为探究木荷幼苗生长对不同氮磷钾配比营养液的响应情况,为木荷壮苗培育合理施肥提供科学的理论依据,选择1月龄木荷幼苗作为试验材料,以Hoagland营养液为母液,进行氮磷钾3因素6水平缺素试验,在水培条件下研究不同氮磷钾配比对木荷幼苗生长的影响.结果表明:不同的氮磷钾营养液配比中,木荷幼苗的各项指标变化趋势基本一致,氮磷钾肥对木荷幼苗生长的影响表现为P>K>N,处理15表现最佳,处理12幼苗长势最差;木荷幼苗的株高、生物量、SOD、POD、CAT、SP均表现出随着施肥水平的提高先升高后降低的变化趋势,Pro和MDA在高水平施肥时显著增加.研究显示,以Hoagland液为水培营养液母液,氮磷钾水平分别为母液中氮磷钾元素浓度的1、1和0.5倍时,木荷幼苗生长最佳.

为测定茶果樟种子的生活力,以2019年、2020年采集的茶果樟种子为研究材料,采用2,3,5-氯化三苯基四氮唑染色法(TTC法),利用3因素3水平的正交试验L9(34)设计,研究不同染色时间、TTC溶液浓度和染色温度对茶果樟种子生活力的影响.结果发现,TCC法能快速测定茶果樟种子的生活力,对2019年、2020年采集的茶果樟种子,生活力检测的最佳条件均为温度35℃+0.7％TTC溶液浓度+浸泡24 h,其中染色时间是显著影响茶果樟种子生活力测定的一个因素.茶果樟种子发芽潜力较好,且2020年的茶果樟种子生活力大于2019年采集的种子,表明茶果樟人工育苗宜采用随采随播的方式.

为了解蒜头果叶绿体基因组密码子的使用偏性,利用Codon W 1.4.2和CUSP软件对蒜头果叶绿体基因组中33个基因的密码子进行中性绘图,应用ENC-plot、PR2-plot绘图分析并确定了其最优密码子.结果 表明:蒜头果叶绿体基因组密码子的第3位碱基GC含量为28.43％,远低于第1位(47.74％)和第2位(41.05％),RSCU值大于1的密码子有30个,其中12个以A结尾,16个以U结尾,说明蒜头果叶绿体基因组的编码基因偏好以A和U结尾.GC12和GC3的相关系数为0.1646,相关性不显著,回归系数为0.0004;有效密码子数(ENC)范围为40.39～54.86,大于45的有25个,ENC比值主要分布在-0.05～0.05之外.蒜头果叶绿体基因组中的大部分基因分布在PR2平面图的下半部或右下半部,说明蒜头果叶绿体基因组密码子偏好性更多地受选择的影响,同时亦受其他因素的影响.蒜头果叶绿体基因组的最优密码子确定为UUU、UUA、GUA等18个.

以云南蓝果树叶绿体基因组为研究对象,采用Codon W 1.4.2等软件,通过分析云南蓝果树叶绿体基因组52个基因密码子的偏好性,探究影响密码子使用偏性形成的主要因素.结果 表明:1)Codon W和CUSP软件分析显示密码子第3位碱基GC含量为28.40％,ENC值＞45的基因有39个,说明密码子偏好以AT结尾,且存在较弱偏性.2)中性绘图分析显示,GC12与GC3的相关系数为0.161 2,相关性不显著,说明密码子第1、第2位与第3位碱基组成存在显著差异.3)ENC绘图结合ENC比值频率表显示,大部分基因距离标准曲线较远,说明密码子偏好性主要受选择的影响.4)PR2-plot分析显示密码子第3位碱基使用频率方面,T＞A,G＞C,说明密码子偏好性受多重因素影响.5)确定UUG、UCA、GCU、AAU、GAU和GGA为最优密码子.综上所述,云南蓝果树叶绿体基因组密码子的使用主要受选择的影响,受其他如突变等因素的影响较弱.

