Effects of Light Stress on Oxygen Evolution and Photochemical Energy Stor age of Hybrid Poplar Clones Determined by Photoacoustic Technique

The oxygen evolution, thermal dissipation, and photochemical energy storage of three hybrid poplar clones, namely the triploid clone B342, the diploid clone B11 [(Populus alba×P. glandulosa)×(P.tomentosa×P.bolleana)], and the triploid clone B346 [(P.tomentosa×P. bolleana)×(P. alba×P.glandulosa)], under light stress were studied using photoacoustics. The oxygen evolution signal and photochemical energy storage varied negatively with the pretreatment_PFD (photon flux density), whereas the thermal signal varied positively with the pretreatment_PFD. Photochemical energy storage was reallocated to PSⅡ more than to PSⅠ, while the photochemical energy storage in PSⅠ was more stable than that in PSⅡ when subjected to light stress. The inhibitors streptomycin (SM), dithiothreitol (DTT) and sodium fluoride (NaF) could all affect the oxygen evolution signal. Clones B11 and B342 were more resistant to light stress than clone B346.

Growth variations and stability analyses of seven poplar clones at three sites in northeast China

Growth characteristics have complex inheritance patterns and genotype(G) by environment(E) interaction make predicting tree response to environmental changes difficult.In this study,the growth of seven poplar clones at three different sites was taken as the research focus,and heights and basal diameters were investigated in the second growing season.An ANOVA showed that all main effects,site,clone number and their interactions were highly significant in the overall F-tests.The coefficients of variation and repeatability of different traits ranged from 15.5 to 43.9%and from 0.549 to 0.912,respectively.AMMI(Additive Main Effects and Multiplicative Interaction) analysis results showed that genotype,environment and G × E interaction were significantly highly correlated.The stability analysis indicated that different clones showed different growth traits on different sites,which suggests that elite clones should be selected separately for different sites.Based on the growth traits,under a 10% selection rate,three clones were selected for different sites and the genetic gains of growth traits ranged from 4.7 to 11.2%.The three selected clones could be used to establish plantations in the future in different sites.

Genetic variation of height growth rhythm between clones of Larix kaempferi × L. gmelini based on logistic models

Fifty-three larch interspecific hybrid clones（Larix kaempferi × L.gmelini） and their parent clones were used for growth curve analysis of height variations.The growth curves of the 55 clones were ＇S＇-shaped and 36 exhibited similar curves as the male parent.The coefficients of the logistic models were higher than 0.943,indicating that our results were effective in the simulation of the growth curves.ANOVA analysis showed significant differences in height of different clones （P／0.01）.Average date of maximum height growth was Day 173,and average duration of rapid growth lasted for 50 days.Annual average increase in height was 9.7cm d^（-1） and daily average increase was 0.2 cm.The ratio of GR to the total annual increase in height ranged from 51.2 to 68.8%,with the average being 59.8%.There was a positive correlation between k values and plant heights which benefited from the evaluation of early plant height.There was also a positive correlation between GR（growth stage）,GD（plant height） and annual increase in height.These results are informative to the evaluation of the elite clone selection and provide a theoretical basis for breeding and management.

Physical Location of the Rice Pi-5(t), Glh and RTSV Genes by ISH of BAC Clones

Mapping of low or single-copy sequences on plant chromosomes has proven difficult because of very low frequency of signal detection. Rice BAC library is being used widely in rice genome research due to its distinctive advantages over other library systems. In this study, two biotin-labeled rice BAC clones closely linked to a rice blast resistance, green leafhopper resistance and tungro spherical virus resistance gene,Pi-5(t), Glh, RTSV, werein situ hybridized to rice chromosomes. They were located on the long arm and short arm of chromosome 4 with FL value of 40%and 100%respectively. The frequency of signal detection reached 46.8%and 59.2%. The signal location were consistent with the selective marker on rice saturated molecular map. The results demonstrated the advantages to locate BAC clones to chromosomes byin situ hybridization and will facilitate the rice low or single-copy gene location by using the BAC library.

