Introduction

Coniferous trees can release large quantities of aromatic resins called rosin,which are important industrial raw materials.Rosin can be separated into turpentine oil and common rosin at a ratio of approximately 1:4(An and Ding 2012;Yang 2014).The main components of turpentine oil are monoterpenes and sesquiterpenes;the resin acid fraction that makes up the rosin is a mixture of isomeric diterpenoid compounds(Wangetal.2002;AnandDing2012).Globally,about20pinespeciesproducehigh-lipidrosin,andinChina,Pinus massoniana L.is the main widely cultivated rosin tree species,accounting for over 90% of total annual turpentine production(Yang 2007;Luo et al.2013).

Terpenoids are composed of two or more isoprene monomers or their derivatives;their synthetic route can be summarized as precursor synthesis followed by cyclization condensation of isoprenoids.Early in 1992,the sesquiterpene synthase gene was fi rst cloned in tobacco(Facchini and Chappell 1992),and during the next two decades more than 200 monoterpene and sesquiterpene synthase genes were cloned in 40 plant species(Degenhardt et al.2009).In conifers alone,70 terpene synthases,including monoterpene,sesquiterpene and diterpene synthases,have been reported,mainly in Abies grandis,Picea abies and Pinus taeda(Bohlmann et al.1999;Martin et al.2004,Ro et al.2005).Up to now,there has been no relevant report of rosin in P.massoniana.

Generally,the product of each terpene synthase is unique,which makes it possible to signi fi cantly regulate a speci fi c target product by applying genetic Engineering to its synthetic enzyme.Earlier studies have shown that when an amorphadiene synthase gene is transferred into tobacco,which lacks an endogenous sesquiterpene synthase,the transformants produced more amorphadiene than in the wild-type donor(Wallaart et al.2001),and similarly,astaxanthin can be detected when beta-carotene ketolase encoded by the alga Haematococcus pluvialis is transferred into tobacco(Mann et al.2000).Additionally,three different lemon monoterpene synthase genes were transferred into wild-type tobaccos,causing the levels of β-pinene,limonene and terpinene in the transformants to increase signi fi cantly(Lucker et al.2004).

2.2 交叉关系一般概念的比较 在这一节中，有许多交叉关系的一般概念，如DNA与RNA、 3种不同的RNA、 mRNA与密码子等。可利用系列生物图对这些交叉概念进行比较，进而弄清楚其间的逻辑关系。

The cyclization condensation stage of terpene synthesis forms a divergent path structure;a common precursor can be used to synthesize hundreds of terpenoids.By regulating only one or a few synthases,the level of the target component can be changed effectively,with limited effects on overall terpenoid levels.Rosin is a mixture of terpene components,so this kind of regulatory model cannot be applied to resin synthesis.Additionally,pine resin composition analysis has shown that diterpene compounds are essential for rosin yield and quality,suggesting that of the three key enzymes GPPS,farnesyl pyrophosphate synthetase(FPPS)and geranylgeranyl pyrophosphate synthetase(GGPPS),GGPPS may be a potential regulatory node.It is therefore important to analyze correlations between GGPPS activity and resin productivity.

GGPP is a key precursor of various isoprenoids that have diverse functions in plant primary and secondary metabolism.The synthesis of GGPP from GPP and two molecules of IPP is catalyzed by GGPPS.GGPPS plays a key role in the synthesis of diterpenoids,carotenoids,chlorophyll,abscisic acid and other important active substances and is an important node in regulating carbon fl ow in the isoprenoid pathway(Aharoni et al.2003).Therefore,the regulation of the precursor could have a direct impact on the subsequent synthesis of terpene components.Using methyl jasmonate-induced GGPPS gene expression in Taxus canadensis,Croteau(Croteau 2002)found that both the gene expression level of GGPPS and the yield of Taxol increased signi fi cantly.When an FPS gene from yeast was inserted into tobacco,the sterol and carotenoid content of the transgenic plant was fourfold higher(Masferrer et al.2002).Induction of the expression of a taxadiene synthase transgene in Arabidopsis thaliana using a glucocorticoidmediated system consistently resulted inmore ef fi cient recruitment of GGPP for the production of taxadiene,which reached levels 30-fold higher than in plants constitutively expressing the transgene(Besumbes et al.2004).Currently,GGPPS has been isolated in a variety of plants,but it has not been reported in P.massoniana.