为了探讨印度紫檀(Pterocarpusindicus)萜类化合物的生物合成,本研究通过对合成萜类物质的MEP途径的第一个限速酶(1-脱氧-D-木酮糖-5-磷酸合酶,DXS)基因进行克隆,获得了印度紫檀DXS的全长cDNA PiDXS基因(NCBI登录号:MK959226),并对其进行生物信息学和组织表达特异性分析.结果显示:PiDXS基因cDNA开放阅读框长2 025 bp,编码674个氨基酸,Blast比对发现,PiDXS具有一个二磷酸硫胺结合位点和一个转酮醇酶结构域;系统进化分析显示,PiDXS基因与大豆DXS和狭叶羽扇豆DXS聚为一类;组织表达特异分析结果显示PiDXS在茎、叶、树皮和根中均能表达,主要在幼嫩的组织中表达.研究结果为确定印度紫檀中DXS的基因功能以及揭示印度紫檀萜类化合物的生物合成提供了基础.

The first complete chloroplast genome (cpDNA) sequence of Pericampylus glaucus was determined from Illumina HiSeq pair-end sequencing data in this study. The cpDNA is 162,450 bp in length, contains a large single-copy region (LSC) of 90,871 bp and a small single-copy region (SSC) of 21,137 bp, which were separated by a pair of inverted repeats (IR) regions of 25,221 bp. The genome contains 130 genes, including 85 protein-coding genes, 8 ribosomal RNA genes, and 37 transfer RNA genes. The overall GC content of the whole genome is 38.0%, and the corresponding values of the LSC, SSC, and IR regions are 36.2, 32.1, and 43.5%, respectively. Further, phylogenomic analysis showed that P. glaucus and Stephania japonica clustered in a clade in family Menispermaceae.

利用石蜡切片法观察极小种群植物—云南蓝果树两性花的小孢子发育过程,以明确小孢子败育的原因和败育时期,为进一步拯救保护和研究云南蓝果树奠定基础.结果表明:(1)云南蓝果树有一部分两性花小孢子正常发育,经过小孢子母细胞、 减数分裂和四分体等时期,最后发育为成熟花粉粒;(2)败育的云南蓝果树两性花在形成四面体型四分体之前都能正常发育,在四分体时期不能分离或分离后不能正常发育,从而败育.研究认为,绒毡层细胞发育异常是云南蓝果树两性花小孢子败育的主要原因,败育的时期是四分体时期和单个小孢子时期.

极小种群野生植物云南蓝果树是国家和云南省实施极小种群野生植物保护工程的代表性物种.为有效保护其遗传资源,本研究通过二代测序技术,对其进行简化基因组测序,开发一批特异性高的单核苷酸多态性标记,分析现存群体的遗传结构和遗传多样性.经过遗传变异检测,本次研究中共获得SNP位点98 498个,通过样品最低测序深度＞2,样品缺失率＜0.5,次要基因型频率(MAF) ＞0.05筛选以后,得到有效SNP位点6 309个.基于过滤后的SNP,运用生物信息学分析方法,对云南蓝果树完成了群体的遗传分析,其中:系统进化树分析将云南蓝果树划分为3大类,研究分析了云南蓝果树各分类的私人等位基因数目(Private)、平均观测杂合度(H0)、平均期望杂合度(He)、核苷酸多样性(π)和平均近交系数(Fs)5个遗传多样性参数;群体结构和主成分分析进一步证明了,云南蓝果树现存植株之间亲缘关系较远,遗传多样性差异较大,具有很高的遗传资源保存价值.本研究结果将为基于遗传管理的云南蓝果树就地保护、遗传资源保存和种群重建等保护工程提供科学依据.

为探讨云南蓝果树(Nyssa yunnanensis W.C.Yin)的濒危机理,运用解析木的原理,结合1979—2009年的气象数据,对两株云南蓝果树风倒木进行研究,分析主要气候因子对云南蓝果树生长指标的影响.结果表明:云南蓝果树的径向、高度和年轮宽度的年增长量呈逐渐下降趋势,而材积年生长量呈上升趋势.云南蓝果树生境自1979—2009年年平均气温缓慢地升高,年总降水量和年平均相对湿度值呈总体减少趋势;年均径向、高度生长量和年轮宽度与年平均气温呈负相关,与年降水量和相对湿度呈正相关;年均材积生长量与年均气温呈正相关,与年降水量和相对湿度呈负相关.

