-片段（DEYGNP）,1个S-片段（SGSSSSSS）,2个K-片段（KIKEKLPG）,属于典型的Y2SK2型脱水素。经Blast比对,发现富含苏氨酸的Thr-区在各物种间变异显著,推测该区为油茶所特有,有利于在被保护的大分子表面形成水合层;同时还发现一个十分保守的基序:EDDGQGGRRKK,可能有利于脱水素的磷酸化和亚细胞定位。通过与柑桔和拟南芥的脱水素比较,提出CoDHNI有可能缓冲种子脱水胁迫响应时钙离子的瞬时增高,并可结合重金属离子,以减轻活性氧的危害。

For a study of aroma components in sand pear juice,solid-phase micro-extraction(SPME) technique was employed to extract some volatile compounds from the juice of such sand pears as Shisho and Atago.23 such volatile compounds were isolated and identified successfully by capillary gas chromatography-mass spectrometry(GC-MS).It's been found out that the main components in Shisho juice are 2-Nonanone(23.08%),Propanoic acid ethyl ester(19.51%),Butanoic acid butyl ester(10.32%),Ethanol(9.82%),Ethyl acetate(8.53%),Anthraoene(4.68%),Acetic acid(4.41%),Cyclohexene(4.29%) etc.;and that the main components in Atago juice are Propanoic acid,2-methyl-ethyl ester(32.53%),Ethyl acetate(24.56%),Isopropyl alcohol(17.81%),1-Undecanol(16.02%) and Butanoic acid butyl ester(4.59%).

以构建的油茶cDNA文库为基础,采用交错延伸PCR技术,分离克隆到一个水通道蛋白基因的编码序列(coding sequence,CDS),它编码一个287aa的跨膜蛋白(GenBank蛋白质id:ACF39901).该蛋白可能由2条基因(GenBank登录号:EU850810、EU850811)编码,这2个基因具有相同的CDS和3'-UTR,但5'-UTR不同,其中之一多了2个小的插入片段.经对该蛋白的同源性比对和特征性基序分析,推测这个水通道蛋白属于质膜内在蛋白成员,定名为CoPIP1-1.通过同源建模,论证CoPIP1-1的水通道活性与通道口的(去)覆盖有关.N端与D环、B环的静电势作用导致了对通道口的(去)覆盖,并受磷酸化,质子化门控机制调控,质子化抑制通道活性,磷酸化则解除抑制.从同源建模的结果和细胞内的氧化状态推测该蛋白为组成型的低活性,这可能是油茶种子近成熟期脱水的成因之一.

Based on a constructed cDNA library of Camellia oleifera,a full-length cDNA(GenBank accession number: EU856537) encoding a dehydrin-like protein(id:ACF72673) composed of 208 amino acids,was cloned from the seeds by 5′-RACE technique.This putative protein should belong to the SK2-type dehydrin by homology analysis,designated as CoDHN2.In its polypeptide the region between the domains of the two similar K-segments is rich in threonine,which is different from the general characteristics of other dehydrins.CoDHN2 dehydrin shared a highly conserved motif of EDDGQGGRRKK,which might contribute to dehydrin's phosphorylation and hence subcellular localization,as another dehydrin do in the seeds of C.oleifera.Prediction of secondary structure via various methods showed that CoDHN2 was intrinsically unstructured,but one of its K-segments can be changed into a α-amphipathic helix.Due to its relatively rich histidine content and good solvent accessibility,It was proposed that CoDHN2 may bind heavy metal ions,and thus eliminate ROS sources caused by these ions and scavenge ROS,or may also serve as buffer for some physiological process under the condition of water deficient.Considering the progress of study on dehydrin,it was speculated that the CoDHN2 was very possible to bind the developing lipid body and protect them against the ROS during rapid biosynthesis of lipid in the C.oleifera seeds,which give a new clue to study the development of lipid body in the seeds of C.oleifera.

FAD2基因控制油酸转化为亚油酸,是油茶油脂合成代谢的关键酶基因。在构建的油茶EST文库基础上,采用5′RACE和交错延伸PCR技术获得了油茶FAD2基因的全长cDNA克隆。该基因序列长1682bp,开放阅读框为1149bp,编码382个氨基酸,并且具有FAD2特有的保守序列和相关特征。经过比对分析,发现油茶的FAD2基因与其他植物的FAD2基因具有较高的相似性。

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.