Identification of differentially expressed genes associated with bud dormancy release in tree peony(Paeonia suffruticosa) by suppression subtractive hybridization

A subtractive cDNA library was developed to study genes associated with bud dormancy release in tree peonies. In order to identify genes that are highly expressed in buds released from dormancy, 588 clones were examined by differential screening. Of these, 185 clones were selected to be sequenced. A total of 37 unique sequences were obtained of which only 31 sequences have matches in the NCBI database or the Arabidopsis thaliana protein database. Semi-quantitative RT-PCR was used to confirm further the expression profiles for 12 transcripts identified within the subtractive cDNA library. Gene ontology analyses indicated that many of the different genes identified have unknown or hypothetical functions while it is speculated that other genes play different mo- lecular roles. In our study, genes involved in bud dormancy release were growth-related or stress-responsive, while low-temperature-induced ribosomal proteins may also play a role in bud dormancy release. Our results provide interesting information for further understanding of the molecular mechanism of bud dormancy release in tree peonies.

Clones No 1333 of Populus alba×P.davidiana and No 1132 of P.davidiana×P.alba×P.daviana

The hybridization experiment was initiated in 1975, in which the parents of P davhaana were collectedfrom Dailing, Heilongiiang Province, P. suaveolens were from Baicheng, P. simonii from Zhaodong of Heilongjiang Prov-ince, and P. tremula from Shanxi Province. Clones No 1333 of P. Alba x P. davidiana and No 1132 of P davidiana x (Palba x P. davidiana F1) had greater genetic variation and heritability in clones tested.

Screening and identification of binding proteins to interferon-α from a cDNA library by yeast-two hybrid system

The aim of this study is to screen proteins interacting with interferon-α （IFN-α）. The IFN-α gene was amplified by polymerase chain reaction （PCR） and cloned into pGBKT-/vector, then the resulted pGBKT-/-IFN-α vector was transformed into yeast strain AH109. The transformed yeast AH109 was mated with yeast Y187 containing liver cDNA library plasmid in 2 × YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade and SD/-Trp-Leu-His） con- taining X-α-gal for selection. After plasmid extraction and enzyme cutting analysis, the blue colonies were subjected to sequence analysis and the results were analyzed by bioinformatics. The results showed that IFN-α was successful cloned into the pGBKT7 vector. IFN-α was expressed and there was no selfactivation and toxicity in AH109. Thirty-four positive colonies were obtained after yeast-two hybrid technique screening. After sequence analysis, eight clones were found to have a binding effect with IFN-α protein. IFN-α was successfully cloned into the pGBKT7 vector. IFN-α protein was expressed and there was no self-activation and toxicity in AH109. Eight proteins that interacted with IFN-α, including vitronectin, fibrinogen A alpha polypeptide, HIV-1 Tat interactive protein 2, arginase, NADH dehydrogenase 1 beta subcomplex, transferrin receptor 2 alpha （TFR2）, HCC-1, alcohol dehydrogenase IB （ADHIB） have been identified as IFN-α-binding proteins.

Improved methods for cloning and detection in the yeast two hybrid assay

The yeast two-hybrid (Y2H) mating assay is a powerful method for detecting protein-protein interactions. Firstly, the gene of interest is cloned into specific Y2H vectors. Although multiple innovations in cloning methods were made in the past two decades, the conventional cloning method of restriction-enzyme (RE) digestion followed by ligation is still widely used. Unfortunately, many researchers, especially new-comers, often encounter difficulties in cloning a gene into a desired vector. Secondly, interaction between two proteins is commonly detected by growth of the diploids in specific media. This step takes about two weeks. Here, we describe improved cloning and detection procedures for the Y2H assay that accelerate the research progress. The changes in procedures involve running an agarose gel after the doubly digested vector and insert are ligated in the cloning step to determine the efficiency of RE digestion and ligation, and performing an additional replica-plating on plates for earlier assessment of interaction in the detection step. We show an example of Y2H interaction between Trs23 and Trs120 (respective subunits of TRAPP I and TRAPP II), as a proof of concept. By following the improved methods described here, the chances of successful cloning increased and the time for the whole Y2H experimental process is significantly shorter.