以野外已经灭绝的极小种群植物富民枳为对象,基于当前建立的种质资源收集圃开展群体遗传分析研究,探讨种质资源收集保存和野外回归的有效途径,为极小种群植物的拯救保护提供科学依据.利用毛细管电泳检测,从已发表的51个柑橘属SSR引物中,筛选出特异性高和多态性好的,对采集自10个种质资源收集地的170份富民枳样本进行遗传多样性分析,采用POPGENE、PIC_Calc、Populations、FigTree和Origin软件分别进行遗传参数估计、 引物多态性信息含量计算、 邻接树构建和聚类图的绘制.结果显示:SSR引物的多态性信息含量(PIC)平均为0.3378;Shannon信息指数(I)平均为0.6428;有效等位基因数(Ne)平均为1.7987;Nei's遗传多样性指数(Nei)平均为0.4300;平均期望杂合度(He)为0.4300;平均观测杂合度(Ho)为0.5687.聚类分析显示,170个富民枳样本共聚类了65个分支末梢,属于同一个分支末梢的被默认为同等的遗传关系,我们称这65个样本为"有效样本".综合评价结果表明:富民枳现存种质资源的遗传多样性水平较低,只有C2和C7在遗传水平上的保护是有效的,即有效样本量大于10,Shannon信息指数又达到该物种遗传多样性水平的90％以上.本研究结果为该物种有效的遗传管理奠定了理论基础.

In this study,controlled pot experiment was carried out to study impacts of different light intensities(100%light, 60%light and 20%light)and different water conditions(100%, 91.68%, 82.85%, 60.00%, 41.86%, and21.28%WHC)(water holding capacity,WHC)on growth and photosynthetic characteristics of N.yunnanensis seedlings.Measure-ments were taken after 6 months of seedlings treated.Both growth parameters,including leaf area,plant height,root bio-mass and aboveground biomass,and photosynthetic characteristics, including Pn, Trand Gsof N.yunnanensis seedlings were decreased with the decrease of soil relative water content.Intense light irradiation(100%light)and deep shade (20%light)were detrimental for the growth and photosynthesis of N.yunnanensis seedlings, but moderate shade(60%light)was most conducive to the individual development of seedlings.When the soil moisture were sufficient,differences in photosynthetic indexes among different light intensity treatments were not significant;however,differences were significant under drought stress conditions.The photosynthetic indexes of 60%light treatments were higher than those of the other two light intensity treatments.The results of comprehensive evaluation demonstrated that the comprehensive evaluation scores of all indexes were the largest in 60%light and soil moisture conditions treatments.The moderate shade could promote growth of N.yunnanensis seedlings,but both the too high or too low of the light intensity were bad for their growth and develop-ment.Established linear regression equation of two variables demonstrated that the effects of soil moisture were more impor-tant than that caused by light on seedling growth,whereas the contribution rate of light intensity was greater for photosyn-thetic indexes.Therefore, adequate soil relative water content(100%WHC)and moderate light intensity(60%light) were best suited for the growth of N.yunnanensis seedlings.

