饱和烷烃处理下紫花苜蓿(Medicago sativa)的转录学特征

为探究紫花苜蓿在石油污染下的耐受机理,采用超声碎促溶的方法,将3种有机物(十二烷、十六烷和二十四烷)配置成质量分数均为1%的混合溶液,模拟饱和烷烃污染对紫花苜蓿幼苗进行处理,分别对污染0,6,24 h的植株取样进行转录组学分析,共获得1431个差异表达基因(DEGs)。GO富集分析表明,这些DEGs主要涉及蛋白结合、代谢途径和催化活性等;KEGG富集分析表明,DEGs主要富集到植物病原体相互作用、MAPK信号通路和光合生物碳固定途径等。qRT-PCR验证转录组结果可靠。研究结果为研究植物降解和耐受原油中饱和石油烃污染机制原理及后续筛选和培育耐石油污染植物提供理论依据。

Analysis of Genetic Relationship among Medicago sativa,Medicago falcate and Trigonella foenum-graecum Using ISSR

[Objective] This study aimed to analyze the genetic relationships among Medicago sativa,Medicago falcata and Trigonella foenum-graecum.[Method] ISSR technique was adopted to determine their genetic relationships.[Result] M.sativa,M.falcate and T.foenum-graecum had a broad genetic base.T.foenum-graecum shared closer relationship with M.falcata rather than M.sativa.The study on relationship between M.sativa and T.foenum-graecum was advantageous to identify disputable transition types.But a boundary should be found to identify species to be M.sativa or T.foenum-graecum.[Conclusion] This study will provide reference for identifying some disputable transition types.

宁南山区紫花苜蓿(Medicago sativa)土壤干层水分动态及草粮轮作恢复效应

以各类作物农田水分为对照，连续两年对宁南山区不同生长年限苜蓿深层土壤水分以及10年生苜蓿地耕翻后轮作不同年份作物农田的水分进行了测定。结果表明，随着苜蓿生长年限的增加，干层深度与厚度先增加后减小。3年生苜蓿干层深度为720cm，6年生干层最深可达1000cm以下，10年生干层深度为920cm，3—12年生苜蓿地0—700cm土层基本上均属于土壤干层范围。苜蓿地0—800cm土壤湿度随生长年限增加而降低，2004年测定的4、7年生和12年生苜蓿地0—700cm土层平均含水率分别为5．30％、5．22％和5．01％；2005年测定的3、6年生和10年生苜蓿地0—800cm土层湿度分别为6．26％、5．60％和5．27％；而800～1000cm土层湿度在一定年限后有恢复趋势。300cm为苜蓿地降水下渗的最大临界深度，300cm以下土壤干层一旦形成，将长期存在，7—12年生苜蓿300～700cm土层湿度仅维持在4．0％左右。苜蓿地和农田的土壤干层厚度与湿度有较大差异，草粮轮作可使苜蓿土壤干层水分基本恢复到农田湿度，而且轮作年份越长，土壤各层次水分恢复效果越好，10年生苜蓿轮作18年后土壤水分基本恢复到农田状态。

几种除草剂和助剂对苜蓿Medicago sative出苗和生长的影响

在温室条件下研究了几种苗前除草剂单用或混用对苜蓿出苗和生长的影响 ,以及不同叶龄苜蓿对苗后除草剂的反应及添加助剂对药效的影响。结果表明 :咪唑乙烟酸在6 0～ 12 0 g/hm2 剂量下 ,于苗前施用 ,对苜蓿出苗和幼苗生长都有明显的抑制作用。咪唑乙烟酸(30 g/hm2 )与二甲戊灵 (495 g/hm2 )混用 ,对苜蓿的药害显著降低。苗后施用除草剂对苜蓿的安全性与叶龄密切相关。咪唑乙烟酸以 30～ 6 0 g/hm2 剂量在苜蓿 3叶期施用 ,对苜蓿安全 ;在12 0 g/hm2 用量下 ,对 5叶期的苜蓿无明显药害。乳氟禾草灵 (130～ 2 6 0 g/hm2 )和丙炔氟草胺(75～ 15 0 g/hm2 )无论是 3叶期还是 5叶期施用 ,对苜蓿幼苗都有严重的药害。与咪唑乙烟酸单用时比较 ,药液中添加 1.0 m L /L的平平加 - 15和 AM- 10 0及 5 .0 m L /L的 SDP。

