植物进化出复杂而精准的信号网络以调控寄主细胞响应病原菌侵染.为探究植物与病原菌互作过程的分子机制,本研究基于立枯丝核菌侵染结缕草的Zoysia·Unigene库与差异表达基因库,利用生物信息学分析主要生物代谢过程并筛选出结缕草与立枯丝核菌互作的分子过程,发现Ca2+调节与磷酸化两条路径与结缕草响应立枯丝核菌密切相关,且侵染24 h是互作的重要时间节点.本研究揭示结缕草与立枯丝核菌互作的分子机制,以期为草坪草在抗褐斑病的分子机制方面的研究提供理论依据.

In order to explore the function of KdSRG1 gene， the gene was cloned from wild-type Kalanchoe daigremontiana， and then analyzed by bioinformatics， subcellular localization， and gene expression analysis. The results show that the open reading frame of KdSRG1 gene was 219 bp， encoding 72 amino acids. KdSRG1 protein is a hydrophilic unstable acidic protein， which is localized in the nucleus and the cell membrane. KdSRG1 protein has no transmembrane structures or signal peptides and species specific. The relative expression of KdSRG1 gene in plantlet and leaf of Kalanchoe daigremontiana was higher than that in roots and stems， which may be involved in the regulation of adventitious bud and leaf growth.By analyzing the cis-acting elements of KdSRG1 promoter， it is speculated that KdSRG1 gene would be involved in plant meristem expression and palisade mesophyll cells differentiation， and the expression of this gene may be regulated by light signal， Abscisic Acid（ABA）， and Methyl Jasmonate （MeJA）. Moreover， KdSRG1 gene may play a role in plant stress regulation. The self-activation detection showed that KdSRG1 protein had no self-activation activity and could be used for subsequent yeast two-hybrid experiments. This study could be used for further research on the mechanism of KdSRG1 gene.

大叶落地生根(Kalanchoe daigremonatiania)主要以叶缘凹陷处产生不定芽的方式实现高效再生,是研究无性繁殖的模式植物.为筛选大叶落地生根不定芽发育过程中重要调控蛋白的互作因子,本研究提取大叶落地生根不同组织来源的植物RNA,利用RNA反转录5'末端交换机制(Switching mechanism at 5'end of RNA transcript,SMART)建库技术和双链特异性核酸酶(Duplex-specific nuclease,DSN)均一化处理技术,构建大叶落地生根酵母cDNA杂交文库,同时构建诱饵载体pGBKT7-KdSAHH,并用营养缺陷法检测其自激活活性.对文库进行质量检测的结果显示:文库滴度为3.2×106 CFU·mL-1,文库总容量为4.8×107 CFU,插入片段长度在250～2 000 bp范围内,重组率为91.7％.诱饵载体pGBKT7-KdSAHH构建成功且在酵母中无转录自激活活性.本研究为后续利用酵母杂交技术筛选大叶落地生根KdSAHH基因的互作蛋白提供基础.

为揭示立枯丝核菌(Rhizoctonia solani)侵染条件下结缕草(Zoysia japonica)中水杨酸(SA)合成的主要途径,通过同源比对法识别关键调控基因,依据病程下的转录模式鉴定响应通路,测试SA浓度及苯丙氨酸解氨酶(PAL)活性验证表达效果.ZjPAL1-ZjPAL5作为典型PAL等位基因,对应Unigene于根部发病后呈现持续上调,在转录水平积极响应侵染.异分支酸合成酶基因中仅监测到ZjICS1表达,且丰度始终不足ZjPAL1-ZjPAL5的10％,并随侵染加深趋于下调.菌丝体侵入根部内皮层后引起PAL活性严重减弱,伴随其减弱程度加剧,途径终产物SA浓度的下降也加快;但同时在尚未感染的叶片中SA浓度却随PAL活性提升保持高水平.PAL基因调控的苯丙氨酸途径是R.solani侵染下结缕草SA合成主导途径,响应强度随侵染深入而增强,PAL活性下降会显著降低SA水平.