For many plants, drought is an important factor affecting the natural regeneration of forest plants.However,the mechanisms by which drought affects natural regeneration of Nyssa yunnanensis are not well known.In this experiment,controlled experiments were conducted to study the effects of drought on water demand limitation and autotoxity on seed germination and early seedling growth of N. yunnanensis. The results showed that the concentration of PEG 6000,the concentration of extract solution and the source of extract or?gans (roots and leaves) had significant effects on seed germination and early seedling growth index of N.yun?nanensis.Morever,the seed germination rate,germination vigor,root length and seedling length decreased with the increase of the concentrations of PEG 6000 and extract solution.However,there was no significant difference between the concentration of 12.5 g/L and 25 g/L extract,and between 10％ and 15％ PEG 6000 concentra?tion.Established linear regression equation of two variables demonstrated that the absolute value of standardized influence coefficient of leaching solution is lower than that of PEG 6000 concentration, suggesting that the effects of water loss caused by PEG was more important than that caused by drought on seed germination. Therefore,water deficiency and aggravating autotoxicity caused by drought,which was induced by the change in original habitat and climate change,had jointly affected the seed germination and early seedling growth of of N. yunnanensis,thus hindering its natural regeneration.The results would illuminate impacts of drought on the nat?ural generation of N. yunnanensis and their mechanisms.Conclusions made from natural regeneration difficulties might partly help reveal endangered reasons of N. yunnanensis,which would provide scientific and theoretic ba?sis for its effective conservation.

Climatic change-induced water stress has been found to threaten the viability of trees, especially endangered species, through inhibiting their recruitment. Nyssa yunnanensis, a plant species with extremely small populations (PSESP), consists of only two small populations of eight mature individuals remaining in southwestern China. In order to determine the barriers to regeneration, both in situ and laboratory experiments were performed to examine the critical factors hindering seed germination and seedling establishment. The results of in situ field experiments demonstrated that soil water potentials lower than -5.40 MPa (experienced in December) had significantly inhibitory effects on seedling survival, and all seedlings perished at a soil water potential of -5.60 MPa (January). Laboratory experiments verified that N. yunnanensis seedlings could not survive at a 20% PEG 6000 concentration (-5.34 MPa) or 1/5 water-holding capacity (WHC; -5.64 MPa), and seed germination was inhibited in the field from September (-1.10 MPa) to November (-4.30 MPa). Our results suggested that soil water potentials between -5.34 and -5.64 MPa constituted the range of soil water potentials in which N. yunnanensis seedlings could not survive. In addition to water deficit, intensified autotoxicity, which is concentration-dependent, resulted in lower seed germination and seedling survival. Thus, seed establishment was probably simultaneously impacted by water deficit and aggravated autotoxicity. Meteorological records from the natural distribution areas of N. yunnanensis indicated that mean annual rainfall and relative humidity have declined by 21.7% and 6.3% respectively over past 55 years, while the temperature has increased by 6.0%. Climate change-induced drought, along with a poor resistance and adaptability to drought stress, has severely impacted the natural regeneration of N. yunnanensis. In conclusion, climate change-induced drought has been implicated as a regulating factor in the natural regeneration of N. yunnanensis through suppressing seed germination and screening out seedlings in the dry season. Based on the experimental findings, habitat restoration and microclimate improvement should both be highlighted in the conservation of this particular plant species.

In order to understand effects of salicylic acid (SA) pretreatment on seed germination and early seedling growth of Nyssa yunnanensis under drought stress, N.yunnanensis seeds were soaked with different concentrations of SA (0g/L,0.25g/L,0.5g/L,0.75g/L,1g/L) and treated with different concentrations of PEG-6000 solutions (0,5%,10%,15%).Results showed that seed germination and early seedling growth were significantly inhibited by mild drought stress without SA pretreatment,and seed germination rate,germination potential,germination index,vigor index,seedling root length,root shoot ratio,root dry weight,root fresh weight,cotyledon fresh weight and cotyledon dry weight were significantly decreased with the increase of PEG-6000 concentration.However,seed germination rate,germination potential,germination index,vigor index,seedling root length,root dry weight,root fresh weight,cotyledon fresh weight and cotyledon dry weight were significantly higher than that of control,and increased with the increase of SA concentration (0.25~1.0g/L) under different degrees of drought stress(PEG-6000 5%~15%).When SA concentration was 0.75g/L,the increase was the most significant.It is indicated that N.yunnanensis has poor tolerance to drought environment,but seed soaking with SA could relieve effects of drought stress on seed germination and early seedling growth.In conclusion,it is suggested that N.yunnanensis seeds should be pretreated with SA at the optimum concentration of 0.75g/L.This study provides a scientific basis to apply SA in the protection and breeding of N.yunnanensis.