Study on Physiological Activity of the Resistance of Medicago sativa to Thrips

[Objective] The experiment explored of resistance of different Medicago sativa through synthetic variations of all physiological indexes and the correlation analysis between physiological activity and damage degree after thrips damage. [Method] According to studying physiological activity (POD activity, SOD activity, PPO activity, PAL activity, MDA content and free proline content) of alfalfa in alfalfa resistance to different number of thrips, the correlation between physiological activity and resistance was investigated. [Result] It was shown that there were negative correlations among POD, SOD, PPO, PAL, MDA, free praline and the number of thrips, and there were negative correlations among POD, SOD, PPO, PAL, MDA, free praline and CAT. The content changes of POD,SOD,PPO,CAT,MDA and free proline in high resistant alfalfa were slow and PAL was quick and the activities of PPO,PAL,CAT were high, and the activities of POD、SOD were low. [Conclusion] The research provided theoretical basis and materials for identifying resistance varieties of alfalfa to thrip.

Bioinformatics Analysis and Homology Modeling Study of Protein Disulfide Isomerase(mPDI) from Medicago sativa L.

pdi gene from Medicago sativa L. ,encoding Protein Disulfide Isomerase（ mPDI ）, has been cloned and sequenced. According to the mRNA and amino acid sequence, the character of mPDI such as the physical and chemical properties, hydrophilicity/hydrophobicity, signal peptide, secondary structure, coiled coil, transmembrane domains, O-glycogylation site, active site, subcellular localization, functional structural domains and three-dimensional structure were analyzed by a series of bioinformatics software. The results showed that mPDI was a hydrophobic and stable protein with 3 coiled coils, 30-glycogylation sites, 2 structural domains of thioredoxin, 2 active sites of thioredoxin, and located in rough endoplasmic reticulum. It has 512 amino acids, the theoretical pl is 4.98, and signal peptide located in 1-24AA. In the secondary structure, a-helix, random coil, extended chain is 26.37%, 53.32%, 20.31% respectively. The validation of modeling accords with the stereochemistry.

Research on Phytoremediation of Heavy Metal Pollution in River Sediment by Medicago sativa L.

[Objective] The aim was to study the phytoremediation of heavy metal pollution in river sediment by Medicago sativa L.,so as to provide reliable references for the phytoremediation of heavy metal pollution in river sediment.[Method] The air-dried,screened and mixed sediment was put in rectangular PVC box（0.6 m×0.5 m×0.4 m） with seepage vent at the bottom,and the water holding capacity（WHC） of sediment was kept at 30%-60% by deionized water.The seeds of Medicago sativa L.were sown in April 2010,and seedlings were thinned after 7 d.Samples were collected from rhizosphere soil every 30 d,and were used to determine the content of heavy metals,bacteria quantity and enzyme activity in sediment.In addition,the accumulation of heavy metals in the roots,stems and leaves of plant was measured after harvest in October.[Result] Different parts of Medicago sativa L.varied in accumulation capacity to different heavy metals.The accumulation amount of Zn in Medicago sativa L.was the highest,especially in roots.Meanwhile,the accumulation amount of heavy metals like Ni,Cr,Cu and Pb in roots was higher than that of stems and leaves.In contrast,Mn was mainly accumulated in leaves and its amount accounted for 42.47% of the total amount in plant.Besides,the accumulation amount of all heavy metals was the lowest in stems.Ni,Cr,Cu and Pb could be degraded more effectively than Mn,and increasing the planting time and sowing times of crop was beneficial to the degradation of heavy metals.After planted Medicago sativa L.,the quantity of microorganisms in sediment went up obviously,and dehydrogenase activity also showed an increaseing trend.[Conclusion] Medicago sativa L.has certain restoring effect on Zn,Ni,Cr,Cu and Pb,and could be used to restore heavy metal pollution in river sediment.