In previous work,high-yield varieties of P.massoniana have been bred mostly at the provenance and family level,but with little assistance at the molecular level.The potential resin yield is mainly affected by basic resin productivity,which is controlled by genetic factors(Lu and Wang 1992).Other investigations in slash pine,Masson’s pine,maritime pine and loblolly pine have also found that genetic factors strongly affect resin yield(Zhuang et al.2007;Tadesse et al.2002;Lombardero et al.2000;Roberds et al.2003).Additionally,another study found that pine resin productivity was positively correlated with tree height,diameter and wood basic density traits but showed a negative genetic correlation to tracheid length(Lian et al.2002)and no genetic correlation to collateral and branching angle(Blinka 2007).It remains unclear whether there is any correlation between pine resin and gene expression levels in the terpene metabolic pathway.The synthesis of diterpenoid compounds plays an important role in the yield and quality of resin,and to some extent,the expression level of GGPPS probably parallels resin productivity.

First,sequence homology analysis of the GGPPS gene was conducted using the NCBI blastn online tool,and open reading frame(ORF)analysis was performed using ORF Finder software and the DNA sequence was translated.In addition,theconservedregionsandtheactivesiteoftheGGPPSprotein were analyzed using blastp,while the transmembrane region andsignalpeptideofthePinGGPPSsequencewerepredicted using the TMHMM and SignalP tools provided by the Technical University of Denmark.The secondary and three-dimensional(3D)structures of PinGGPPS encoding protein were predicted using the PSIPRED service in ExPASy and SWISS-MODEL software,respectively.A phylogenetic tree for the PinGGPPS and GGPPS sequences from other species was constructed using the neighbor-joining method and Clustal W in Molecular Evolutionary Genetics Analysis(MEGA)software(v.5.0)(Tamura et al.2011).

Materials and methods

Plant material

Seeds of Gupeng provenance were collected from Xincheng County of Guangxi Gupeng Town,planted in the experimental nursery of Guangxi Forestry Research Institute for 6 month,which were used for gene cloning and to test gene expression in provenance offspring.Seeds of the superior groupwerecollectedfromprimaryseedorchardsofanational improved variety of Masson pine seed base in Guangxi Nanning Forestry Science Research Institute.Likewise,they were sown in the experimental nursery of Guangxi Forestry Research Institute and grown for 6 months to use for gene expression of superior group offspring.

从林木生长发育阶段的不同，可以看出其内在价值的不同。因此，核算林木资源资产经济价值时，应选择与之相适应的核算方法。

The experimental nursery of Guangxi Forestry Research Institute is located in north latitude 22°93′,longitude 108°35′,at an altitude of 110 m,with mean annual precipitation of 350 mm and mean temperature of 21.8°C,in latosolic red soil.Adult pine trees were from Guangxi State-owned Daguishan Forest Farm,Liupai breakout,which was afforested in 1992.The woodland is located in north latitude 24°48′,longitude 111°88′,at an altitude of 272 m,with mean annual precipitation of 1535 mm and mean temperature of 19.9°C,in latosolic red soil.

Using RT-PCR with speci fi c primers based on analysis of GGPPShomologsinotherplants,weobtainedaGGPPSgene sequence from P.massoniana,which we named PinGGPPS and submitted to an online database for domain analysis.The 1143-bp full-length cDNA of PinGGPPS contained an ORF that encoded a 380-amino-acid protein.Online analysis showed that PinGGPPS included a domain belonging to the IPP synthase superfamily,namely,domain Trans IPPS(Fig.1).ThededucedaminoacidsequenceofPinGGPPSwas compared with other species,and two Asp-rich regions at residues 183–189 and 324–328 were found with typical sequences DDXXXXD and DDXXD,called FARM(the fi rst aspartate-rich motif)and SARM(the second aspartate-rich motif),which are binding sites for allylic substrates and IPP,respectively(Hemmi et al.2003).All these characteristics correspond to previously reported GGPPS proteins.

Cloning of the GGPPS gene

Total RNA was isolated from 6-month-old conifer seedlings using an RNA Extraction Kit(Takara,Japan)according to the manual.First-strand cDNA was synthesized from total RNA using a RevertAid First Strand cDNA Synthesis Kit(Thermo Fisher Scienti fi c,Waltham,MA,USA)following the manufacturer’s protocol.The cDNA products were stored at-80°C until use.

