An improved draft genome sequence of hybrid Populus alba×Populus glandulosa

Populus alba × P.glandulosa clone 84 K,derived from South Korea,is widely cultivated in China and used as a model in the molecular research of woody plants because of hi gh gene transformation efficiency.Here,we combined63-fold coverage Illumina short reads and 126-fold coverage PacBio long reads to assemble the genome.Due to the hi gh heterozygosity level at 2.1% estimated by k-mer analysis,we exploited TrioCanu for genome assembly.The PacBio clean subreads of P.alba × P.glandulosa were separated into two parts according to the similarities,compared with the parental genomes of P.alba and P.glandulosa.The two parts of the subreads were assembled to two sets of subgenomes comprising subgenome A(405.31 Mb,from P.alba)and subgenome G(376.05 Mb,from P.glandulosa) with the contig N50 size of 5.43 Mb and 2.15 Mb,respectively.A high-quality P.alba × P.glandulosa genome assembly was obtained.The genome size was 781.36 Mb with the contig N50 size of 3.66 Mb and the longest contig was 19.47 Mb.In addition,a total of 176.95 Mb(43.7%),152.37 Mb(40.5%)of repetitive elements were identified and a total of 38,701 and 38,449 protein-coding genes were predicted in subgenomes A and G,respectively.For functional annotation,96.98% of subgenome A and 96.96% of subgenome G genes were annotated with public databases.This de novo assembled genome will facilitate systematic and comprehensive study,such as multi-omics analysis,in the model tree P.alba X P.glandulosa.

Survival and development of immature Harmonia axyridis (Pallas) feeding on Chaitophorus populeti (Panzer) propagated on transgenic Populus alba × P. glandulosa

Laboratory feeding experiments with the poplar aphid, Chaitophorus populeti （Panzer）, feeding on transgenic poplar （P. alba × P. glandulosa） varieties C13-5 and C013-5, were carried out to study the effect of transgenic poplar on the ladybird Harmonia axyridis （Pallas）. The mortality and development time of the immature stages, the eclosion rate and body mass of H. axyridis were measured. The results indicated that C. populeti feeding on different varieties of transgenic plants had no statistically significant ef- fect on the mortality ofH. axyridis larvae. The development time of larval and pupal stages were not significantly different between the two transgenic poplars and a non-transgenic poplar. Furthermore, the body mass and eclosion rate did not show any difference between the H. axyridis feeding on aphids reared on transgenic plants and those from non-transgenic plants. It is suggested that transgenic plants have no deleterious effect on the predatory ladybird.

Waterlogging tolerance and wood properties of transgenic Populus alba×glandulosa expressing Vitreoscilla hemoglobin gene(Vgb)

Because overexpression of Vitreoscilla hemoglobin gene(Vgb)gene in plants can enhance tolerance to waterlogging,here Vgb was inserted into Populus alba×glandulosa to investigate its expression and effects on growth and physiological responses to waterlogging stress in the transgenic poplars.Southern blotting and RT-PCR analysis of Vgb-transgenic P.alba×glandulosa showed that the Vgb gene was integrated into the genome of the V13-81 and V13-85 transgenic lines and expressed.In greenhouse waterlogging stress tests,mortality of the transgenic poplar was significant lower than that of nontransgenic plants with increasing treatment time from 2 to 22 days.The transgenic plants had higher chlorophyll content and less chloroplast damage than in the control plants.Additionally,starch accumulation increased,and growth was enhanced in the transgenic plants,suggesting that the Vgb-expressing lines had improved energy reserves.Field trials of the transgenic poplar suggested that Vgb expression promotes growth and influences wood quality.Taken together,our results suggest that the expression of Vgb can increase the accumulation of chlorophyll and starch in the transgenic poplar,improve its ability to endure flooding,and improve growth and wood quality of the transgenic plants.

