日本落叶松种子萌发过程中18个miRNAs的表达变化

运用qRT-PCR技术,检测日本落叶松种子萌发过程中的5个阶段——干种子 (萌发培养0 d)、吸胀(1 d)、萌动(5 d)、胚根外露(9 d)和子叶展开(28 d),miR156、miR159和miR160等18个miRNAs在其种胚内的表达模式,并对其目标基因进行生物信息学分析,探讨miRNA在针叶林木种子萌发过程中的基因表达模式。结果表明:(1)从整个萌发过程分析,18个miRNAs的表达水平皆以干种子阶段最高,吸胀和萌动阶段迅速下降;干种子阶段的表达水平分别是吸胀、萌动、胚根外露和子叶展开阶段的4.8 43.5、17.2 1 000.0、45.5 1 000.0、62.5 1 862.2倍。(2)从各个阶段分析,miR894的表达水平在5个阶段均最高,miR408均最低;其余16个miRNAs在5个阶段内的表达水平各有变化,综合排序结果从高到低依次为:miR951、miR950、miR156、miR159、miR396、miR398、miR390、miR160、miR947、miR165、miR319、miR162、miR171、miR397、miR395和miR172。(3)miRNA目标基因预测结果显示:目标基因主要是ATP/DNA结合蛋白、ARF等转录因子、质膜和核糖体等细胞组分及MAP等激酶活性蛋白等蛋白编码基因。(4)miR160对其目标基因JR160237 具有切割作用,切割位点发生在miR160与目标基因互补结合位点第10与11位碱基之间。JR160237在种子吸胀阶段表达水平迅速升高,在萌动、胚根外露和成苗阶段有所回落。     

落叶松体胚发育中12个针叶树特异microRNAs表达分析

以落叶松体细胞胚胎发育8个阶段的材料构建小RNA文库,进行了Solexa测序、miRNA鉴定、靶基因预测、qRT-PCR定量分析等研究。结果表明:发现的87个miRNAs中,29个为针叶树特异的,来自12个家族,分别为miR946、miR947、miR950、miR951、miR1311、miR1312、miR1313、miR1314、miR1316、miR3701、miR3702和miR3704,其中25个长度为22nt,且在小RNA文库中的最低拷贝数为18(miR1311),最高拷贝数为33 009(miR950)。发现12个miRNA家族的34个靶基因可能参与生长发育、抗病、AGO反馈调节等多方面遗传调控;qRT-PCR分析表明:72%miRNAs表达次高峰在后期单胚或早期子叶胚,在中期子叶胚最低,最高峰在后期子叶胚,分别与子叶形成、胚胎后熟、胚胎休眠的生理活动相吻合,而miRNA前体的表达模式不同于miRNA,前体变化幅度小于成熟体。     

黑杨三倍体与二倍体叶片中miRNA表达差异研究

本文以改进CTAB法提取的总RNA为模板,通过设计茎环反转录引物,利用定量RT-PCR技术从黑杨幼嫩叶片、成熟叶片中均检测到miRNA。结果表明,miR159、miR167、miR171、miR319在三倍体幼叶中表达量均高于二倍体,miR164在三倍体成熟叶片中的表达量高于二倍体表达量。除miR319外,其余miRNA与其相应的靶基因在二倍体和三倍体黑杨不同阶段叶片中表达的消长关系都较为典型。暗示这些miRNA 靶基因可能与黑杨三倍体的速生性状形成相关。     