大叶落地生根（Kalanchoe daigremontiana）为景天科多年生肉质草本植物,是研究无性繁殖的模式植物。SOC1 （Suppressor of overexpression of constans 1）基因是植物成花过程中MADS-box基因家族中的关键转录因子。为深入研究大叶落地生根KdSOC1基因的功能,于大叶落地生根中克隆KdSOC1基因,进行生物信息学分析,构建酵母双杂交诱饵表达载体pGBKT7-KdSOC1,并采用LiAc法将重组质粒pGBKT7-KdSOC1转化至Y2HGold酵母菌株中,通过营养缺陷型培养基培养进行自激活检测。结果表明,成功克隆了KdSOC1基因,该基因开放阅读框共有684 bp。生物信息学分析结果显示,Kd SOC1蛋白共有227个氨基酸残基,分子量为25.88 kD,大叶落地生根SOC1蛋白与牡丹的亲缘关系最为接近。双杂交诱饵表达载体pGBKT7-KdSOC1构建成功,并在酵母细胞中正常表达,表达产物对报告基因无自激活作用。本研究为筛选大叶落地生根中与其互作的蛋白及后续实验提供了帮助。

旨为获取机械损伤引起的AP2/EREBP、NAC以及WRKY转录因子相关的差异基因.通过将转录组测序后组装的序列转化为Mapman可识别的探针ID,对这三类转录因子进行筛选分析,加上转录组中Swiss-Prot和Nr数据库的注释.获得AP2/EREBP类转录因子差异基因13个,NAC类差异基因8个,WRKY类差异基因11个,在T8和T9时刻时,AP2/EREBP类、NAC类和WRKY类分别出现一个与对照(T7)相比明显下调的差异基因,而在NAC这一类转录因子中,与对照相比,有2个差异基因先下调,再上调,其余三类转录因子的差异基因均处于上调状态.通过数据库注释表明日本结缕草在遭受机械损伤时AP2/EREBP和WRKY类转录因子能够增强对非生物胁迫和生物胁迫的应答能力,增强抗逆性,并且NAC类转录因子传递信号至茎端分生组织并调控其生长.

In order to explore the molecular mechanism of Zoysia japonica tissue and organ regeneration after cutting,and analyze the effects of auxin (IAA) and cytokinin (CTK) on this process,Z.japonica cultivar 'Zenith' at 0 h,2 h,6 h and 24 h after cutting was photoed and observed by microscope to analyze its morphology and microstructural changes in stem apex meristem.Enzyme-linked immunosorbent assay (ELISA) and real-time fluorescent quantitative PCR (qRT-PCR) were used to explore IAA,CTK and the response models of related genes.The results showed that the polar transport of IAA regulated by ZjPIN1 responded after cutting,which was involved in the formation of organ primordia at 2 h and 24 h in the SAM of 'Zenith',and IAA signal transduction pathway was activated after 6 h,and involved in the rapid growth of plants.The ZjWUS gene participated in the proliferation of SAM at 2 h and 24 h,whereas CTK signal became active at 6 h,which inhibited the expression of ZjWUS and maintained the stability of SAM.These results indicated that ZjPIN1 and ZjWUS,combined with IAA and CTK signal transduction pathways,participated in tissue and organ regeneration of Zoysia japonica after cutting.This study could provide a theoretical basis for the study of the molecular mechanism of plant tissue and organ regeneration after injury.

以紫花苜蓿为试材,采用RT-PCR技术克隆出一个与拟南芥SAG113同源的基因,利用无缝连接酶将其与3302Y连接构建过量表达的植物表达载体,通过花序浸泡法转化拟南芥获得具有草铵膦抗性的植株,以期为研究MsSAG113的功能提供参考依据.结果表明:MsSAG113基因编码区长849 bp,编码283个氨基酸,PCR和RT-PCR检测显示成功获得了转MsSAG113基因拟南芥植株,初步证明在转基因拟南芥中外源SAG113基因能够转录表达.

