DROUGHT-INDUCED UNKNOWN PROTEIN 1 positively modulates drought tolerance in cultivated alfalfa(Medicago sativa L.)

Alfalfa is the most widely cultivated perennial legume forage crop worldwide.Drought is one of the major environmental factors influencing alfalfa productivity.However,the molecular mechanisms underlying alfalfa responses to drought stress are still largely unknown.This study identified a drought-inducible gene of unknown function,designated as Medicago sativa DROUGHT-INDUCED UNKNOWN PROTEIN 1(MsDIUP1).MsDIUP1 was localized to the nucleus,chloroplast,and plasma membranes.Overexpression of MsDIUP1 in Arabidopsis resulted in increased tolerance to drought,with higher seed germination,root length,fresh weight,and survival rate than in wild-type(WT)plants.Consistently,analysis of MsDIUP1 over-expression(OE)alfalfa plants revealed that MsDIUP1 also increased tolerance to drought stress,accompanied by physiological changes including reduced malondialdehyde(MDA)content and increased osmoprotectants accumulation(free proline and soluble sugar),relative to the WT.In contrast,disruption of MsDIUP1 expression by RNA interference(RNAi)in alfalfa resulted in a droughthypersensitive phenotype,with a lower chlorophyll content,higher MDA content,and less osmoprotectants accumulation than that of the WT.Transcript profiling of alfalfa WT,OE,and RNAi plants during drought stress showed differential responses for genes involved in stress signaling,antioxidant defense,and osmotic adjustment.Taken together,these results reveal a positive role for MsDIUP1 in regulating drought tolerance.

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.

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.

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.

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.

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.

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。

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。

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.

Comparison Test on Adaptability of Six Medicago sativa (Alfalfa) Varieties in Guangxi

In order to screen the Medicago sativa(alfalfa)varieties suitable for planting in Guangxi,six domestic and foreign heat-resistant alfalfa varieties,namely WL903,WL712,WL525HQ,59N59,Elite 9 and Longwei 6010,were selected and tested for growth performance,nutritional value and over-summer rate by the random block method.The results showed that the alfalfa varieties with better plant height and fresh grass yield were 59N59 and WL903,which showed the plant heights reaching 62.01 and 62.53 cm,respectively,and the fresh grass yields up to 40.93 and 38.38 t/hm 2,respectively,exhibiting extremely significant differences from the poorly performing Longwei 6010(P<0.01).The nutritional value of each tested variety was good,and the crude protein content was more than 20%.Among them,Elite 9 had a crude protein content as high as 28.43%.The over-summer rates of the six alfalfa varieties were all higher than 75%.It showed that the alfalfa varieties with fast growth,high yield,high nutritional value and high over-summer rate in this experimental area were 59N59 and WL903,which are suitable as the first varieties for planting in Guangxi.

Effects of EDTA and Zn Stress on Physiological Characteristics of Medicago sativa L.

[Objectives]The paper was better understand the mechanisms of Zn plant uptake in the presence of EDTA and to evaluate the contributions of Zn-EDTA complexes to Zn uptake.[Methods]Three alfalfa cultivars were cultivated for 60 d before exposure to 0,250μg Zn and 250μg Zn+10μg EDTA per kg soil for 50 d.Zn concentrations in tissues were analyzed by flame atomic absorbance spectrometry.Subsequently,Zn amount per plant,translocation factor(TF)and bio-concentration factor(BCF)were calculated.Nonenzymatic compounds in tissues were analyzed spectrophotometrically.[Results]Application of Zn+EDTA expressively increased biomass of different tissues of three alfalfa cultivars.Among the three alfalfa cultivars,Medicago sativa ssp.displayed the highest Zn concentration in tissues,the largest Zn amount in aerial parts,and the highest BCF in aerial parts under Zn+EDTA exposure.Under Zn+EDTA stress,increases in free proline in roots,stem,and leaves of M.sativa ssp.were found.Inhibited O_(2)^(·-)production in stem and leaves,increases in soluble sugar,but decreases in soluble protein were observed in M.sativa ssp.[Conclusions]M.sativa ssp.is superior to other two cultivars for Zn phyto-remediation,and its well-coordinated physiological changes under Zn+EDTA exposure confer the great Zn tolerance of this cultivar.

Genetic Diversity of Tunisian and Chinese Alfalfa (Medicago sativa L.) Revealed by RAPD and ISSR Markers

Medicago sativa L.) is one of the most important forage crops in the world. The genetic variability analysis of 19 alfalfa populations collected from three sites in South Tunisia (Gabes, Kebili, Tozeur) and 1 from North West China were carried out using Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers. Five RAPD primers amplified 44 bands of which 22 were polymorphic;and five ISSR primers amplified 51 bands of which 33 were polymorphic. The percentage of polymorphic bands detected by RAPD and ISSR was 50% and 64.7%, respectively. The resolving power (Rp) varied between 0.6 and 4.1 with an average of 2.02 for RAPD marker and between 0.7 and 6.5 with an average of 2.28 for ISSR marker. However the Average Informativeness band (AvIb) was ranged from 0.2 to 0.9 with an average of 0.5 in RAPD marker and from 0.29 to 0.7 with an average of 0.624 in ISSR marker. The RAPD marker revealed less within population genetic diversity than ISSR marker. Although Cluster (UPGMA) and Correspondence Factorial Analyses (CFA) indicate that populations’ clustering made independently both from the geographical origin.