中间锦鸡儿生长发育过程中5个miRNAs及其靶基因的表达模式分析

利用qRT-PCR技术分析了5个miRNAs(cin-miR157a、cin-miR159a、cin-miR165a、cin-miR172b和cin-miR396a)及其靶基因在中间锦鸡儿(Caragana intermedia)不同发育阶段不同器官的表达变化。结果显示,5个miRNAs的表达模式均存在时空特异性,其中cin-miR159a、cin-miR165a、cin-miR172b、cin-miR396a在成年期营养器官中强烈表达,在幼年期和成年期生殖器官中弱表达;cin-miR157a在幼年期和成年期生殖器官中强烈表达,在成年期营养器官中弱表达。除cin-miR159a的靶基因MYB和cin-miR165a的靶基因HD-Zip Ⅲ外,其它被检测的miRNAs靶基因与相应的miRNAs的表达呈现出明显的相互消长的表达模式。该结果表明中间锦鸡儿miRNA可通过调控其靶基因的表达,进而调控发育阶段转变、器官形态建成、细胞分化等生物学过程。研究结果将为中间锦鸡儿的定向改良工作提供理论依据和基因资源。     

核果类果树中microRNAs的生物信息学预测及验证

microRNA(miRNAs)是一类非编码小分子RNA,在植物生长发育、新陈代谢以及逆境生理中起重要作用。基于生物信息学的基因搜索和同源搜索,从NCBI数据库中登录的核果类果树的EST和GSS中寻找miRNAs。从核果类果树的107982条ESTs和48273条GSSs中搜寻到了11条miRNAs前体序列,编码11条成熟体序列,并预测到19个靶基因,分别编码与生长发育、新陈代谢和转录调节等相关的蛋白。利用miRbase数据库进一步分析表明:11条miRNAs属于8个microRNA家族,分别来自桃和梅,其大小为20～23bp,其中6条为21bp,而且ppe-miR162a和ppe-miR164f保守性最高。采用poly(A)RT-PCR方法随机对预测的ppe-miR156h,pmu-miR482,ppe-miR399a,ppe-miR171a进行组织表达验证,发现ppe-miR156h在果梅花芽中表达,pmu-miR482,ppe-miR399a,ppe-miR171a在桃叶片中表达。     

辣椒miR169家族在非生物胁迫下的表达及启动子顺式作用元件分析

miRNA169(miR169)是植物中保守且规模较大的一类miRNA基因家族,其在非生物胁迫下的功能被广泛报道。为深入研究辣椒miR169基因家族(Can-miR169)在非生物胁迫下的表达及上游转录调控机制,以‘遵辣一号’(Zunla-1)为研究对象,利用生物信息学手段分析辣椒与其他植物miR169成熟体和前体保守性和进化关系。采用real-time PCR技术对干旱、低温、高盐、高温胁迫下辣椒miR169基因家族前体进行应答检测。克隆辣椒Can-miR169基因家族12个成员启动子,进行顺式作用元件预测及元件功能验证。结果显示：(1)miR169成熟体在植物中保守性强。(2)4种非生物胁迫下,辣椒miR169家族成员前体表达量相对于对照组差异显著。(3)成功克隆了Can-miR169基因家族12个启动子,顺式作用元件预测其家族启动子中含有大量与非生物胁迫相关的元件,如MYB、MYC、MBS、ABRE、LTR、DRE等。(4)经验证明,这些元件为干旱、低温、高盐等胁迫应答元件。本研究结果为进一步挖掘非生物胁迫下调控Can-miR169的转录因子,明确其上游调控机制提供新信息,为辣椒...     

miR396在落叶松体细胞胚胎中的功能研究

[目的]探讨miR396在落叶松体细胞胚胎发生中的功能.以期该研究能为解决体胚发育中子叶畸形胚与生根率低的问题提供新的视角,同时也为进一步揭示体胚发育的分子调控网络做基础铺垫.[方法]构建pre-miR396超表达载体,通过农杆菌介导侵染,将pre-miR396转入落叶松胚性细胞中,筛选稳定抗性细胞系.在DNA与RNA水平上,对抗性细胞系进行的鉴定、检测,同时统计比对其与野生细胞系间发芽率、叶片数目以及生根率的差异.[结果]在抗性细胞系基因组中,扩增出HTP与miR396的特异性片段,且无农杆菌Virg基因片段.RNA表达水平上,抗性系pre-miR396与miR396表达皆增加,而靶基因LaGRFs的表达量下调.统计表明抗性系胚的发芽率、生根率、叶片数目畸形率分别为70.60%、0.60%、37%,而野生型分别为92.50%、10.55%、4%,抗性系胚的发芽率、生根率显著降低(Sig.=0.000),叶片数目畸形率显著增加(Sig.=0.000).[结论]miR396在落叶松体细胞胚胎发生过程中,可能通过负调节靶基因LaGRFs的表达或者通过LaGRFs影响与它相互作用的基因,参与体胚子叶原基细胞代谢,从而影响了子叶与叶片的发育;通过负调节LaGRFs或者其它与根部发育相关的靶基因,影响了根端分生区细胞的活动,参与调控根的发育.     