Effects of pruning intensity on nonstructural carbohydrates of Populus alba×P.talassica in the arid desert region of Northwest China

Although pruning is important to obtain highquality,large-diameter timber,the effects of pruning on nonstructural carbohydrates(NSC)in aboveground organs of many timber species are not well understood.Three intensities of pruning(none,moderate and severe)were tested on poplars(Populus alba×P.talassica)in the arid desert region of northwest China to compare the concentrations of soluble sugar(SS),starch(ST)and total nonstructural carbohydrate(TNC)in leaves,branches and trunks during the growing season.The concentration of NSC components after different pruning intensities varied similarly in seasonal patterns,increasing slowly at the beginning of the growing season,continuously declining in the middle,then gradually recovering by the end of the growing season.The monthly mean NSC concentration in poplar differed significantly among the three pruning intensities(p<0.05).The SS concentration in pruned trees was higher than in unpruned trees(p<0.05).For moderately pruned trees,the concentrations of ST and TNC in trunks and branches were higher than in unpruned and in severely pruned trees(p<0.05).Compared with no pruning,pruning changed the seasonal variation in NSC concentration.The orders of SS and TNC concentrations in aboveground organs were leaf>branch>trunk,while the order of ST concentration was trunk>leaf>branch,which was related to functional differences of plant organs.The annual average growth in height of unpruned,moderately pruned,and severely pruned poplars was 0.21±0.06,0.45±0.09 and 0.24±0.05 m,respectively,and the annual average growth in DBH were 0.92±0.04,1.27±0.06 and 1.02±0.05 cm,respectively.Our results demonstrate that moderate pruning may effectively increase the annual growth in tree height and DBH while avoiding damage caused by excessive pruning to the tree body.Therefore,moderate pruning may increase the NSC storage and improve the growth of timber species.

胡杨PeERF1基因提高转基因银腺杨84K耐旱性研究

[目的]分析PeERF1基因在胡杨干旱胁迫下的作用和PeERF1转基因植株抗旱的生理适应机制,为进一步研究该基因在木本植物中的抗旱调控机制奠定基础。[方法]以胡杨为材料进行20%PEG6000模拟干旱(0、12和24 h)处理,对胡杨PeERF1基因进行时空表达模式分析。以非转基因(WT)、过表达35S::PeERF1转基因植株(PE)、显性抑制35S::PeERF1-SRDX转基因植株(SE)为试验材料,采用不同浓度PEG6000(对照组,20%)处理WT、PE和SE模拟干旱胁迫,并对其进行表达模式、生长性状和生理指标分析。[结果]研究结果表明PeERF1基因在胡杨叶中的表达水平最高,其次是茎和根。在正常状态下,转基因植株和WT生长性状、叶绿素含量、过氧化氢酶(CAT)、丙二醛(MDA)和过氧化物酶(POD)含量变化不大。在20%PEG6000处理后,PE转基因植株比WT表现出更好的生长状态,PE转基因植株的叶绿素含量、CAT和POD含量高于WT,PE转基因植株MDA含量低于WT。而SE转基因植株则表现出相反的性状。[结论]本研究结果初步显示干旱胁迫下,PeERF1基因转基因植株生长状态、叶绿素含量、过氧化氢酶、丙二醛和过氧化物酶等相关生理指标均发现显著变化。PeERF1对转基因杨树响应干旱起到了正向调控的作用。

银腺杨转Cry I Ac和API双价抗虫基因的研究

在建立以银腺杨（84K）叶片为外植体的再生系统基础上，通过农杆菌介导法把CryⅠ4c和API双价抗虫基因导人银腺杨（84K）基因组中。转化杨树叶片，在含有卡那霉素的培养基上诱导不定芽和诱导生根，获得了400株卡那霉素抗性转化再生植株。抗性植株经PCR检测，有70株呈阳性。通过Southern杂交和ELISA检测进一步证明CryⅠ4c和API双价抗虫基因已整合到银腺杨（84K）基因组中，拷贝数1～2个，并得到了表达。转化植株用杨扇舟蛾幼虫进行饲虫试验，结果表明昆虫幼虫的死亡率高达60．0％～80．0％。同时，存活幼虫的生长发育也受到了明显抑制。本研究结果为杨树抗虫育种提供了新的种质资源。