Conservation of plant species with extremely small populations (PSESP) is a focus of wild plant conservation in China at present. A relevant strategy for PSESP conservation requires improvement from previous programs for rare and endangered plants and national key protected plants. An integrated strategy for PSESP conservation of the Nyssa yunnanensis was initiated and applied over a 7-year period (2009–2015). Here, we reviewed the processes to implement the strategy: resource inventory, formulation of conservation action plan (CAP), in situ conservation, seedling propagation, near situ conservation, ex situ conservation and scientific research. Major concerns and technical requirements for each action are described and further analyzed within a broad scope to conserve PSESPs. A detailed resource inventory that highlights both the change in population status and the participation of local residents is recommended before the formulation of the CAP. Techniques for determination of the area of a mini-reserve is developed for in situ conservation of N. yunnanensis. Near situ conservation is a novel approach whereby establishing new viable population in contiguous areas with a similar climate, habitat and community, in which techniques for seedling preparation, soil preparation, and early management are introduced. A population-based species conservation strategy for N. yunnanensis may aid additional PSESP conservation, so as to contribute to overall wild plant conservation.

In order to take reasonable and effective protection of Poncirus polyandra,SSR markers was used as genetic integrity detection markers to study effective sample size of different gradient on genetic diversity index in microsatellite analysis.Five pairs of SSR primers were used to detect the genetic integrity of the 65 germplasm resources,and to analyze the genetic diversity of the germplasm resources.Results demonstrated that the effective sample size had significant positive correlation effects on genetic diversity index (effective number of alleles,observed heterozygosity,expected heterozygosity,polymorphic information content and Shannon's information index) of P.polyandra and the genetic diversity index increased with the increase of the effective sample size.When the effective number of alleles,polymorphic information content (PIC) and Shannon's information index were measured,the effective sample size was 29,which represented more than 95％ of the level of genetic diversity.When the effective sample size was 25,the total genetic diversity level was above 95％.The comprehensive analysis showed that the effective sample size of 25-29 was more suitable,and the germplasm conservation of P.polyandra was reasonable and effective.

The objective of this study was to verify the effects of arbuscular mycorrhizal fungi (AMF) on drought resistance of Nyssa yunnanensis, and to explore the mycorrhizal ways of plant conservation. A pot experiment was conducted to study the effects of AMF on anatomical structure characteristics of N. yunnanensis seedlings and their drought resistances under different water conditions. Six water conditions (soil water content) were designed in this pot experiment:32.32%, 29.63%, 25.86%, 19.39%, 12.93% and 6.46%, and at each water treatment, both sterilization (Low AMF) and no sterilization ( High AMF) were contained through adding fungicide benomyl to control AMF. The results showed that AMF colonization rate were significantly decreased in “Low AMF” treatment under different water treatments. Moreover, AMF colonizationrate of N. yunnanensis roots significantly decreased with the intensity of increased drought. No significant difference was found in anatomical structure characteristics under mild drought stress conditions ( soil water content was 29.63%) whereas N. yunnanensis seedlings showed higher resistances under severe drought stress conditions ( soil water content was less than 25.86%) . Benomyl treatment significantly affected seven leaf structure indices, such as the leaf cuticle thickness, palisade tissue thickness, upside epidermal thickness, plisade tissue / spongy tissue ratio, tightness of leaf tissue structure, sponge tissue thickness and leaf institutions looseness when soil water content was less than 25. 86%, suggesting that high AMF could enhance leaf structure traits on behalf of the drought resistance of N. yunnanensis seedlings when under severe drought stress conditions. Effects of AMF on N. yunnanensis seedlings under 25.86%, 19.39% and 12.93% were more significant than under 6.46%water content of soil. That was because AMF colonization was severely restrained by 6.46%water content of soil. Thus, effects of AMF on plant probably positively related to the colonization rate. Based on principal component a-nalysis of N. yunnanensis 10 structure’s index of leaves, and the method of membership function value, leaf traits of main structure index were comprehensively evaluated. The results demonstrated that N. yunnanensis seedlings showed stronger drought resistance under high AMF conditions. The experimental results provided the theoretical basis for the reasonable use of AMF in the protection of endangered species N. yunnanensis.