紫花苜蓿(medicago sativa)抗旱相关促分裂原活化蛋白激酶MAPK基因的克隆

用异硫氰酸胍方法提取紫花苜蓿超级阿菠萝(Apollo Supreme)植株嫩叶和幼根的总RNA;采用RT-PCR的方法获得紫花苜蓿超级阿菠萝抗旱相关促分裂原活化蛋白激酶MAPK基因中MMK4基因的cDNA序列全长。cDNA序列测定表明:我们克隆到的MMK4基因的cDNA序列全长1116bp,由此推导的氨基酸序列与GeneBank上MMK4(X82270)基因的cDNA序列所编码的氨基酸序列完全相同,但其中有14个氨基酸的密码子有所不同。

紫花苜蓿(Medicago sativa)超氧化物歧化酶的电泳分析

采用聚丙烯酰胺凝胶电泳对不同品种苜蓿的超氧化物歧化酶进行电泳分析。结果显示 ,不同苜蓿品种具有6条共同的SOD同工酶酶带。适应高寒地区气候条件的当地品种 (系 )同德杂种苜蓿表现出与其它苜蓿品种较大的特异性 ,与其它苜蓿品种相比缺少Rf0 .5 7和Rf0 .60两条酶带 ,而具有两条Rf0 .66和Rf0 .79酶带。抑制试验表明苜蓿超氧化物歧化酶为CuZn SOD。

陇东地区油污土壤紫花苜蓿(Medicago sativa)和金鸡菊(Coreopsis lanceolata)——植物修复效应比较

对陇东黄土高原地区油污土壤紫花苜蓿(Medicago sativa)和金鸡菊(Coreopsis lanceolata)植物修复效应进行分析比较。经5个月场地修复试验,相关植物生理、土壤理化及微生物指标分析结果表明:(1)金鸡菊和紫花苜蓿根区土壤总石油烃(Total Petroleum Hydrocarbons,TPH)去除率最高可达75.33%和69.88%;(2)7%和9%油污土壤对金鸡菊生理特性的抑制作用相对较小,且其根区土壤pH、脲酶和碱性磷酸酶活性显著高于紫花苜蓿(P<0.05);(3)金鸡菊根区土壤中食烷菌属(Alcanivorax)、嗜盐单胞菌(Halomonas)和类诺卡氏菌属(Nocardioides)等相对丰度显著高于紫花苜蓿(P<0.05);(4)油污浓度增加抑制了紫花苜蓿植物生理进而限制其根区TPH去除率,而土壤理化和微生物的共同作用使得金鸡菊根区土壤TPH去除率高于紫花苜蓿。

Comparative transcriptome analyses reveal that the MsNST1 gene affects lignin synthesis in alfalfa(Medicago sativa L.)

As the second most abundant natural polymer,accounting for approximately 30%of the organic carbon in the biosphere,lignin plays an essential role in plant development.However,a high lignin content affects the nutritional quality of alfalfa(Medicago sativa L.),the most widely cultivated perennial legume forage crop.Histological analysis indicated that G-lignin and S-lignin were present in the stem,leaf,and petiole of alfalfa,and the deposition of lignin increased gradually in descending internodes.Neutral detergent fiber(NDF),acid detergent fiber(ADF),and acid detergent lignin(ADL)contents continually increased from the top to the bottom of the stem,and ADL content showed a similar trend in leaves.Alfalfa leaves and stems from five different nodes(1,2,4,6,and 8)were used as materials to investigate molecular regulatory mechanisms in lignin synthesis by RNA sequencing.Respectively 8074 and 7752 differentially expressed genes(DEGs)were identified in leaves and stems,and 1694 DEGs were common to the two tissues.‘‘Phenylpropanoid biosynthesis”was the most enriched pathway in both leaves and stems,and 134 key regulatory genes in lignin synthesis were identified by a weighted gene co-expression network analysis.The NAC family transcription factor MsNST1 gene was highly expressed in old leaf and stem tissues.The deposition pattern of G-and S-lignin differed among M.truncatula wild-type,nst1 mutants,and overexpression lines,and the transcription levels of lignin synthesis genes such as HCT,F5H,and COMT in these three materials also differed.These results suggest that MsNST1 affects lignin synthesis in alfalfa.These findings provide a genetic basis and abundant gene resources for further study of the molecular mechanisms of lignin synthesis,laying a foundation for low-lignin alfalfa breeding research.

Effects of Modulation Methods on the Drying Characteristics and Nutritional Quality of Medicago sativa Hay

Two-year-old Medicago sativa at budding initial stage was taken as research materials.Five methods were used to make green hay,including flatting stems ＋ spraying 2.5% K2CO3,flatting stems,sun curing,drying in shade and drying under 105 ℃ condition（CK）.Besides,effects of different green hay making methods on dry characteristics and nutritional quality of M.sativa green hay were studied,and a comprehensive evaluation of M.sativa green hays was conducted.Results showed that,except CK,the drying rates in other making methods were all fast at first,and then slow down.Both of drying under 105 ℃ condition and flatting stems ＋ spraying K2CO3 could speed up drying rate and reduce nutritional losses of green hay.Sun curing could also speed up drying rate,but it could not maintain the quality of green hay.The results of Grey Relational Analysis on five green hay making methods indicated that CK had the best comprehensive performance,followed by green hays made by flatting stems ＋ spraying K2CO3.Therefore,flatting stems ＋ spraying K2CO3 was a quick and easy method to make green hay,and it was worth to be recommended in practical production.