川黔紫薇LeAGL11基因克隆表达分析及转录自激活检测

【目的】探究转录因子AGL11在紫薇属紫薇自交及川黔紫薇与紫薇杂交过程中不同时间的差异性表达及其在酵母中的转录自激活特性,为研究紫薇远缘杂交结实率低的分子机制奠定基础。【方法】以‘紫韵’紫薇自交授粉和 ‘紫韵’紫薇ב川黔1号’川黔紫薇杂交授粉后 24、48 和72 h的雌蕊为材料,克隆LeAGL11基因,通过生物信息学分析其理化性质等,采用实时荧光定量方法分析该基因在不同授粉方式和不同阶段的相对表达量,通过DNA重组技术构建LeAGL11的酵母表达载体,转化至Y2HGold感受态细胞内进行转录自激活检测。【结果】从川黔紫薇中克隆得到LeAGL11基因,该基因的编码序列长666 bp,编码221个氨基酸,LeAGL11蛋白无信号肽和跨膜结构域,不属于膜蛋白,属于亲水性蛋白。氨基酸序列比对和进化树分析显示其与紫薇、石榴和葡萄的AGL11蛋白都有较高的相似度;蛋白质结构预测和序列分析表明其具有MADS-box基因家族中典型的MADS-box和K-box保守结构域。实时荧光定量试验分析不同阶段的相对表达量表明LeAGL11基因在杂交后呈现出上调的趋势,在自交后呈现出先下调后上调的趋势,在自交授粉24 h时相对表达量最高,自激活检测发现pGBKT7-LeAGL11重组质粒在缺色氨酸的培养基中生长,在SD/-Trp+AbA+X-α-gal培养基中没有发生颜色变化。【结论】LeAGL11基因在紫薇自交过程及川黔紫薇和紫薇杂交过程中的相对表达量具有一定的差异性,且LeAGL11无转录自激活活性,可用于后续试验。

杂交兰ChCAO基因克隆及其在叶艺品系中的表达特征

【目的】叶绿素酸酯a加氧酶(CAO)是叶绿素b形成过程中的关键酶,对杂交兰CAO基因进行克隆及表达特征分析可为探究其在杂交兰叶艺形成中的调控作用奠定基础。【方法】以杂交兰‘紫妍氏’(K21)及其叶艺品系‘中透紫妍氏’(K21-3)为试验材料,用RT-PCR和RACE技术从叶片中克隆获得ChCAO基因,对ChCAO进行结构特征、理化性质、序列比对以及系统进化关系等分析;用qRT-PCR法对ChCAO在不同组织及叶艺品系叶片中的表达特性进行分析;并用VIGS技术对ChCAO进行沉默表达。【结果】ChCAO基因编码区长1608 bp,编码535个氨基酸,ChCAO与墨兰CAO亲缘关系最近,并与其他兰科植物CAO聚为一类。qRT-PCR结果显示,ChCAO基因表达具有组织特异性,在叶中相对表达量最高,根中相对表达量最低;此外,ChCAO在K21绿叶和K21-3绿叶区域叶片中的相对表达量显著高于K21-3叶艺区域叶片中的相对表达量。构建该基因的VIGS沉默载体转化烟草,发现沉默ChCAO后烟草叶片呈黄化状态,叶片中的叶绿素含量及ChCAO基因的相对表达量也显著降低。【结论】克隆获得了杂交兰ChCAO基因,并对其功能进行了初步鉴定,为进一步研究杂交兰叶艺形成机理提供重要依据。