采用盆栽试验与称重控水法,将土壤相对含水量分别控制在田间最大持水量的100％、91.68％、82.85％、60.00％、41.86％和21.28％,并在这6个不同的土壤相对含水量条件下,分别设添加苯菌灵(杀真菌剂)(低AMF)和不添加苯菌灵(高AMF)处理,研究干旱胁迫下AMF对极小种群野生植物云南蓝果树幼苗生长和光合特征的影响,揭示云南蓝果树濒危的微生物学机制,为云南蓝果树保护措施的制定与实施奠定基础.结果表明,添加苯菌灵处理显著降低了不同水分条件下的AMF侵染率,说明试验中AMF处理的实生苗在生长和光合特征上的差异是苯菌灵处理下侵染率下降导致的;随着干旱胁迫的加剧,云南蓝果树幼苗的根部AMF侵染率显著降低、叶面积等生长指标和净光合速率(Pn)等光合参数都发生显著变化;高AMF处理可以显著增加水分充足和轻度干旱胁迫条件下云南蓝果树幼苗的大部分生长指标和光合参数,而对重度胁迫下的云南蓝果树幼苗没有显著影响,说明重度干旱胁迫对其影响大于AMF的影响;另外,整合了可塑性指数分析和隶属函数分析两种方法对其抗旱性进行评价,云南蓝果树幼苗基本上无法通过调节形态和光合能力来适应水分环境的变化,但是高AMF处理可使云南蓝果树幼苗具有较强的可塑性和更强的抗旱性.实验结果为云南蓝果树的科学保育及种苗繁育提供了理论依据.

为探究水分对毛枝五针松(Pinus wangii)幼苗生理特性的影响,对不同土壤含水量下幼苗松针的抗氧化活性进行了研究.结果表明,土壤含水量为田间持水量的40％～ 80％时,其POD、SOD和CAT活性较强;随着处理时间的延长,细胞质膜渗透性减弱,抗逆性变弱;脯氨酸、可溶性糖和蛋白质含量均以20％～40％的田间持水量较高;处理30 d后丙二醛含量随着土壤含水量的增加而提高,在土壤含水量为田间持水量的40％时,丙二醛含量最低,抗逆性最强.因此,人工培育幼苗时,土壤水分过多的时间尽量不要超过30d,控制土壤水分为田间最大持水量的30％～50％,这样能提高幼苗存活率.

利用不同质量浓度(0、0.25、0.50、0.75、1.00g·L-1)的水杨酸(SA)对云南蓝果树种子进行浸种处理和利用不同质量分数(0、5％、10％、15％)的PEG-6000溶液模拟不同强度的干旱胁迫(模拟的水势分别为0、-0.1、-0.2、-0.4 MPa),研究干旱胁迫下水杨酸浸种处理对云南蓝果树早期幼苗叶片生理指标的影响.结果表明:未经SA浸种处理时,随着PEG-6000质量分数的增加,云南蓝果树早期幼苗叶片的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性,脯氨酸(Pro)、可溶性糖质量分数显著降低,而丙二醛(MDA)质量摩尔浓度显著升高.随着SA质量浓度的升高,SOD、POD、CAT活性,Pro、可溶性糖质量分数显著升高,而MDA质量摩尔浓度显著降低,意味着水杨酸对云南蓝果树种子的预处理一定程度上增强了其早期幼苗的抗旱性.因此,水杨酸浸种可减轻干旱胁迫对云南蓝果树早期幼苗的伤害.而且,云南蓝果树早期幼苗在水杨酸质量浓度高于0.75 g·L-1、水势低于-0.1 MPa时,防御作用较强.建议育苗前对云南蓝果树种子进行SA预处理,最适质量浓度为0.75 g·L-1.