Both LEAFY COTYLEDON 1-LIKE and LEAFY COTYLEDON1 are AHAP3 subunit of the LEAFY COTYLEDON1 type,but LEAFY COTYLEDON 1-LIKE is capable of converting the cells of vegetable stage into the cells of embryo periods in the early stages of somatic embryogenesis,differing from the LEAFY COTYLEDON1 that plays an important role in the early stages of embryonic development. LEAFY COTYLEDON2,which has the same specific B3 domain as the LEAFY COTYLEDON gene,can participate in creating the environment of embryogenesis in somatic tissue by IPA-YUC auxin synthesis pathway. Besides,LEAFY COTYLEDON2 promotes the occurrence and development of embryos by regulating the ratio of ABA/GA. The expression of WUSCHE and SHOOTMERISTEMLESS is independent but coordinated to maintain the function of shoot apical meristem(SAM). The KNOX pathway that is independent from the WUSCHE expression but interacts with plant hormone can maintain the environment of high-concentration CK and low-concentration GA for embryonic SAM,which is beneficial to the pathway of WUSCHE -CLAVATA responding to CK and continuously produce stem cells. In summary,it is feasible to improve the understanding of the molecular regulation network of embryogenesis,and to lay the molecular basis for further studying embryogenesis.

Part of conservative sequence of Japan zoysia‘ Zenith’ NCED gene was screened and cloned on the basis of the transcriptome.Time-space expression pattern of NCED gene was studied under Rhizoctonia solani early invasion(0～48 h)in ‘Zenith’ roots through real-time quantitative PCR.Results indicated that the NCED gene expression after the stress treatment was higher than that without infection,and showed an increasing tendency before the decrease,the gene expression achieving the highest at 36 h.Further test of ABA level in‘ Zenith’ roots showed the same trend with NCED gene expression from 0～48 h and flattened out after 48 h.It suggested that NCED gene regulated the synthesis of phytohormone ABA and was probably further involved in plant disease resistance.This paper provides a theoretical basis for Japan zoysia resistance mechanism and breeding research.

S-腺苷-L-高半胱氨酸水解酶(S-adenosyl-L-homocysteine hydrolase,SAHH)是已知的唯一能够水解S-腺苷高半胱氨酸(S-adenosylhomocysteine,SAH)成半胱氨酸和腺苷的酶.SAH作为转甲基反应的抑制剂,与S-腺苷甲硫氨酸(S-adenosyl-L-methionine,SAM,体内甲基供体)的比值常用来作为衡量植物整体甲基化水平的重要指标.为鉴定大叶落地生根(Kalanchoe daigremontiana) SAHH的功能,本研究根据已知的大叶落地生根SAHH基因(KdSAHH)序列(GenBank登录号:KF953475)设计引物,对其进行干旱胁迫下的表达分析、亚细胞定位分析以及烟草(Nicotiana tobacum)转化,并对T1代苗用20％ PEG6000模拟干旱处理,测定0、12和24 h的相对含水量(relative water content,RWC)、叶绿素(chlorophyll,Chl)含量、丙二醛(malondialdehyde,MDA)含量、过氧化物酶(peroxidase,POD)含量、超氧化物歧化酶(superoxide dismutase,SOD)含量等与抗旱相关的生理指标.结果表明,KdSAHH基因在轻度(20 d)、中度(35 d)、重度(50 d)干旱胁迫下表达量依次升高,表明该基因与抗旱性相关;亚细胞定位结果显示,该蛋白定位于细胞质与细胞核;对转基因烟草进行PCR检测,获得9株阳性植株;测定干旱胁迫下的相关生理指标,结果显示,与野生型烟草相比,转KdSAHH烟草在干旱胁迫下能够保持相对稳定的RWC、Chl含量以及相对较低的MDA、POD含量.与野生型相比,转KdSAHH烟草受伤害轻,抗旱性增强,表明KdSAHH基因能够提高烟草植株的抗旱性.研究结果丰富了KdSAHH基因的功能,为后续研究该基因提供了借鉴和参考.