4.相关工作人员要对业务知识进行不断学习，努力提高自身业务水平，在工作中要时时刻刻保持一个良好的态度进行工作，通过知识充电的方式来提高自身技能，履行在工作中的职责。

Basedonhomologoussequenceanalysis,theGGPPSgene sequence was obtained from NCBI,and primers were designedaccordingtotheconservedregionusingVectorNTI(v.10.0).The primer sequences were sense primer GGPPSF1 5′-ATGGCTTACAGTGGTAGAC-3′ and anti-sense primer GGPPS-R1 5′-TCAGTTTTGTCGAATGCAA-3′.Utilizing the cDNA as a template,PCR was conductedusing the following program:predenaturation at 95°C for 5 min;28 cycles of the standard thermal ampli fi cation cycling with denaturation at 94 °C for 30 s,annealing at 60 °C for 30 s and extension at 72°C for 1 min;continuing extension at 72 °C for 10 min;and fi nal hold at 8 °C for 30 min.

首先，应加强宣传教育，调动农民参与培训的主动性与积极性。许多农民对农业机械技术培训工作缺乏必要了解，配合度不高。只有农民了解农业机械化的重要性及好处后，才能积极主动地参与到培训工作中。因此，相关部门应告知农民这些必要的信息，打消农民疑虑，以实现农业机械技术培训效果的提升。其次，将理论与实践有机结合起来。大部分农民文化素质不高，很难迅速接受高深艰涩的理论知识。农业机械化培训要将理论与实践结合起来，为农民创造更多的实践机会。

Sequence analysis of GGPPS

In this study we report for the fi rst time the cloning and characterization of a P.massoniana GGPPS homolog(PinGGPPS).We found a correlation between resin productivity and the level of gene expression,which would provide support for breeding strains of P.massoniana highly productive for resin.

RT-qPCR conditions

Quantitative RT-PCR primers for GGPPS were designed using Vector NTI (qRT-PCR-F: 5′-AGGCACTGGAAAGGG-3′;qRT-PCR-R:5′-AATGCACAGAACAGG-3′).The housekeeping gene actin was selected as the most stably expressed reference gene in P.massoniana and its primer sequences are as follows:P-actin-F 5′-CTGGAATC CATGAGACTACTTACAA-3′;P-actin-R 5′-AACCGCC ACTGAGCACAATA-3′.

Plant tissues from P.massoniana were collected and immediately stored in liquid nitrogen until use.Total RNA was isolated using an RNAprep Pure Plant Kit(Polysaccharides and Polyphenolics-rich)(Tiangen,Beijing),and then cDNA was synthesized using an RNA LA PCR Kit(Takara).

在潮湿的季节，当水塘满水时，红树林鳉鱼迅速孵化。随后它们快速生长，很快成熟并开始产卵繁殖，直到水塘开始干涸。它们将产下的卵留在泥土中，当下一个雨季降临时，这些卵将会迅速孵化，一个新的生命周期就此开始。

Real-time fl uorescent quantitative PCRs were conducted using a Roche Light Cycler 480 System.Each reaction contained 1 μL of cDNA template and 9 μL of the reaction mixture,which were used in 2×SG Fast qPCR Master Mix(BBI).Cycling conditions were as follows:95°C for 3 min;40 cycles at 95 °C for 7 s,57 °C for 10 s,and 72°C for 15 s.Each reaction was repeated for at least three technical and biological replicates.

Analysis of PinGGPPS gene expression pattern

Samples were collected from four different tissues from one 6-month-old P.massoniana seedling from Gupeng provenance for analysis of the PinGGPPS gene expression pattern.Four pine needles each from the upper,middle and lower part of the plant were bulked as the respective needle samples,and the root samples were root tissues of plant.After removing needles and roots,the 1/3 upper stems were considered as immature stem samples and the 1/3 lower stems as the semiligni fi ed stem samples.All the samples were collected and stored immediately in liquid nitrogen until use.All total RNAs were extracted using an RNAprep Pure Plant Kit(Polysaccharides and Polyphenolics-rich)(Tiangen,Beijing),and then cDNA was synthesized using an RNA LA PCR Kit(Takara).Subsequently,real-time fl uorescent quantitative PCRs were conducted.Setting three repetitions,all data were treated by SPSS 19.0 software(SPSS Chicago,IL,USA),and were carried out with one-way ANOVA,as well as Duncan’s multiple range test.