Photosynthesis and Transpiration Characters of Alfalfa (Medicago sativa) and Their Relationship with Relevant Factors during Branching Stage

[ Objective] The paper presents the diumal changes of photosynthesis and transpiration of different alfalfa varieties and their relationship with the associated physiological and ecological factors during branching stage, so as to provide a basis for the development, utilization, and breed- ing of alfalfa. [ Method] Under natural conditions, the diurnal changes of net photosynthetic rate （Pn）, transpiration rate （Tr）, the relevant physio- logical factors including leaf temperature （TI）, stomatal conductance （Gs） and intemal COn concentration （Ci）, as well as the relevant physiologi- cal factors including photosynthetic available radiation （PAR）, CO2 concentration in field （Ca） and air temperature （Ta） were measured in four al- falfa varieties （Algonguin, WL323 HQ, WL414, and Millionaire）. The water use efficiency （WUE） and light use efficiency （LUE） were calculated, and the correlation among them was also analyzed. [Result] The Pn, Tr, PAR and Ta of the four varieties appeared to vary in a single-peak curve; the sequence of WUE was WL323 HQ ~ Algonguin ~ WL414 ~ Millionaire; there was no significant difference in LUE of the four alfalfa varieties; coef- ficient analysis showed that Pn was mainly affected by PAR, Gs, and Ci, while Tr by PAR and Ta. [ Conclusion] WL323 HQ is the variety with high Pn, high WUE and low Tr, and it has strong adaptability to drought. In four alfalfa varieties, PAR, Ta, Gs, and TI are the primary determining fac- tors while Ca and Ci the limiting factors of Tr; Gs is the primary determining factor while Ci the limiting factor of Pn.

Primary Study on ISSR Molecular Makers of Resistant Gene against Sclerotinia trifoliorum in Medicago sativa L.

[ Objective] The paper was to study ISSR molecular makers of resistant gene against Sclerotinia trifoliorum in Medicago sativa L. [ Method] Using mi- crosatellite markers （ISSR） molecular maker technology, combined with bulked-segregant analysis （BSA） method, the molecular makers for gene linkage with re- sistance against S. trifoliorum were screened from five resistant plants and seven susceptible plants. Leaf in vitro inoculation method was used to carry out resistant verification on 94 hybrid plants in Ft generation of high resistant No. 83 ~ high susceptible No. 4. [ Result] Among 93 ISSR primers, 35 primers could produce clear and stable amplification bands, and six of them could produce 9 specific bands between resistant and susceptible DNA pools. Resistance verification result showed that 825 - 1400, 831 - 1480, 850 - 1800, 858 - 1600, 866 - 1900, 888 - 1400 could be used as ISSR molecular makers of the resistant gene against S. trifoliorum in M. sativa. [Conclusion] The results provided basis for the further research on mapping,, cloning and genetically modified of resistant gene against S. trifoliorum in M. sativa.

Effect of Different Kinds of Growth-promoting Rhizobacteria on Physiological and Biochemical Characteristics of Medicago sative Linn. Seedlings under the Stress of Na_2CO_3

[Objective] The study aimed to explore the influences of Plant growth-promoting rhizobacteria（PGPR）on salt tolerance and physiological effect of seedlings of Medicago sative L.[Method] Three different kinds of growth-promoting rhizobacteria solutions were used to spray on M.sative seedlings and the influences of different kinds of growth-promoting rhizobacteria on physiological and biochemical characteristics of M.sative seedlings under 75 mmol/L of Na2CO3 were studied.[Results] Compared with the control group,the chlorophyll,free proline,soluble sugar and soluble protein content of M.sative seedlings treated with different kinds of growth-promoting rhizobacteria were increased,while the effect of CS3 treatment was the best.[Conclusion] The growth-promoting rhizobacteria could promote growth and development of M.sative as well as increased its resistance.