猕猴桃Ac-miR160d启动子区的克隆及表达分析

【目的】为深入研究猕猴桃miR160d转录调控机制提供线索,也为猕猴桃分子育种及品种改良提供参考。【方法】根据‘红阳’猕猴桃基因组信息,设计引物克隆miR160d(Ac-miR160d)前体基因及其启动子序列,并对其前体序列进行二级结构验证,对启动子序列进行保守结构域和顺式作用元件分析。同时,对猕猴桃果实进行非生物(NaCl、GA_3)和生物(灰霉菌)胁迫处理,利用qRT-PCR技术检测miR160d在不同胁迫处理后0～6 d时的表达情况。【结果】获得的Ac-miR160d前体序列具有稳定的茎环折叠的RNA二级结构以及miR/miR*二聚体结构,Ac-miR160d启动子序列不仅具有TATA-box、CAAT-box基本元件,还包含参与激素(GA_3、水杨酸、脱落酸、茉莉酸等)应答、厌氧响应、干旱诱导等逆境胁迫以及光响应等多种顺式作用元件。猕猴桃果实内AcmiR160d的表达分析结果显示:当猕猴桃果实受到盐胁迫时,Ac-miR160d的水平在胁迫后1 d时即发生显著下调,胁迫后3～6 d时下调更为明显;当猕猴桃果实受到赤霉素胁迫和灰霉菌侵染时,Ac-miR160d的水平出现先升高、后降低的趋势,在处理后1～2 d时急剧升高,随后逐渐降低。【结论】Ac-miR160d启动子与猕猴桃植物激素信号及胁迫信号响应密切相关。通过非生物和生物胁迫试验,证实Ac-miR160d参与猕猴桃植物激素信号响应,并在其抗逆境胁迫过程中发挥重要调控作用。     

胁迫条件下毛竹miR164b及其靶基因PeNAC1表达研究

MicroRNAs(miRNAs)在植物生长发育以及抗逆等过程中起着重要的调控作用。miR164 作为植物特有的miRNA,其主要的靶基因是植物NAC转录因子。为揭示毛竹 miR164 对其靶基因的调控机制,通过茎环引物法和RT-PCR技术,从毛竹(Phyllostachys edulis)中克隆出miR164b成熟序列及其靶基因PeNAC1。序列分析表明miR164b的靶点位于PeNAC1 编码区,RLM-5'RACE PCR产物测序结果证实切割位点位于靶点的第10-11位碱基之间。组织特异性表达分析表明,毛竹 miR164b和PeNAC1在根、茎、叶及鞘中均表达,其中miR164b在根中表达丰度最高,在茎中表达丰度最低;而PeNAC1的表达丰度恰好与miR164b 相反。实时定量PCR结果显示,NaCl(250 mmol·L^-1)、低温(4℃)和强光(1 500 μmol·m^-2·s^-1)处理后毛竹叶片中 miR164b 的表达均明显下调,GA₃(100 μmol·L^-1)处理后 miR164b表达量明显上调;而在同样处理条件下,PeNAC1的表达恰好呈现出与其完全相反的趋势。由此表明,miR164b对靶基因PeNAC1 具有表达调控作用,可能与毛竹响应非生物胁迫的抗逆过程密切相关,这为利用miRNA开展竹子抗逆分子育种提供了参考。     