PCR products were validated using 0.8%agarose gel electrophoresis,and then the DNA fragment was inserted into the pMD-18T vector at 16°C overnight.The recombinant was inserted into E.coli DH5α competent cells,followed by overnight cultivation at 37°C.After overnight growth,three randomly selected E.coli transformants were con fi rmed by PCR ampli fi cation using GGPPS-F1/GGPPSR1 primers,and the positive clones were sequenced by Shenzhen Genomics Biotechnology Co.

Correlation analysis of GGPPS gene expression levels and different resin productivity in adult plants

Pine needles were collected from 20 adult trees on a clear morning in mid-September.Five bundles of needles were collected from four directions(east,west,north,south)in the upper,middle and lower part of the trees,then all samples were immediately placed into liquid nitrogen until use.All total RNAs were extracted using an RNAprep Pure Plant Kit(Polysaccharides and Polyphenolics-rich)(Tiangen,Beijing),and then cDNA was synthesized using an RNA LA PCR Kit(Takara).Subsequently,real-time fl uorescent quantitative PCRs were conducted to analyze the GGPPS gene expression levels and resin productivity for each plant.

Fig.1 Protein domains of PinGGPPS.The PinGGPPS domain was analyzed using the blastn online tool of the NCBI(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSea rch&LINK_LOC=blasthome),identifying a domain belonging to the IPP synthase superfamily,namely domain Trans IPPS)

Meanwhile,the corresponding pine resins were harvested from mid-August to mid-September for 28 successive days and weighed.Resin was collected continuously for 3 years as three biological repetitions.All data were analyzed with one-way ANOVA and Duncan’s multiple range test using SPSS 19.0 software(SPSS,Chicago,IL,USA).Subsequently,gene expression levels and resin productivities were analyzed with Bivariate Correlations using SPSS 19.0 software(SPSS,Chicago,IL,USA).

GGPPS gene expression analysis of seedlings of fi lial generation with different resin productivity

Twenty 6-month-old seedlings from superior samples with high-yield resin productivity were randomly selected as the superior group,while the provenance control consisted of 20 6-month-old seedlings from provenance seeds.All pine needles were collected from the above trees on a clear morning in mid-May.Four bundles of needles were collected in the upper,middle and lower part of each tree,then immediately placed into liquid nitrogen until use.Subsequently,total RNA and qPCR were carried out using these samples.Three repetitions were carried out,and the data were statistically compared with Independent-Samples T Test using SPSS19.0 software(SPSS Chicago,IL,USA).

Results

To identify whether the protein PinGGPPS contains transmembrane and signal peptides,we used the tools TMHMM and SignalP,but no transmembrane or signal peptide sequence was detected in PinGGPPS,indicating that this protein is not a membrane protein or a secreted protein,and must be located in the cell matrix,which coincides with its predicted function.

该阶段需要完成的工作主要包括以下几个方面：接收相关的设计委托书，并签订合同；在规定时间内，有序地完成各项设计工作。设计难度由浅入深，并将各个工种区分开来，按照工种合理分配设计任务；设计团队需要定期召开交流会，协调好各方面的设计关系，保证设计进度的同时，把握住设计的大方向；在提升商场内部美观程度的同时，要严格按照各项标准进行设计，使建筑稳定性得到有力保证[1]。

Cloning and sequence analysis of PinGGPPS

Secondary structure elements of the deduced PinGGPPS were predicted using PSIPRED on the ExPaSy website(http://www.expasy.org/),and 16 α-helices and no β-sheets were found(Fig.2).The 3D structure of PinGGPPS modeled using SWISS-MODEL Workspace con fi rmed this result(Fig.3).Using the large subunit of heterotetrameric GPPS from mint(Mentha piperita)as the modeling reference template(Chang et al.2010),the result showed a coverage rate of 77%,from residue 86 to 380,with 66.67%sequence identity to mint GPPS,suggesting that PinGGPPS may have the same catalytic function.