Degradation of TCP in Soil Planted with Alfalfa(Medicago sativa L.)

[Objective] The research aimed to study the degradation of 2,4,6-trichlorophenol （TCP） in soil planted with alfalfa （Medicago sativa L.）,as well as to provide references for the Chlorophenols phytoremediation technology in the practical application.[Method] By the use of pot culture experiment in greenhouse,the phytoremediation effect of alfalfa on TCP-contaminated soil,the growth conditions of alfalfa,as well as the effect of TCP on the activity of soil polyphenol oxidase,dehydrogenase and catalase were studied.[Result] After the alfalfa was grown for 75 d,the TCP content in soil of three different concentrations treatments （low,middle and high） decreased dramatically within 15 d,and then the decreasing rate was gradually slow; on the 30^th d of cultivation,the fresh weight of treated alfalfa showed no significant difference with the control （P〈0.05）,indicating that TCP in soil had inhibition effect on the growth of alfalfa; alfalfa could significantly enhance the activities of polyphenol oxidase,dehydrogenase and catalase,thus raising the degradation capability of soil plants and microorganisms on pollutants in soil.[Conclusion] There results indicated that alfalfa could enhance the degradation rate of organics in the contaminated soil and enhance soil enzyme activity,so the alfalfa could be used for the bioremediation of TCP contaminated soil.

紫花苜蓿复合体(Medicago sativa complex)叶片形态特征及数量分类研究

运用比较形态学和比较解剖学方法,使用扫描电镜和光学显微镜对紫花苜蓿复合体（Medicago sativa complex）6个分类群的叶片形态特征和叶片解剖结构进行了观察和比较,并以15个叶片表征形态性状为基础,采用聚类分析法（UPGMA）和主成分分析方法（PCA）对6个分类群进行了数量分类研究。观察结果表明：各分类群叶片的上、下表皮多为不规则形细胞;垂周壁呈深浅不一的波状;气孔器为不规则型,具有蜡质气孔盖,气孔密度有一定差异。6个分类群的叶片均为薄纸质型,厚度130~170μm,表皮细胞切面近圆形或近长方形;栅栏组织细胞1~2层,厚度41~68μm,细胞排列紧密;海绵组织厚度32~75μm,细胞排列疏松;不同分类群叶片的组织疏松度和组织紧缩度有一定的差异,大花苜蓿（M.trautvetterii Sumnev.）叶片的组织疏松度最高,紫花苜蓿叶片的组织紧缩度最高。UPGMA结果显示：在结合线1.53处可将6个分类群划分为2支,其中,黄花苜蓿（M.falcataL.）独立为一支,其余5个分类群聚成另一支;在结合线1.18处,第2支又被分成2个亚支,其中一个亚支包含紫花苜蓿和天山苜蓿（M.tianschanica Vassilcz.）,另一个亚支则包含西锡金苜蓿（M.schischkinii Sumnev.）、座垫苜蓿（M.rivularis Vassilcz.）和大花苜蓿。PCA结果表明：对紫花苜蓿复合体而言,叶片表皮细胞形状、垂周壁式样、轴性分化特征、组织疏松度和气孔密度等特征具有较好的分类价值;基于主成分分析的Q分布图与聚类分析结果也具有较高的一致性。根据本研究结果及前人的研究结果,认为国产的紫花苜蓿复合体应包含3个分类群,即紫花苜蓿、黄花苜蓿及多变苜蓿（M.varia Martyn）。此外,西锡金苜蓿、座垫苜蓿、天山苜蓿和大花苜蓿等杂交后代分类群的性状分化不稳定,应属于多变苜蓿的同种异名植物。