银中杨、玉簪落叶生物质炭对Pb^(2+)、Cd^(2+)、Cr^(6+)吸附的影响因素

为探讨银中杨、玉簪落叶所制备生物质炭对水体Pb^(2+)、Cd^(2+)和Cr^(6+)吸附规律的差异及影响因素,采用限氧裂解法将银中杨及玉簪落叶制成生物质炭,并以此为吸附载体研究其在不同初始离子质量浓度、pH值、Na+浓度及接触时间等因素影响下对Pb^(2+)、Cd^(2+)和Cr^(6+)的吸附。结果表明:随着初始Pb^(2+)、Cd^(2+)和Cr^(6+)质量浓度的增加(0~800 mg·L^(-1)),落叶生物质炭对相应重金属离子的吸附量也增加。将初始质量浓度设置在0~200 mg·L^(-1),生物质炭对3种金属离子的吸附量由大到小表现为Pb^(2+)、Cd^(2+)、Cr^(6+),然而,将初始离子质量浓度提升至300~800 mg·L^(-1),吸附量由大到小表现为Pb^(2+)、Cr^(6+)、Cd^(2+);溶液pH值由2增至8,可使Pb^(2+)和Cd^(2+)在生物质炭表面的吸附率得到迅速提升,然而,生物质炭对Cr^(6+)的吸附率在整个pH值变化范围则呈渐趋降低的趋势;随着Na+浓度增加(0~0.6 mol·L^(-1)),落叶生物质炭对3种金属离子所表现的吸附规律各不相同,其中,对Pb^(2+)的吸附量先下降而后渐趋升高,对Cd^(2+)的吸附量逐渐下降,而对Cr^(6+)的吸附量则表现为先增加而后下降。Na+离子浓度由0 mol·L^(-1)提升至0.6 mol·L^(-1)可使生物质炭对Pb^(2+)和Cd^(2+)的吸附量分别降低16.8%和97.1%,相反,对Cr^(6+)吸附量却有所促进,使其增加55.6%;生物质炭对初始质量浓度为400 mg·L^(-1)的Pb^(2+)、Cd^(2+)和Cr^(6+)吸附的数量随接触时间延长(0~1 440min)而逐渐增加,相同条件下由大到小表现为Pb^(2+)、Cr^(6+)、Cd^(2+);生物质炭对Pb^(2+)、Cd^(2+)的吸附主要以电性吸附为主,而专性吸附则为生物质炭吸附Cr^(6+)的主要机制。

刺激植物响应蛋白基因Epl1克隆、原核表达及功能初探

【目的】研究刺激植物响应蛋白Epl1对木本植物山新杨的刺激响应功能,为开发新一代提高植物免疫的新型诱导剂奠定基础。【方法】从棘孢木霉ACCC30153菌株中克隆获得一个刺激植物响应蛋白基因Epl1,并进行序列分析和原核表达,将获得的原核重组蛋白rEpl1与山新杨组培苗进行互作,初步探讨rEpl1蛋白对山新杨生长、生理指标、以及生长和防御相关基因转录水平的影响。【结果】刺激植物响应蛋白基因Epl1的cDNA序列全长为417bp,预测蛋白分子量12.6 kDa,属于Cerato-platanin家族;通过构建原核表达载体,成功获得重组蛋白rEpl1;在1μg/mL rEpl1诱导下,山新杨组培苗生长较快、根系旺盛,生物量明显增加,CAT活性在诱导第1 d时为对照的3.51倍,可溶性糖含量和脯氨酸含量在诱导初期急剧积累;山新杨生长素基因LAX2/AUX和水杨酸防御基因JAR2的转录水平分别在蛋白诱导2 d和12 h时达到峰值,分别为对照的873.10和388.02倍。【结论】Epl1基因的克隆、序列分析和原核表达为进一步研究Epl1蛋白诱导植物系统抗病性提供了理论基础;重组蛋白rEpl1与山新杨组培苗互作实验,初步Epl1蛋白能够刺激木本植物在生理及分子水平上的响应,从而促进生长,提高抗性。

杨树PtRRI基因的组织特异性表达模式分析

[目的]模式植物在木本植物中鉴定的许多重要调控因子家族在木本植物中出现了基因家族成员扩张,但ARRs家族作为细胞分裂素响应调节因子在杨树基因组中成员数量反而减少,其在木本植物中如何行使功能需要进一步研究。[方法]本研究通过生物信息学构建PtRRI启动子与GUS融合表达载体,检测植物激素处理后PtRRI表达量和检测PPtRRI::GUS转基因植株在生根过程中GUS信号等方法,对杨树PtRRI基因的组织特异性表达模式进行分析。[结果]表明:PtRRI在杨树根部、形成层、木质部表达量相对较高,PtRRI转录受6-BA激素诱导,与其在不定根发育过程中激素调控下的表达相一致。[结论]PtRRI基因可能参与杨树的次级生长。