比如,“石榴裙”本为“服饰”类,由于调整其使用范围,而增加相应的成分,便可以直接联系到其领有者或者使用者这样的主体对象,也就是某种特定的“人”类,于是其借代的语义格式便可以描写成[+像石榴一样红+裙子+领有者/可以穿着的人]。

Analysis of PinGGPPS expression patterns

To clarify the patterns of PinGGPPS expression in the plant,we investigated transcript levels in roots,needles,immature stem,and semiligni fi ed stems of 6-month-old seedlings of P.massoniana using qPCR,with actin as the reference gene.The results showed the highest transcript level of PinGGPPS in the needles,and the level differed

slightly between immature stem and semiligni fi ed stems,although the level in each was signi fi cantly lower than in the needles.The lowest level was found in roots(Fig.4).Meanwhile,a one-way ANOVA demonstrated that there were signi fi cant differences among the tissues,we also found that the differences in the expression levels in these four tissues were highly signi fi cant differences in a Duncan’s multiple range test(Fig.4).

Fig.2 Secondary structure of PinGGPPS.Secondary structure elements of the deduced PinGGPPS were predicted using PSIPRED on the ExPaSy website,showing 16 α-helices,but no β-sheets

Fig.3 Predicted three-dimensional structure of PinGGPPS.The 3D structure of PinGGPPS modeled using SWISS-MODEL Workspace,also showing 16 α-helices,but no β-sheets

Fig.4 PinGGPPS expression in root,leaf(needle),immature stem and semiligni fi ed stem tissue from 6-month-old seedlings of Pinus massoniana revelaed by qRT-PCR.Values are mean±SE calculated from three repetitions;different letters represented a highly signi fi cant difference at 0.01 level

Because pine needles are the main site of resin synthesis and also the center of plant photosynthesis,the high expression level of PinGGPPS observed in pine needles is consistent with their physiological function.Physiological processes in the stem mainly involve internode elongation and ligni fi cation,which have less correlation with the physiological function of PinGGPPS,which agrees with the signi fi cantly reduced expression seen in stem tissue.Furthermore,the physiological structure and function of roots differ remarkably from those of stems and needles,and the low level of PinGGPPS in roots is consistent with our expectations.

Analysis of resin productivity and PinGGPPS gene expression level in different plants

To analyze the correlation between PinGGPPS gene expression level and resin productivity,pine resin yield and the PinGGPPS gene expression levels in pine needles were measured in 20 adult plants for 28 days in succession.The average gene expression level in these 20 adult plants was 0.88±0.45(Fig.5),and the average resin productivity was 776.71±64.38 g(Fig.6).Simultaneously,one-way ANOVA results showed that the resin productivities among the tested plants differed signi fi cantly.Additionally,except for two plants(nos.14 and 19),others differed signi fi cantly at 0.01 level in Duncan’s multiple range test(Fig.6).The gene expression levels among the tested plants also differed signi fi cantly,and Duncans multiple range test also showed that the level in most plants differed remarkably at 0.01 levels(Fig.5).

目前，高校的教育对象主要是“90后”。他们思维活跃，好奇心强，判断和自律能力弱，易受大数据的不利影响，从而加大了思想政治教育的难度。大数据信息的多样性和复杂性使得大学生容易沉迷于网络游戏，严重依赖信息网络等网络成瘾现象，不利于大学生思想心理的健康发展。同时，在大数据时代，一些罪犯利用它进行欺诈活动等不法行为,从而对大学生的人生观和价值观的正确形成产生不良影响，甚至有可能发生行为失控、道德观念模糊等问题酿成悲剧，影响思想政治教育实效性的提升。

Gene expression levels of eight plants(sample nos.10,9,16,13,14,8,4,11)were higher than average,and their corresponding resin productivities were also higher than average,accounting for 72.7% of the plants with high resin yield(11 plants:nos.10,13,16,11,20,8,9,14,19,15,4).In addition,PinGGPPS gene expression levels in the nine plants with lower than average resin yield were also below average,accounting for 75% of all plants with lower expression(12 plants).Overall,the consistency rate of these two sets of data was slightly more than 70%.

We found that the PinGGPPS gene expression levels present a substantially linear distribution when plotted against their corresponding resin yield(Fig.7).All data were entered into SPSS(v.19.0)for analyses of the correlation between these two groups;the correlation coef ficient was 0.733,with p＜0.01,showing a moderate positive correlation.