白菜种子cDNA酵母文库的构建及BrTTG1互作蛋白的筛选及分析

【目的】通过构建白菜种子的cDNA文库,筛选WDR 40蛋白TRANSPARENT TESTA GLABRA 1(TTG1)的互作蛋白,探究TTG1参与MBW三元复合体调控种皮原花青素形成的分子机制。【方法】以棕籽白菜自交系‘B147’的种子为材料,提取总RNA并建立cDNA文库,通过gateway技术构建诱饵载体pGBKT7-TTG1并进行酵母双杂交筛库。【结果】酵母文库库容为1.2×10^(7)CFU,文库滴度是5.0×10^(7)CFU/mL,插入片段平均长度大于1000 bp,诱饵载体在酵母中无自激活活性。通过构建的诱饵载体pGBKT7-TTG1与构建的cDNA文库杂交,共获得了38个阳性互作蛋白,功能预测显示其中一个蛋白注释为MYB转录因子,注释为MYB73,序列分析结果显示该基因含有R2R3-MYB型抑制子保守基序C1和C2,推测该基因为白菜中参与种皮颜色形成的R2R3-MYB型抑制子,暗示着白菜中可能存在不同MYB转录因子参与的调控网络,影响着原花青素的形成。【结论】本研究构建了白菜种子组织的酵母双杂交cDNA文库,获得了38个TTG1阳性互作蛋白,首次挖掘到了可能影响白菜种皮颜色原花青素形成的R2R3-MYB型抑制子MYB73,为后期探究白菜种皮原花青素的调控网络奠定良好的基础。

钩藤UrLAMT基因及其启动子的克隆与分析

【目的】克隆钩藤(Uncaria rhynchophylla)中马钱苷酸甲基转移酶(loganic acid methyltransferase,LAMT)基因及其启动子,对UrLAMT基因的组织表达及其对生物和非生物胁迫的响应进行分析,并对UrLAMT及其启动子的生物信息学进行分析,为进一步研究UrLAMT转录调控奠定基础。【方法】基于钩藤的转录组数据设计引物,采用PCR从钩藤cDNA中克隆UrLAMT序列,并对其进行生物信息学分析;采用实时荧光定量PCR,分析UrLAMT在钩藤不同组织(根、茎、叶、花、果实、钩)中的表达,以及对外源茉莉酸甲酯(MeJA)、遮光/复光胁迫的响应;利用FPNI-PCR技术克隆UrLAMT上游启动子序列,并验证其活性,结合酵母单杂交试验,分析相应转录因子与UrLAMT启动子的调控关系。【结果】克隆得到钩藤UrLAMT序列,其长度为1137 bp,共编码378个氨基酸。UrLAMT蛋白质相对分子质量为42.64 ku,理论等电点为5.76,为亲水性蛋白,无信号肽,不含跨膜结构,定位在细胞质中;UrLAMT基因在钩藤叶中表达量最高,其次为根;UrLAMT均能响应MeJA和光;其与短小蛇根草和长春花的进化关系较近。UrLAMT启动子序列长度为1141 bp,除核心响应元件外还含有多个光响应元件,UrLAMT启动子在酵母中与光调控转录因子HY5存在相互作用。【结论】获得了UrLAMT基因及其启动子序列,其在钩藤叶中表达量最高,可能受光诱导。

天香百合、药百合黄酮醇合成酶FLS基因克隆和表达分析

以天香百合(Lilium auratum)和药百合(L.speciosum var.gloriosoides)为研究材料,分别克隆获得黄酮醇合成酶(flavonol synthase, FLS)基因,命名为LaFLS和LsFLS。实验结果表明,LaFLS和LsFLS基因均含完整的开放阅读框1 035 bp,均编码344个氨基酸,氨基酸序列高度保守,均具有DIOX-N结构域和2-酮戊二酸和铁(Ⅱ)依赖性双加氧酶结构域,属于2-酮戊二酸和铁(Ⅱ)依赖性双加氧酶超家族;系统进化分析表明,LaFLS和LsFLS除与东方系百合西伯利亚和索邦的FLS亲缘关系最近外,与百合科郁金香(Tulipa fosteriana)等亲缘关系较近;生物信息学分析显示,LaFLS和LsFLS蛋白无信号肽序列和跨膜结构域,均为亲水性蛋白,亚细胞定位结果显示二者主要定位在细胞质中。基因表达分析结果表明,在花蕾发育过程中,LaFLS和LsFLS随花蕾发育出现先上升后下降再上升的趋势,而且在花被片无色区的表达量显著高于有色区域。