木霉诱导下山新杨PodaPIN9基因的组织表达调控

PIN蛋白具有多个跨膜结构域,影响着高等植物生长素的外向运输和众多生长发育过程。木霉菌是能促进植物生长、提高其对多种病害防御作用的生防因子。研究木霉菌对木本植物山新杨生长素的极性分布的影响有重要意义。克隆了山新杨PodaPIN9基因,对其核酸和蛋白序列进行分析;同时,构建的进化树显示PodaPIN9与6个物种的9条PIN基因具有高度一致性(> 80%)。qRT-PCR分析表明,PodaPIN9在山新杨茎尖、成熟叶和根中均有表达。在根中表达量极低;在茎尖和叶中的表达量极高,分别是根中的503和346倍。该基因受木霉影响在茎尖和叶中的表达量均显著下调;而根中的表达量显著上调,达到对照的32. 01倍。并发现根际接种木霉48h会使杨树茎尖、叶和根中生长素含量降低。说明木霉菌能影响杨树茎尖、叶和根中IAA水平及PodaPIN9的表达量。并且Pearson相关性分析表明在茎尖、叶和根中,PodaPIN9表达量与IAA水平具有不同的相关性。

银白杨×马氏杨杂交子代及亲本的AFLP分析

为了探讨银白杨、马氏杨及银白杨×马氏杨杂交子代间的遗传多样性参数及个体聚类关系,利用8对引物对银白杨、马氏杨及银白杨×马氏杨杂交子代4个样本进行了AFLP分析。结果表明,8对引物共扩增出780条扩增谱带,其中多态性条带数为715条,多态性比例为91.65%,平均有效等位基因数为1.5017,Nei’s平均遗传多样性指数为0.3065,Shannon平均信息指数为0.4679。不同杂交子代在E-AAC/M-CAG引物对上均有特异性位点,这些特异位点可用于品种鉴定。聚类研究发现,在相似系数为0.76时,试验样品分成2个AFLP群,其中马氏杨为1个AFLP群,银白杨及2个杂交子代为1个AFLP群;当相似系数为0.78时,银白杨和2个杂交子代的AFLP群又划分为2个亚群,其中杂交子代1和杂交子代2聚合为亚群1,银白杨为亚群2。本研究的结果多态性比例很高,对样品的区分率达到100%,为杨树AFLP分析检测提供了指导借鉴价值。

C/N、pH对银中杨–玉簪落叶及草坪碎屑混料腐解的影响

【目的】以银中杨Populus alba×P.berolinensis–玉簪Hosta plantaginea落叶和草坪碎屑为基础材料,通过对混料C/N、pH的调控,筛选混料腐解的最佳条件。【方法】采用室内培养法对银中杨–玉簪落叶及草坪碎屑混料进行60 d恒温好氧腐解,设置物料C/N为10、15和20以及pH为4、7和10,探讨其对混料腐解过程中水溶性有机碳(WSOC)、可提取腐殖酸碳(HEC)、胡敏酸碳(HAC)、富里酸碳(FAC)及胡敏素碳(Hu C)含量的影响。【结果】将C/N设为10或15时,培养结束后混料HEC及HAC均有所累积,而将混料C/N设为20则更有利于微生物对WSOC、HEC及Hu C的矿化及周转。C/N为15或20时,培养结束后混料腐殖质品质有所改善,而在混料中添加更多氮素使C/N为10时,则在一定程度上阻碍了FAC向HAC的转化。混料C/N为20更有利于其胡敏酸(HA)分子结构的简单化,其次是C/N为10,而C/N为15时更有利于HA分子结构的稳定;混料pH为7有利于微生物对Hu C的矿化,使HEC和HAC含量分别增高30.0%和69.0%。混料pH为10时对HAC/FAC的促进程度最大,使之增加78.3%,pH为4或7时对混料HAC/FAC的促进程度较小。【结论】C/N为15、pH为7时,对银中杨–玉簪落叶及草坪碎屑混料腐解的促进作用最为显著。