高强度微波辐照和汽蒸处理对落叶松木材材色的影响

为了揭示高强度微波辐照处理、汽蒸处理和微波-汽蒸处理对落叶松(Larix spp.)木材色度学参数的影响及其与微波功率和汽蒸处理时间的关系,进而评价微波汽蒸处理对落叶松木材材色的影响,采用同一树干的落叶松板材生材,测量并计算其在微波功率5.54、12.92和24,与汽蒸2、4、6、8、12、16 h处理下同一位置处理前后,色度学参数L*、a*、b*并通过公式计算出C*、Ag*和ΔE*等参数,系统分析处理前后色度学参数的变化情况。结果表明,素材气干后,L*值和Ag*值升高,a*、b*和C*值降低,总体色差ΔE*升高;微波处理并气干后,试材的色度学参数变化趋势与素材一致,但其数值均略大于素材值的绝对值;汽蒸处理并气干后,试材的L*值和ΔE*值升高,b*值和C*值降低,a*值在汽蒸2 h和4 h时升高,其余各处均降低,Ag*值在汽蒸2、4 h时降低,其余时间段为升高或不变;微波-汽蒸处理并气干后,试材受微波处理和汽蒸处理的共同影响,L*值和ΔE*值升高,变化趋势相似,b*值和C*值降低,a*值在12.92 kW-汽蒸8 h和12 h处升高,其余各处均降低,Ag*值在5.54kW-汽蒸2 h、4 h和6 h,12.92 kW-汽蒸8 h和12 h,24kW-汽蒸4、6、12 h处为降低。     

Estimated amount of carbon accumulation of hybrid larch in three 31-year-old progeny test plantations

Hybrids generated by crossing Kuril larch (Larix gmelinii var. japonica) and Japanese larch (L. kaempferi) are expected to have high carbon accumulation ability because of fast growth and high wood density in Hokkaido, Japan. We estimated the amount of carbon accumulation of the hybrid larch in three progeny test plantations consisting of 21 full-sib families and compared the results to the carbon accumulation of open-pollinated progenies of Japanese larch plus-trees (improved Japanese larch). Gene-environment interactions were not observed for tree height and diameter at breast height but were seen for wood density (area-weighted density, AWD). The amount of carbon accumulated per unit area (C_stand) positively correlated with the stand volume. The AWD did not correlate with the C _stand; therefore, families with high wood density can be selected independent of the C _stand. The C _stand of the best full-sib family, female parent half-sib family, and male parent half-sib family at three sites were 106.1, 84.6, and 93.2 Cton·ha⁻¹, respectively. All these values exceed the mean for the improved Japanese larch, which has a C _stand value of 82.5 Cton·ha⁻¹.     

Color and surface chemistry changes of extracted wood flour after heating at 120 °C

To investigate the effect of heat on color and surface chemistry of wood flour (WF), unextracted, extracted and delignified samples of commercial WF were heated at 120 °C for 24 h and analyzed by colorimetry, diffuse reflectance visible (DRV), attenuated total reflectance Fourier transform infrared (ATR-FTIR) and Fourier transform Raman (FT-Raman) spectroscopies. Unextracted samples showed a slight increase in CIEL^*a^*b^* color coordinates, a ^* and b ^*. Compared with unextracted samples, color changes of the extracted samples varied with composition of the extraction solvents with generally smaller increases in a ^* and larger decreases in b ^* values. Delignified samples were marked by even larger increases in both a ^* and b ^* values. The color changes could be explained by analysis of the respective DRV, FTIR and FT-Raman spectra of the samples before and after heating. Heating of the extracted WF at 120 °C resulted in a red shift of the absorption at 430 nm and increase in absorption in the violet-blue spectral region of visible light. Delignified samples showed even more pronounced absorption in this spectral region after heating.     