PinGGPPS gene expression in F1 seedlings with different resin productivities

The superior group was compared with the provenance group,as shown in Fig.8,and average expression level of PinGPPS was 1.108 and 1.029,respectively,indicating that expression in the superior group was slightly higher than in the provenance group,but the difference was not signi fi cant.

In the superior group,the number of plants with moderate expression levels(mean±30%)was in the majority,about 55%;fewer plants showed higher(＞30%above the mean)and lower(＞30%below the mean)expression levels.However,in the provenance group,the number of plants in these three categories was fairly evenly distributed(35,40 and 25%,respectively),illustrating that PinGPPS gene expression and resin productivities in the superior group are stable.In comparison with the superior group,the range in the high and low expression levels observed in provenance offspring was much greater,and the distribution of offspring showed no obvious trend,indicating that the provenance group was presumably a mixed population with low selective breeding,and therefore represents the natural range of resin productivities.

Fig.5 PinGGPPS gene expression in different plants.Pine needles from 20 adult plants were analyzed for GGPPS gene expression using real-time fl uorescent quantitative PCR.Different letters represent a highly signi fi cant difference at 0.01 level

Fig.6 Resin productivity in different plants.Resin was collected continuously from 20 adult plants for 28 days and weighed.Different letters represent a highly signi fi cant difference at 0.01 level

Discussion

The condensation reaction catalyzed by GGPPS is a node for the synthesis of the common diterpene precursor from terpene precursors and is thus an important target for the regulation of diterpene biosynthesis.GGPPS genes have also been identi fi ed in fungi,archaea,eubacteria,protozoa,and mammals(Soderberg et al.2001;Artz et al.2010;Ling et al.2007).

江泽民新安全观阐明了人民民主专政在对外关系领域的特定职能，是对人民民主专政职能理论的新发展。为充分发挥人民民主专政对外职能提供了理论依据和行动指南，对维护国家安全，巩固人民民主专政的国家政权，为国内发展营造良好的国际安全环境具有重要的实践意义。

Phylogenetic analysis indicates that GGPPS protein sequences of different species form distinct clusters;i.e.,GGPPSs from animals,plants,bacteria,fungi and archaea belong to different branches.Furthermore,GGPPSs from bacteria and archaea are more closely related to those from plants,and those from fungi and animals are closely related to each other.Currently,GGPPSs from different sources are divided into three types:I(archaea),II(plants and eubacteria)and III(fungi,insects and mammals)(Hemmi et al.2003;Ling et al.2007;Sagami et al.1994).Although the fi nal product of GGPPS in all organisms is GGPP,kinetic studies show that type I and II GGPPSs preferentially catalyze the condensation of DMAPP or GPP with IPP to form GGPP,while type III GGPPSs prefer to use FPP as their main substrate.

Fig.7 Correlation analysis of PinGGPPS gene expression levels and resin productivity.Gene expression levels and resin productivities of the above 20 adult plants were analyzed using SPSS,as was the corresponding correlation analysis

Previous research has found that the enzymes involved in the synthesis of GGPP,monoterpenes,diterpenes and tetraterpenes are mainly located in plastids(Tholl,2006).In the plant cytoplasm,FPPS catalyzes the synthesis of FPP from IPP,and sesquiterpenes,triterpenoids and other terpenoids are then successively synthesized,but in plastids,GGPPS catalyzes the synthesis of GGPP from GPP and DMAPP.GGPP then takes part in the production of monoterpenes,diterpenes,chlorophyll,and carotene.GGPPS is the primary enzyme responsible for catalyzing the formation of GGPP in plastids,providing raw materials for the synthesis of diterpene,chlorophyll and other active substances.In addition,plastids serve as factories for the synthesis of pine resin,and investigation into ultrastructural changes during the secretion of pine resin found that with the development of resin ducts,the more the number of plastids increased,the more pine resin synthesis increased(Li et al.2008).

Fig.8 PinGPPS gene expression levels in offspring with different resin productivities.Twenty 6-month-old seedlings from superior samples with high-yield resin productivity were randomly selected as the superior group,while the provenance control consisted of 20 6-month-old seedlings from provenance seeds.All pine needles from the above plants were analyzed by qPCR

In the present work,we analyzed the correlation between PinGGPPS gene expression level and resin productivity and found that gene expression levels corresponded to resin productivity,but that consistency was very low when the results were examined at the level of individual plants.We speculate that this is because resin productivity data were collected continuously over an extended period,while gene expression data were collected one time.If we were to collect data over a long period,the results might correspond more closely.In addition,the correspondence between gene expression and resin productivity will be more obvious for samples with greater differences from the mean,but smaller differences might be more easily masked by the environment and other factors.Our results show a correlation coef fi cient of 0.733(p＜0.01),indicating that the PinGGPPS gene expression level is signi fi cantly correlated with resin productivity.

