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.

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.

煤气化渣及燃煤固废种植基质对紫花苜蓿生长影响试验

为了探究煤气化及燃煤固废制备种植基质可行性,以宁夏回族自治区宁东能源基地煤气化产生的气化细渣和气化粗渣、燃煤产生的粉煤灰及炉渣制备种植基质,采用盆栽法,比较其与3种土壤(砂土、壤土及黏土)对紫花苜蓿成苗和生长的影响。分析种植前后土壤和基质的物理化学性质变化。结果表明,砂土有利于紫花苜蓿种子萌发及生长;粉煤灰板结严重影响出苗;气化细渣孔隙度较小以及磷含量过高造成了出苗率低和苗株枯黄。出苗率以砂土最高,其次是炉渣、然后是气化粗渣、壤土及黏土。气化粗渣和砂土除有机质含量及磷含量差异较大外,其他物化性质相似,二者培养的紫花苜蓿株高根长的数值接近且最大。炉渣种植紫花苜蓿的株高及根长低于气化粗渣和砂土。研究表明,气化粗渣和炉渣与砂土类似,可作为土壤基质用于牧草种植,对宁夏矿区土壤修复及煤基固废利用具有重要意义。

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.

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.

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.

基于“3414”的沧州市苜蓿肥料效应试验

为了寻求沧州市苜蓿(Medicago sativa L.)生产最佳养分施用方案,对苜蓿施肥提供科学技术指导。采用“3414”试验设计,以中苜3号为试验材料,利用不同方法对肥料施用量及苜蓿产量进行回归分析,得出在苜蓿生产中N、P_(2)O_(5)和K_(2)O的最佳用量。结果表明,不同施肥处理对苜蓿产量有一定影响,除不施肥处理外,施肥处理的苜蓿产量均有所提高。PK处理互作效应最强,NPK和NK处理互作效应其次,NP的互作效应最弱。由三元二次回归方程计算得出N、P_(2)O_(5)和K_(2)O的最佳施用量分别为4.26、50.03、147.15kg/hm^(2),最佳产量为23 467.6 kg/hm^(2)。3种肥料含量与产量间得出的一元二次回归方程虽然拟合度较高(R2为0.985 3-0.997 3),但3种肥料与产量的相关关系均未达到显著水平。所以,推荐使用三元二次效应方程拟合结果作为沧州市苜蓿生产施肥推荐量。

朔州市紫花苜蓿生长期气候条件分析

研究山西省朔州市紫花苜蓿(Medicago sativa L.)生长发育期的温度、水分、光照等气象要素,发现朔州市年积温、年平均气温和日照等基本符合紫花苜蓿的生长需求,而降水量不能满足紫花苜蓿正常生长需求,在灌溉能够得到保证的前提下可以在地势平坦的土地上进行规模化种植。朔州市平川区比山区更适宜种植紫花苜蓿,平川区紫花苜蓿1年适宜刈割4次,山区1年适宜刈割3次。6月中旬至7月中旬为朔州市播种紫花苜蓿最佳时期,如果4—6月土壤墒情好或有灌溉条件的可以播种苜蓿种子建植。朔州市种植紫花苜蓿具有较好经济价值,可大面积推广种植。

紫花苜蓿花期调控基因MsCOL2的克隆及功能研究

开花是植物由营养生长过渡到生殖生长的转折点,紫花苜蓿(Medicago sativa L.)的产量和品质与开花时间密切相关。光周期是影响植物开花的重要因素之一,CONSTANS-like(COL)基因家族在光周期开花调控途径中发挥重要的作用。为揭示COL家族基因在紫花苜蓿开花调控中的功能,本研究克隆了MsCOL2基因,该基因编码401个氨基酸;组织差异性表达分析显示,MsCOL2在花中表达量最高;花不同发育时期表达分析显示,MsCOL2在花芽分化期表达量最高;在不同光周期条件下,MsCOL2的表达水平具有不同的节律性表达;外源激素50μmol·L^(-1)赤霉素和100μmol·L^(-1)水杨酸处理后,MsCOL2表达水平呈现不同的变化趋势。为了验证过表达MsCOL2对开花相关基因表达的影响,分析了过表达MsCOL2株系中开花相关基因的表达水平,结果显示促进开花基因的表达水平下调,抑制开花基因的表达水平上调。表型观察显示,过表达株系延迟开花,表明MsCOL2对延迟开花有重要的调节作用。

紫花苜蓿(Medicago sativa L.)Tify基因家族的鉴定、生物信息学及表达模式分析

Tify基因家族是植物特有的基因家族,在植物生长发育、响应逆境胁迫和信号转导等方面发挥着重要作用。为了研究紫花苜蓿中Tify基因家族及其在非生物胁迫下的响应,本研究在紫花苜蓿中鉴定出84个Tify基因并对其进行染色体定位、基因结构分析等方面的生物信息学分析,并随机挑选Ms Tify6、Ms Tify32、MsTify46、MsTify48、Ms Tify65五个基因进行盐、碱、干旱胁迫下的RT-qPCR分析。理化性质及结构分析表明:除MsTify1、Ms Tify2、MsTify3外的其余81个基因在除7号染色体外的7条染色体上呈不均匀分布。RT-qPCR结果显示了MsTify基因对盐、碱、干旱等非生物胁迫有明显的响应,在调控逆境胁迫中发挥着关键性作用。为后续研究Tify基因提供了新的理论基础。

In vitro Evaluation of Seed Germination in Twelve Alfalfa Cultivars under Salt Stress

Alfalfa(Medicago sativa L.),when exposed to abiotic stress such as salinity,suffers significant losses in yield and productivity.The present study evaluated the salinity tolerance of 12 alfalfa cultivars in vitro using five concentrations of sodium chloride(NaCl),ranging from 0 to 250 mmol L^(−1).The results obtained in the current study revealed that the Saudi cultivars,Kasimi and Hassawi,and the German cultivar(Berlin)had the highest salinity tolerance in terms of germination percentage(GP),corrected germination rate index(CGRI),days to reach 50%germination(GT_(50)),and ability to form cotyledonary and true leaves.Under mmol L^(−1) NaCl,the Saudi cultivar Kasimi cultivar showed GP,CGRI,and GT_(50) of 55.20%,123.15,and 3.77 days,respectively.Similarly,the German cultivar(Berlin)showed GP,CGRI,and GT_(50) of 50.06%,86.61,and 5.17 days,respectively.These findings might reveal a pivotal aspect in salt tolerance in alfalfa.Our results will help to select salt-tolerant alfalfa cultivars that could thrive in arid and semi-arid areas with salinity problems.