不同品种紫花苜蓿(Medicago sativa)的化感效应研究

用砂培法研究紫花苜蓿(Medicago sativa)的六个品种苗期地上部分水浸提液对鸭茅(Dactylis glomerataL.)的化感效应。结果表明,紫花苜蓿六个品种茎叶水浸提液对鸭茅的化感效应在供试品种间差异显著。游客、苜蓿对鸭茅的种子发芽及幼苗生长有抑制作用,但游客却对鸭茅的苗干重有促进作用;三得利和赛迪对鸭茅的种子发芽、根长及根干重有抑制作用,却对苗生长的影响无显著差异。此外,供试紫花苜蓿浸提液对鸭茅的化感作用,随着浓度的升高,抑制作用随之增强。

In silico genome-wide identification,phylogeny and expression analysis of the R2R3-MYB gene family in Medicago truncatula

The R2R3-MYB genes make up one of the largest transcription factor families in plants, and play regulatory roles in various biological processes such as development, metabolism and defense response. Although genome-wide analyses of this gene family have been conducted in several species, R2R3-MYB genes have not been systematically analyzed in Medicago truncatula, a sequenced model legume plant. Here, we performed a comprehensive, genome-wide computational analysis of the structural characteristics, phylogeny, functions and expression patterns of M. truncatula R2R3-MYB genes. DNA binding domains are highly conserved among the 155 putative MtR2R3-MYB proteins that we identified. Chromosomal location analysis revealed that these genes were distributed across all eight chromosomes. Results showed that the expansion of the MtR2R3-MYB family was mainly attributable to segmental duplication and tandem duplication. A comprehensive classification was performed based on phylogenetic analysis of the R2R3-MYB gene families in M. truncatula, Arabidopsis thaliana and other plant species. Evolutionary relationships within clades were supported by clade-specific conserved motifs outside the MYB domain. Species-specific clades have been gained or lost during evolution, resulting in functional divergence. Also, tissue-specific expression patterns were investigated. The functions of stress response-related clades were further verified by the changes in transcript levels of representative R2R3-MYB genes upon treatment with abiotic and biotic stresses. This study is the first report on identification and characterization of R2R3-MYB gene family based on the genome of M. truncatula, and will facilitate functional analysis of this gene family in the future.

Synonymous codon usage pattern in model legume Medicago truncatula

Synonymous codon usage pattern presumably reflects gene expression optimization as a result of molecular evolution. Though much attention has been paid to various model organisms ranging from prokaryotes to eukaryotes, codon usage has yet been extensively investigated for model legume Medicago truncatula. In present study, 39 531 available coding sequences （CDSs） from M. truncatula were examined for codon usage bias （CUB）. Based on analyses including neutrality plots, effective number of codons plots, and correlations between optimal codons frequency and codon adaptation index, we conclude that natural selection is a major driving force in M. truncatula CUB. We have identified 30 optimal codons encoding 18 amino acids based on relative synonymous codon usage. These optimal codons characteristically end with A or T, except for AGG and TTG encoding arginine and leucine respectively. Optimal codon usage is positively correlated with the GC content at three nucleotide positions of codons and the GC content of CDSs. The abundance of expressed sequence tag is a proxy for gene expression intensity in the legume, but has no relatedness with either CDS length or GC content. Collectively, we unravel the synonymous codon usage pattern in M. truncatula, which may serve as the valuable information on genetic engineering of the model legume and forage crop.

新疆大叶紫花苜蓿(Medicago sativa.L)子叶、下胚轴、根的植株再生

随着植物抗逆性研究和植物转基因技术的发展,通过异源目的基因转化培育耐盐碱苜蓿品种的研究已引起人们的关注,植物受体高频再生体系的建立是异源转化高效的基础。选取新疆大叶紫花苜蓿种子萌发5～7d无菌苗的子叶、下胚轴及根为外植体,诱导愈伤培养基为MS+2,4-D0.1～3.0mg/L(8种不同水平)或MS+2,4-D2.0mg/L+KT0.01～0.5mg/L(10种不同水平),诱导芽培养基为MS+6-BA0.5mg/L+NAA0.05mg/L,生根培养基为MS。结果表明,外植体在MS+2,4-D2.0mg/L+KT0.2mg/L培养基中能够产生状态较好可再分化的愈伤组织,子叶、下胚轴、根的平均出愈率分别为93.1%、100%、100%。愈伤组织在MS+6-BA0.5mg/L+NAA0.05mg/L培养基中培养40～80d中均可分化芽,子叶、下胚轴、根的芽平均分化率为50%、78%、50%,将2cm以上的芽转入MS培养基中诱导生根,14d后,生根的小植株炼苗移入花土中,成活率达90%以上。子叶、下胚轴、根在该体系中均能获得再生植株,根也是一种较好的植株再生材料,以根为外植体进行植株再生的研究报道还较少。