We analyzed PinGGPPS gene expression patterns in seedlings of different fi lial generations.Compared with the provenance group,the overall expression level in the superior group was slightly higher,but the difference was not signi fi cant.PinGGPPS gene expression and resin productivity in the superior group were stable,and most plants had moderate expression levels,because the superior group consisted of the offspring of plants with high resin yield after many generations of screening.In contrast,expression levels in the provenance group were not clearly distributed in relation to resin productivity,and its resin productivity re fl ected the natural range,perhaps because the offspring were from a mixed population with low selective breeding.

2.6 在2#仓滑模施工时为增加上人梯的稳定，也须进行连墙件预埋钢板的埋设，其水平间距L1=1.2m，每层5个，竖向间距为 H1=3.6m。

In this study,we characterized the gene PinGGPPS in P.massoniana for the fi rst time,and we attempted to establish a correlation between its expression level and resin productivity at a molecular biological level.Detection of gene expression has many advantages,including the lack of an age requirement,so that seedlings can be tested,allowing a short life cycle as well as high throughput.Resin productivities can be measured using the target gene PinGGPPS as an index,rather than using traditional direct measurement methods.Combining molecular biology techniques and traditionalbreeding methods enables breeding requirements to be achieved easily and would provide support for breeding high resin productivity strains of P.massoniana,underlining the importance of theory and production practice.

2.5 保守性分析 通过多序列比对发现果蝇、斑马鱼、老鼠、大象、猪、苏门达腊猩猩、人类、黑猩猩共8个物种的WDR62蛋白，发现患儿携带的2个WDR62基因突变位点所对应的蛋白C376及Q1207在物种间具有较高保守性(图2)。

Acknowledgements This research was supported by the Guangxi Natural Science Foundation No.2014GXNSFBA118106.

References

Aharoni A,Giri AP,Deuerlein S,Bouwmeester HJ(2003)Terpenoid metabolism in wild-type and transgenic Arabidopsis plants.Plant Cell 15(12):2866–2884

An N,Ding GJ(2012)Study on chemical constituents of oleoresin from Pinus msssoniana in Guangxi.J Cent S Univ For Technol 3:59–62

Artz JD,Wernimont AK,Dunford JE,Hui R(2010)Molecular characterization of a novel geranylgeranyl pyrophosphate synthase from Plasmodium Parasites. J Biol Chem 286(5):3315–3322

Besumbes O,Sauret-Greto S,Phillipa MA,Boronat A(2004)Metabolic engineering of isoprenoid biosynthesis in Arabidopsis for the production of taxadiene,the fi rst committed precursor of Taxol.Biotechnol Bioeng 88(2):168–175

Blinka KW(2007)Resin fl ow in clonal Loblolly Pine.North Carolina State University,Raleigh

Bohlmann J,Phillips M,Ramachandiran V,Croteau R(1999)cDNA cloning,characterization,and functional expression of four new monoterpene synthase members of the Tpsd gene family from Grand Fir (Abies grandis). Arch Biochem Biophys 368(2):232–243

Chang TH,Hsieh FL,Ko TP,Wang AH(2010)Structure of a heterotetrameric geranyl pyrophosphate synthase from Mint(Mentha piperita)reveals intersubunit regulation.Plant Cell 22(2):454–467

Croteau R(2002)Biosynthesis and catabolism of monoterpenoids.Chem Rev 87(5):929–954

Degenhardt J,Kollner TG,Gershenzon J(2009)Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants.Phytochemistry 70(15–16):1621–1637

Facchini PJ,Chappell J(1992)Gene family for anelicitor-induced sesquiterpenecyclase in tobacco.Proc Natl Acad Sci USA 89(22):11088–11092

Hemmi H,Noike M,Nakayama T,Nishino T(2003)An alternative mechanism of product chain-length determination in type III geranylgeranyl diphosphate synthase. Eur J Biochem 270:2186–2194