银腺杨不同部位叶片形态、生理及转录差异分析

本研究以银腺杨成年树木为对象,测定了不同类型叶片形态、生理和基因表达差异,为揭示银腺杨的成熟效应提供参考。主要研究结果如下:(1)长枝叶和短枝叶的叶片形态差异显著,不同部位的叶片有很大程度的重叠。(2)不同部位叶片所含化合物差异显著,即:下部叶片的叶绿素a和叶绿素总含量显著大于上部和中部;下部叶片的SOD酶活性显著高于上部和中部,MDA含量表现为中部>上部>下部;下部叶片的淀粉含量显著低于上部和中部;不同部位叶片的IAA含量随着高度的降低而上升,ABA含量表现为中部>上部>下部;长枝叶的IAA和IAA/ABA显著大于短枝叶,ZR含量呈现相反的变化趋势。(3)基于转录组数据对差异显著基因进行筛选,不同部位叶片之间的差异显著基因数量少于不同类型叶片之间差异显著基因数量。(4)对各比较组的差异显著基因进行GO和KEGG富集分析,结果显示上部长枝叶相对于下部长枝叶(SC-vs-XC)、上部短枝叶相对于下部短枝叶(SD-vs-XD)差异显著基因显著富集到光合作用通路上,对其中的2个基因进行基因注释,发现这2个基因均编码PsbR蛋白,且下部叶片中基因表达量上调;对生长素转导过程进行分析,发现短枝叶中相关基因表达量均上调。

Single-cell RNA sequencing reveals a high-resolution cell atlas of xylem in Populus

High-throughputsingle-cellRNAsequencing(sc RNA-seq) has advantages over traditional RNA-seq to explore spatiotemporal information on gene dynamic expressions in heterogenous tissues. We performed Drop-seq, a method for the dropwise sequestration of single cells for sequencing, on protoplasts from the differentiating xylem of Populus alba × Populus glandulosa. The sc RNA-seq profiled9,798 cells, which were grouped into 12 clusters.Through characterization of differentially expressed genes in each cluster and RNA in situ hybridizations,we identified vessel cells, fiber cells, ray parenchyma cells and xylem precursor cells. Diffusion pseudotime analyses revealed the differentiating trajectory of vessels, fiber cells and ray parenchyma cells and indicated a different differentiation process between vessels and fiber cells, and a similar differentiation process between fiber cells and ray parenchyma cells. We identified marker genes for each cell type(cluster) and key candidate regulators during developmental stages of xylem cell differentiation. Our study generates a high-resolution expression atlas of wood formation at the single cell level and provides valuable information on wood formation.

转果聚糖蔗糖转移酶基因银腺杨的获得(英文)

采用农杆菌介导的遗传转化方法,将来自枯草杆菌的果聚糖蔗糖转移酶基因(SacB)导入银腺杨,以提高杨树对水分胁迫的抗性。以来自无菌培养的叶片为外植体,通过大约10 0 0个叶盘与农杆菌LBA4 4 0 4共培养,将植物双元表达载体pKP中SacB基因导入银腺杨基因组,经卡那霉素筛选后,共获得10 2株卡那霉素抗性植株。经PCR特异性扩增和Southern点杂交分析,证明其中97株再生植株基因组DNA中整合了SacB基因。对其中的6 2个无性系进行RT PCR分析,结果表明SacB基因在其中的5 0个无性系中获得表达。温室生长观察表明,转基因无性系外部形态与对照相比没有稳定的显著差异,少数部分转基因无性系的生长明显受到抑制,其他转基因无性系生长正常。这些转基因无性系的获得为培育抗旱转基因杨树奠定了基础。

银腺杨与毛新杨正反交三倍体选育

采用花粉染色体人工加倍获得的银腺杨 (Populusalba×P .glandulosa)、毛新杨 (P .tomentosa×P .bolleana)未减数 2n花粉 ,施加一定剂量的辐射处理后杂交 ,两杂交组合共获得 16株三倍体 .其中银腺杨×毛新杨组合以1470rad处理最佳 ,三倍体得率 3 .8% ;而毛新杨×银腺杨 (16 80rad)则获得了高达 12 .9%的三倍体 .三倍体整体生长水平优势明显 ,单株生长更为突出 ,2年生实生苗的苗高和地径最高 4.9m和 5 .8cm ,分别超出群体平均值的5 8%和 176 % ;并且正反交三倍体的遗传效应差异明显 .利用未减数 2n花粉 ,经花粉辐射处理杂交选育杂种三倍体 ,将是今后白杨乃至杨属植物育种的最有效途径之一 .