杂交马褂木扦插苗优良无性系筛选试验

本试验选取闽亚美马褂木5号等8个一年生杂交马褂木优良无性系,以普通马褂木扦插苗和杉木3代容器苗为对照,对比分析其造林成活率、树高和胸径等生长性状。结果表明,对照组普通马褂木扦插苗成活率91.4%,杉木3代容器苗成活率92.7%,一年生杂交马褂木扦插苗成活率92.5%,与对照差异无统计学意义(P>0.05);一年生杂交马褂木优良无性系的平均树高2.8m、平均胸径3.1cm,与普通马褂木和杉木3代容器苗的树高、胸径差异均具有统计学意义(P<0.05);8个优良无性系中,筛选出无性系品种闽亚美马褂木5号、闽亚美马褂木6号和闽亚美马褂木9号的生长情况较好。研究结果为杂交马褂木优良无性系推广应用提供参考。

尾叶桉×细叶桉杂种无性系扦插生根和生长性状的研究

对尾叶桉×细叶桉5个杂种组合的181个无性系扦插生根和生长性状的研究表明：每穗根数、每穗最长根长和生根率3个扦插生根性状，杂种组合间和杂种组合内无性系间的差异均达0．01显著水平；46个月生树高、胸径和单株材积3个生长性状，杂种组合问差异不显著，杂种组合内无性系间的差异达0．01显著水平。无性系扦插生根性状和生长性状的广义遗传力介于0．32（每穗最长根长）～0．94（生根率）之间，表明各性状受中等到高等程度的遗传控制。无性系扦插生根性状平均值与生长性状平均值的表型相关均不显著。无性系各生长性状在13、18、46个月生之间的年年相关均达到0．01显著水平，表明在较早生长期进行无性系选择具有一定的可行性。本研究初选出易扦插、速生的尾细桉杂种无性系24个，无性系间单株材积株间变异系数为13．87％～68．55％，无性系内变异可能与母株年龄效应、位置效应和立地环境条件等C-效应因子有关。对桉树杂交育种而言，杂交制种后直接进行育苗、扦插和无性系试验是一条快捷有效的无性系培育途径。

白杨派杂种无性系植株早期性状变异与选择研究

以30个白杨杂种无性系为材料,测定树高、胸径、树形、叶片、树皮特性等17个指标。方差分析与遗传参数计算结果表明,白杨杂种无性系各指标差异极显著(P<0.001),各指标表型变异系数变化范围为15.63%～57.50%,遗传变异系数变化范围为8.99%～52.57%,重复力高(0.7734～0.9848)。说明白杨杂种无性系各性状存在广泛变异,且这种变异受较强的遗传因素控制。表型相关结果表明,白杨杂种无性系树高、地径、胸径、单株材积和冠幅之间均达显著相关水平,遗传相关系数接近表型相关系数。主成分分析结果显示,白杨杂种无性系14个性状前3个主成分的累积贡献率为84.93%,其中第1主成分为生长量等性状,第2主成分为干形等性状,第3主成分为叶片性状。利用综合评定法对无性系进行评价,当入选率为10%时,无性系BL204、BL206和BL207入选,入选无性系树高、地径、胸径和单株材积的遗传增益分别为27.41%、31.73%、32.90%和102.37%。这些分析结果为下一步选育优良无性系提供了重要的信息和依据。