Li AM,Wang YR,Wu H(2008)Initiation and development of resin ducts in major organs of Pinus massoniana.Sci Silvae Sin 44(9):36–40

Lian HM,He BX,Zeng LH,Qin J(2002)Study on the comprehensive election of half-sib families of Masons pine.For Sci Technol Guangdong Prov 18(1):1–6

Ling Y,Li ZH,Miranda K,Moreno SN(2007)The farnesyldiphosphate/geranylgeranyl-diphosphate synthase of Toxoplasma gondii is a bifunctional enzyme and a molecular target of bisphosphonates.J Biol Chem 282(42):30804–30816

Lombardero MJ,Ayres MP,Lorio PL,Ruel JJ(2000)Environmental effects on constitutive and inducible resin defences of Pinus taeda.Ecol Lett 3(4):329–339

Lu PX,Wang Y(1992)Variation of oleoresin yield among high-gum yielding slash pine families and their realized genetic gain.For Res 5(6):700–705

Lucker J,Schwab W,van Hautum B,Verhoeven HA(2004)Increased and altered fragrance of tobacco plants after metabolic engineering using three monoterpene synthases from lemon.Plant Physiol 134(1):510–519

Luo LP,Shu WB,Nie HQ,Yan ZY(2013)Comparison on growth volume and resin-producing capacity of tugong provenance between pure forest and mixed forest of Pinus massoniana.Guangxi For Sci 42(1):66–70

Mann V,Harker M,Pecker I,Hirschberg J(2000)Metabolic engineering of astaxanthin production in tobacco fl owers.Nat Biotechnol 18(8):888–892

Martin DM,Fa ldt J,Bohlmann J(2004)Functional characterization of nine Norway Spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily.Plant Physiol 135(4):1908–1927

Masferrer A,ArróM,Manzano D,Ferrer A(2002)Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase(FPS1S)in transgenic Arabidopsis induces a cell death/senescence-like response and reduced cytokinin levels.Plant J 30(2):123–132

Ro DK,Arimura G,Lau SY,Bohlmann J(2005)Loblolly pine abietadienol/abietadienal oxidase PtAO(CYP720B1)is a multifunctional,multisubstrate cytochrome P450 monooxygenase.Proc Natl Acad Sci 102(22):8060–8065

Roberds JH,Strom BL,Hain FP,Lott LH(2003)Estimates of genetic parameters for oleoresin and growth traits in juvenile loblolly pine.Can J For Res Rev 33:2469–2476

Sagami H,Morita Y,Ogura K(1994)Puri fi cation and properties of geranylgeranyl-diphosphate synthase from bovine brain.J Biol Chem 269(32):20561–20566

Soderberg T,Chen A,Poulter CD(2001)Geranylgeranylglyceryl phosphate synthase.Characterization of the recombinant enzyme from Methanobacterium thermoautotrophicum.Biochemistry 40(49):14847–14854

Tadesse W,Nanos N,Auñon F,Gil L(2002)Evaluation of high resin yielders of Pinus pinaster Ait.For Genet 8(4):271–278

Tamura K,Peterson D,Peterson N,Kumar S(2011)Mega5:molecular,evolutionary,genetics,analysis,using maximum,likelihood,evolutionary,distance,and maximum,parsimony,methods.Mol Biol Evol 28(10):2731–2739

Tholl D(2006)Terpene synthases and the regulation,diversity and biological roles of terpene metabolism.Curr Opin Plant Biol 9(3):297–304

Wallaart TE,Bouwmeester HJ,Hille J,Maijers NC(2001)Amorpha-4,11-diene synthase:cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin.Planta 212(3):460–465

Wang YS,Zeng LH,Luo M,Qin J(2002)Analysis of tapping resin trial in natural stands of masson’s pine.For Sci Technol 18(2):1–4

Yang ZQ(2007)Status and countermeasures about developing pine resin in Guangxi.Guangxi For Sci 36(3):143–146

Yang ZQ(2014)Comparative study on the resin yield and rosin components of Pinus massoniana superior provenances among different ages.Sci Silvae Sin 50(6):147–151

Zhuang WY,Zhang YY,Zou YX(2007)Selection for high-resin yield of Slash Pine and analysis of factors concerned.Acta Agric Univ Jiangxiensis 29(1):55–60