日本落叶松 UDPGDH基因的cDNA克隆和表达分析

以日本落叶松杂交Solexa转录组测序获得的数据为依据,经拼接后获得尿苷二磷酸葡萄糖脱氢酶(UDPGDH)原序列,以该序列两端设计引物,在落叶松亲代和子代幼嫩针叶中成功克隆了UDPGDH基因cDNA序列,序列长1 443 bp,编码480个氨基酸。qRT-PCR结果分析表明:UDPGDH基因的表达量在落叶松杂交子代中远远高于亲本,正交子代的表达比反交子代高,说明UDPGDH在形成细胞壁半纤维素前体物过程中起着非常重要的作用,暗示UDPGDH在子代的高表达有利于杂种优势的形成。     

Enhanced seed production and metabolic alterations in Larix leptolepis by girdling

The demand for Japanese larch (Larix leptolepis) seeds has increased in Korea but their supply has been limited due to sporadic natural seed production. Among the various flower induction techniques, girdling woody stems has been recognized as a simple and efficient method to enhance seed production in a number of conifers. In this study, stem girdling was applied to 42-year-old Japanese larches, resulting in remarkable enhancement of seed cone production with no morphological changes in the seed characteristics. Metabolic alterations in needles of the girdled and the control trees were interrogated by using 23 individual metabolites including soluble sugars, polyamines and amino acids. In the girdled trees, the contents of both soluble sugars and polyamines were higher from June to July and then became similar to those in the control. The levels of amino acids were significantly increased only in August by girdling compared to the non-girdled trees. In control larch trees, the dominant amino acids were changed from Glu and Ala until July to Asp and Tyr in August. Interestingly, the concentrations of Asp-derived amino acids such as Lys, Met and Thr, were increased by girdling and they also showed significant correlations with the number of seed cones per trees, suggesting that the enhanced levels of three amino acids might positively influence on the development of seed cones in girdled larch trees. Although the role of Asp-derived amino acids on enhanced seed cone production remains unclear, the approach presented in this study might provide useful information in elucidating metabolic network modulation induced by girdling and will be further applied for enhanced seed cone production in Japanese larch trees.     

Differences in spectral trajectory with stand volume development between Japanese larch and Japanese oak in Hokkaido,Japan

Our objective was to investigate the differences in the spectral trajectory of the LANDSAT Thematic Mapper with stand volume development between Japanese larch (Larix kaempferi) and Japanese oak (Quercus mongolica var. crispula) in the experimental forest of Kyushu University, Hokkaido, Japan. We used nonlinear regression and a categorical variable to investigate the relationship between spectral reflectance factor and stand volume for each forest type. This research showed that the reflectance factor of Japanese larch and Japanese oak reached saturation and that both forest types had different spectral trajectories with stand development. In addition, we found that middle of development stage (stand volume was between 100 and 200 m³/ha) of each forest type had similarity in reflectance factor.     

Heat-induced colour changes of pine (Pinus pinaster) and eucalypt (Eucalyptus globulus) wood

Heat treatment of Pinus pinaster and Eucalyptus globulus wood was carried out by hot air in an oven for 2–24 h at 170–200°C and by steam in an autoclave for 2–12 h at 190–210°C. The colour parameters L*, a* and b* were determined by the CIELAB method on radial, tangential and transverse sections of untreated and treated wood, and their variation with regard to the treatment (ΔL*, Δa* and Δb*) were calculated in percent. For untreated eucalypt wood, lightness (L*) varied between 54.1 and 63.8% with a* between 7.4 and 8.5, and b* between 15.7 and 19.9. For untreated pine wood, L* varied between 67.3 and 76.1%, a* between 6.9 and 7.6 and b* between 16.3 and 24.1. Oven heat-treated wood became darker (ΔL* about 50% for 4% mass loss), and this was more for eucalypt wood under the same treatment conditions. In general, the contribution of red (a*) and yellow (b*) colour decreased with heat treatment. The transverse section of the two species darkened less for both the treatments with small differences between radial and tangential sections. Lightness decrease was related to chemical changes; with good correlations with glucose (R ² = 0.96), hemicelluloses (R ² = 0.92) and lignin (R ² = 0.86). As regards colour, the heat treatments showed an interesting potential to improve the wood quality for solid timber products from pine and eucalypt.