外源SacB基因在银腺杂种杨基因组中的表达及抗旱性分析

利用转基因技术培育抗旱杨树新品种是改善干旱地区生态环境的有效途径之一.该研究对已整合外源SacB基因的银腺杂种杨株系进行半定量RT-PCR和温室水分胁迫试验.研究结果表明,在所测定的7个转基因株系中,外源SacB基因均成功转录mRNA;转基因株系叶片中积累了SacB基因表达产物(果聚糖);在干旱胁迫条件下,转基因株系的生长量、生物量和叶片含水量明显高于对照.相关分析表明转基因株系生长量、生物量和叶片含水量与果聚糖积累量呈极显著正相关,说明SacB基因的导入提高了转基因杨树对水分胁迫的抗性.

转基因银腺杂种杨对土壤微生物的影响

In this study,genetic stability of foreign genes was checked by using PCR technology. A total of 4 lines of soil samples from transgenic and non-transgenic control Populus alba×P.glandulosa and the soil samples from the stands were collected in two successive years,and the bacteria,fungi and antinomyces were isolated by using flat dilution method to determine their quantities. The results demonstrated that target genes were stable present in the genome of transgenic poplar. We found that three kinds of soil microorganism appeared different trends within different months and different years. The ANOVA and multiple comparison analyses revealed that there was no significant difference in quantity of the soil microorganisms among most poplar lines at the same time point. Meanwhile,significant differences of the soil microorganism community were found among several transgenic lines,and between a few transgenic and non-transgenic lines,but they did not show any relation to the type and number of foreign genes. Our preliminary results indicated that the 2-year-old transgenic poplar growing in field had no significant effects on soil microorganisms.

转vgb基因银腺杂种杨的耐涝性

对导入透明颤菌血红蛋白(vitreoscilla hemoglobin,VHb)基因vgb银腺杂种杨进行Southern印记杂交和RT-PCR检测,证实vgb基因已整合到杨树基因组中,并在转录水平上得到表达。在温室对4个转基因株系及对照盆栽扦插苗进行模拟持续淹水胁迫试验,研究淹水条件下植株的生长及生理性状。结果表明,随着淹水时间的延长苗木逐渐死亡,对照植株在淹水17天后死亡率达66.67%,而4个转基因株系死亡率均低于50.00%,其中T81最低,只有25.00%;同时4个转基因株系在淹水胁迫时能够保持较高的叶绿素含量,并且膜质过氧化物MDA含量均低于对照植株;涝渍胁迫下转基因株系生长受抑制程度低于对照植株。说明在淹水胁迫下vgb基因的表达增强了银腺杂种杨叶绿素含量的积累,进而促进了转基因植株的生长,提高了转基因株系对淹水的忍受能力。

一株能拮抗3种土传病害病原真菌的长枝木霉

为了丰富生防木霉菌(Trichodermaspp.)种资源,在园林植物八宝景天(Sedum spectabile)根际土壤中分离得到一株优势木霉。根据该菌株菌落的形态特征、形态学显微观察、菌株rDNA-ITS序列分析以及邻接法同源性比对等结果判定木霉菌株种类。采用平板对峙法将该菌株与3种土传病害病原菌[(Sclerotinia sclerotiorum)、立枯丝核菌(Rhizoctonia solani)和细链格孢菌(Alternaria alternata)]进行对峙培养。并用该木霉诱导山新杨(Populus davidiana×P.albavar.Pyramidalis)组培苗,观察离体叶片抵抗细链格孢菌侵染的能力。结果表明,此木霉为长枝木霉(Trichoderma longibrachiatum),命名为Tl-70。该菌株对3种土传病害病原真菌均有较明显的拮抗作用。对核盘菌的抑菌率最高为77.71%,显著高于对细链格孢菌和立枯丝核菌的抑制率(P<0.05);对立枯丝核菌的抑菌率为58.56%,显著高于对细链格孢菌的抑制率(P<0.05);对细链格孢菌的抑菌率最低,为53.32%。并且,该木霉能提高山新杨组培苗叶片拮抗细链格孢菌侵染的能力。说明此菌株是具有潜力的生防菌株。