Zoysia japonica brown spot was caused by necrotrophic fungus Rhizoctonia solani invasion, which led to severe financial loss in city lawn and golf ground maintenance. However, little was known about the molecular mechanism of R. solani pathogenicity in Z. japonica. In this study we examined early stage interaction between R. solani AG1 IA strain and Z. japonica cultivar “Zenith” root by cell ultra-structure analysis, pathogenesis-related proteins assay and transcriptome analysis to explore molecular clues for AG1 IA strain pathogenicity in Z. japonica. No obvious cell structure damage was found in infected roots and most pathogenesis-related protein activities showedg a downward trend especially in 36 h post inoculation, which exhibits AG1 IA strain stealthy invasion characteristic. According to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database classification, most DEGs in infected “Zenith” roots dynamically changed especially in three aspects, signal transduction, gene translation, and protein synthesis. Total 3422 unigenes of “Zenith” root were predicted into 14 kinds of resistance (R) gene class. Potential fungal resistance related unigenes of “Zenith” root were involved in ligin biosynthesis, phytoalexin synthesis, oxidative burst, wax biosynthesis, while two down-regulated unigenes encoding leucine-rich repeat receptor protein kinase and subtilisin-like protease might be important for host-derived signal perception to AG1 IA strain invasion. According to Pathogen Host Interaction (PHI) database annotation, 1508 unigenes of AG1 IA strain were predicted and classified into 37 known pathogen species, in addition, unigenes encoding virulence, signaling, host stress tolerance, and potential effector were also predicted. This research uncovered transcriptional profiling during the early phase interaction between R. solani AG1 IA strain and Z. japonica, and will greatly help identify key pathogenicity of AG1 IA strain.

Kalancho? daigremontiana reproduces asexually by forming plantlets on the leaves. During the developing process,the plantlets experience the various stages similar to that of zygotic embryo. The development of the plantlet share features of both organogenesis and embryogenesis. Research has shown that SHOOT MERISTEMLESS(STM)gene is required for the formation of plantlet. LEAFY COTYLEDON 1(LEC1)gene is expressed during the development of plantlet,however,it is not needed in the formation of the plantlet of K. daigremontiana. The functions of FUSCA3(FUS3),SAHH and other relevant genes in the development of the plantlet of K. daigremontiana still needs further investigation. This paper summarized the development of K. daigremontiana plantlet and relevant genes,aiming at providing some references for related researches.

【目的】了解立枯丝核菌（Rhizoctonia solani）AG1 IA对日本结缕草（Zoysia japonica steud.）的侵染过程及其引起的病害症状,为进一步从分子水平研究该菌的病理学侵染机制和草坪草抗病分子育种提供理论基础。【方法】通过根部接种法促使立枯丝核菌与日本结缕草无菌苗建立侵染关系,从而对其病叶率、病株率及病情指数进行统计分析,同时结合组织染色透明技术及植物组织石蜡切片对立枯丝核菌的侵染过程、感染方式进行研究。【结果】立枯丝核菌AG1 IA的侵染过程主要为:菌丝吸附在植物组织表面,并沿组织表面定向生长,形成侵染结构——侵染垫与组织建立密切的侵染关系。菌丝通过细胞间隙侵入植物组织内部,主要侵染植物皮层细胞及除木质部导管以外的整个维管束系统。结缕草的地上部分与地下部分组织对立枯丝核菌的侵染显现不同的寄主反应。【结论】立枯丝核菌的侵染过程主要包括吸附、定向生长、渗透、定殖4个部分;立枯丝核菌的侵染主要引起结缕草叶片病症;结缕草病变与菌丝直接侵染无直接联系,表明该菌具有复杂的侵染机制。

In order to understand the function ofSAHHinKalanchoe daigremontiana, a cDNA sequence, named asKdSAHH, was cloned by RT-PCR and RACE-PCR. The results showed that the full-length ofKdSAHHcDNA was 1748 bp, encoding 485 amino acids. The predicted molecular weight (MW) of KdSAHH was about 53 kDa with estimated pI of 5.59–5.682. The Scanprostie and DNAstar prediction showed that KdSAHH protein with two conserved motifs was very conservative in evolution. There were high homology between KdSAHH and SAHHs in other species by amino acid alignment, and which had the closest relationship with that inMedicago sativa. Lupinus luteusused as template, there were differences in three-dimensional structure of KdSAHH simulated by SWISS-MODLE and Phyre. These would lay out basis for research in expression and function of KdSAHH.