Expression of Cold Resistant Gene CAS19 of Gongnong No.2 Medicago sativa L. in Tobacco

[Objective] The aim was to study the expression of cold resistant gene CAS19 of Gongnong No.2 Medicago sativa L. in tobacco. [Method] A pair of primers was designed according to nucleotide sequences of cold resistant gene CAS19 of M. sativa,and then RT-PCR was used to amplify the protein gene of CAS19,which was then cloned into pMD18-T vector and subcloned into expression vector PBI121. The recombination expression plasmid PBCAS was constructed. And then it was transferred into tobacco genome via Agrobacterium,and Southern-blotting analysis was used for detecting transgenic plants. [Result] CAS19 gene was integrated into the tobacco genome and highly expressed. [Conclusion] This study had provided theoretical basis for further exploring the expression mechanism of cold resistant gene CAS19 in tobacco.

西北地区苜蓿属(Medicago L.)的种类和分布

通过对西北地区苜蓿属植物的调查、研究,初次阐述了西北地区苜蓿属植物的种类、地理分布和生活环境,简要描述了每种的主要特征及用途,编出了分种检索表。

Iron Deficiency Tolerance at Leaf Level in <i>Medicago ciliaris</i>Plants

Iron deficiency is an important environmental factor restricting plant productivity. Selecting tolerant genotypes is one of the possible ways to solve this problem. Many studies reported the effects of Fe deficiency on photosynthesis and anti-oxidative defense system. Yet, there is little information available on the use of these attributes as selective criteria. In the present study, we aim to determine some physiological and biochemical traits conferring Fe deficiency tolerance at leaf level in two lines of Medicago ciliaris. Our results showed that Fe deprivation had a lowering effect on photosynthesis (chlorophyll, photosynthetic electron transport activity and chlorophyll fluorescence) in both lines studied. However, the sensitive line TN8.7 was more affected. Hydrogen peroxide concentration was negatively correlated with the activities of antioxidant enzymes and with the concentration of some non-enzymatic antioxidant. The tolerant line TN11.11 was characterized by a more efficient antioxidant defense system in comparison with the sensitive line TN8.7. The main conclusion of this study is that photosynthesis and antioxidant defense system could be used as physiological and biochemical indicators of Fe deficiency tolerance in Medicago ciliaris plants.

Phytosulfokine-α Promotes Root Growth by Repressing Expression of Pectin Methylesterase Inhibitor (PMEI) Genes in Medicago truncatula

Phytosulfokine-α(PSK-α),a sulfated pentapeptide with the sequence YIYTQ,is encoded by a small precursor gene family in Arabidopsis.PSK-αregulates multiple growth and developmental processes as a novel peptide hormone.Despite its importance,functions of PSK-αin M.truncatula growth remains unknown.In this study,we identified five genes to encode PSK-αprecursors in M.truncatula.All of these precursors possess conserved PSK-αsignature motif.Expression pattern analysis of these MtPSK genes revealed that each gene was expressed in a tissue-specific or ubiquitous pattern and three of them were remarkably expressed in root.Treatment of M.truncatula seedlings with synthetic PSK-αpeptide significantly promoted root elongation.In addition,expression analysis of downstream genes by RNA-seq and qRT-PCR assays suggested that PSK-αsignaling might regulate cell wall structure via PMEI-PME module to promote root cell growth.Taken together,our results shed light on the mechanism by which PSK-αpromotes root growth in M.truncatula,providing a new resource for improvement of root growth in agriculture.