白杨双交杂种无性系抗旱性鉴定指标分析

以白杨双交杂种无性系为材料 ,在水分胁迫条件下 ,对无性系生理和生长差异进行研究的基础上 ,建立了抗旱性筛选的指标体系 ,对无性系进行了抗旱性鉴定 ,并对代表干旱适应能力指标及抗旱生产力指标进行了比较分析 ,筛选出了叶片失水率、渗透调节能力、蒸腾速率、净光合速率、苗高和单叶面积 6项指标 。

白杨杂种无性系的抗寒性

以９个白杨杂种无性系为材料，测定了相对电导率、钾离子相对渗出率、失水率、水分饱和亏缺，并进行了苗木生长恢复试验，根据各项指标的测定结果对各无性系抗寒性进行了综合评定。结果表明，无性系间抗寒性差异较大，尤其Ｂ４２９，易县毛白杨、Ｂ４３３与其它５个无性系相比抗寒能力较差，各无性系抗寒性由大到小依次是：ＭＸ１＞Ｂ４３２＞Ｂ４３０＞ＭＸ２＞Ｂ４３１＞Ｂ４１４＞Ｂ４２９＞易县毛白杨＞Ｂ４３３。

杨树新杂种无性系生长与材性的联合选择

以青海省西宁市湟水林场9年生10个杨树新杂种无性系为材料，对生长和材性等9个性状进行了方差分析和遗传参数估算。研究结果表明，树高、胸径、材积、Pilodyn值、南向外侧基本密度、整株木材基本密度、纤维长度、纤维宽度、纤维长宽比在无性系间存在极显著或显著差异。无性系8-25的材积最高，为0．213 m3；8-24的最低，为8-25的14．5％。各性状的重复力在0．711～0．977，其中树高重复力最高，为0．977，纤维长度的最低，为0．711，胸径和材积的均高于0．900。 Pilodyn测定结果与木材密度的相关分析结果表明无性系Pilodyn平均值、南向外侧木材基本密度平均值和整株木材基本密度平均值间存在极显著的相关关系，相关系数为-0．846～0．981。胸径与树高、材积、南向外侧木材基本密度单株间存在极显著和显著的相关关系，相关系数分别为0．852、0．962和0．462。通过对10个无性系生长和材性之间的相关分析，发现杨树新杂种无性系胸径、材积、纤维长与南向外侧整株木材密度显著相关；参试无性系与对照北京杨和青杨比较，杂种无性系都超过青杨对照，有两个超过了北京杨，整株木材密度与对照相近，差异不显著。利用多性状主成分分析，选出8-6、8-16、8-25综合性状表现优良的无性系，其中8-25生长最好，胸径分别是对照青杨、北京杨的1．14、1．61倍，单株材积分别是对照青杨、北京杨的1．80、5．76倍。

水分胁迫下白杨双交杂种无性系苗木生长研究

以白杨双交杂种无性系为材料 ,在水分胁迫条件下 ,对无性系幼苗的生长进行了研究。结果表明 ,水分胁迫对叶片、苗高和生物量生长均产生显著影响。轻度水分胁迫即可使苗木叶面积生长受到抑制 ,随着水分胁迫的加剧 ,叶片速生期有提前的趋势 ,速生期持续时间有延长趋势 ,但受胁迫的苗木绝对生长速率较低 ,没有明显的生长高峰期出现 ,造成单叶面积随水分胁迫的发展而显著减小。水分胁迫对叶面积的影响大于对苗高生长的影响 ,在供水量为 6 0 %时 ,苗高生长才受到明显抑制。根、茎、叶干物质积累以供水量 80 %最为适宜 ,供水量进一步下降 ,各部分干物质积累逐渐受到抑制 ,茎和叶生物量受抑制最明显 ,根生物量受影响相对较小 ,因此随着水分胁迫的发展 ,茎 根比值表现为明显的下降趋势。不同无性系在各生长指标上和各指标随水分胁迫下降幅度上均存在着显著差异。大部分指标间表现为正相关关系 。