The study was researched about ultrasonic extraction process of flavones from Xanthoceras sorbifolia Bunge leaf. The content of flavones in Xanthoceras sorbifolia Bunge leaf was determined by four factors including ethanol volume ,solid -to -liquid ratio ,extraction tempreture ,extraction time and ultrasonic power. Based on the single factor,the study determined the optimal process conditions by response surface experiments. The results showed that the optimal process conditions were percent of ethanol volume 60% , solid-to-liquid ratio 1∶10(g/mL),extraction tempreture 65℃ ,extraction time 31min and ultrasonic power as 70Hz. Under these conditions,the flavones extracted from Xanthoceras sorbifolia Bunge leaf yields was 4.06%.

The liquid extracting from cassava leaves and its effects on turtles' growth are optimized in the study.Firstly,the optimal process of extracting liquid from cassava leaves is determined.The extracting conditions include ethanol density,solid-to-liquid ratio,extracting temperature and extracting time.Secondly,the effects of the liquid of cassava leaves on the growth of soft-shelled turtle by determining the survival rate,growth performance,feed conversion ratio,trophic component of turtles,blood biochemical and immunity indicators are analyzed.The result shows that the optimal process conditions are percent of ethanol density(50%),solid-to-liquid ratio(40:1),extracting temperature(70 ℃),extracting time(2 h).In conclusion,it indicates that the feed with the extracting liquid could improve the turtles' growth rate,decrease the feed coefficient,decrease the content of blood sugar,cholesterol and triglyceride of turtle and decrease MDA of turtle serum,so that the liquid could promote the glycometabolism,fat metabolism and immunity of turtle.Thus,this study not only firstly adds the liquid extracting from the cassava leaves into the turtles' feed,but also provides the theoretic reason for using the liquid as natural and low-toxic feed additive.

In order to optimize the extract condition,total alkaloids in Nitraria sibirica was extracted by ultrasound and the content of total alkaloids was determined by the acidic dye colorimetry,the effects of solid-liquid ratio,temperature,time and ethanol concentration on the optimum extraction parameters were investigated,thereby determining the optimal extraction parameters;the best one of isolation and purification from seven kinds of resin were screened out to research its effects on the alkaloids of static,dynamic adsorption.The results show that with the conditions of solid-liquid ratio of 1:20,temperature 60 ℃,time 40 minutes,ethanol concentration of 70%,the highest total alkaloid extract rate was 217.372 9 μg/g;the adsorption and resolution effects of D101 on N.sibirica’s total alkaloids were the best,the dynamic adsorption tests proved that the optimum process conditions were: sample solution pH 5,flow rate 0.75mL/min,ethanol concentration 80%.

Thoughts and perspectives on bilingual teaching of Genetics and Breeding for Turfgrasses and Forage Grasses were proposed based on two years-teaching-practice. Suggestions are made as following. At first, appropriate learning goals should be established. Text book, lectures, teaching resources and materials should be designed carefully and gradually according to three levels. The three levels are transi- tion level, bilingual level, and the English level. Interactions between students and teachers are crucial in promoting critical thinking for students. In addition, discussion and communications between students for a given topic in either English or Chinese should be arranged. Scientific papers focused on breakthrough in turfgrass breeding history could be used in the teaching process. Finally, incorporating oral English test, lab skills assessment, and discussion performances into the evaluation tests is effective in stimulating learning motive for students.

This article uses Siberian nitraira seed oil as materials,and uses the supercritical CO2 fluid extraction technology to extract nitraria seed oil.It also uses the orthogonal test to study the effects of pressure,temperature,and time.It confirms the best conditions of nitraria seed oil's extraction are that: the pressure is 30 MPa;the temperature is 30 ℃;and the time is 150 minutes.By analyzing the supercritical extraction of nitraria seed oil's physical and chemical properties and GC/MS,it can show that: the acid value is 3.36 mgKOH/g;the peroxide value is 0.48 g/100 g;the iodine value is 143.2 g/100 g;the saponification value is 196 mg/g;and the moisture and volatile matter content is 0.12 g/100 g.There are 11 kinds of fatty acids detected,and the quality occupies 70.75% of the total fatty acids,which indicates that supercritical CO2 extraction process of the nitraria seed oil is stable and reliable,and it has a very high application value,providing a reference for the further development and utilization.