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.

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.

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.

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.

Research Advances of Root Rot in Medicago sativa L.

As the most famous perennial leguminous forage in the world, Medicago sativa L. ranks the first in both yield and nutritional value, and can be used for many years once planted in field production. However, root rot has become a very important reason for yield decrease and plant de-cline of alfalfa due to long service life. According to the research progress at home and abroad, the main pathogen distribution, harms and control methods of root rot in M. sativa at home and abroad were systematically expounded, in order to provide a theoretical basis for further research of the disease.

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。

Effects of Transparent Testa8(TT8) gene and Homeobox12(HB12) gene silencing in alfalfa(Medicago sativa L.) on molecular structure spectral profile in relation to energy,degradation,and fermentation characteristics in ruminant systems

Alfalfa(Medicago sativa L.) is a legume forage that is widely cultivated owing to its high biomass yield and favorable nutrient values. However, alfalfa contains relatively high lignin, which limits its utilization.Downregulation of two transcriptional factors, Transparent Testa8(TT8) and Homeobox12(HB12), has been proposed to reduce lignin content in alfalfa. Therefore, silencing of TT8(TT8i) and HB12(HB12i) in alfalfa was achieved by RNAi technology. The objective of this project was to determine effect of gene modification through silencing of TT8 and HB12 genes in alfalfa plants on lignin and phenolic content,bioenergic value, nutrient supply from rumen degradable and undegradable fractions, and in vitro ammonia production in response to the silencing of TT8 and HB12 genes in alfalfa. All gene silenced alfalfa plants(5 TT8i and 11 HB12i) were grown under greenhouse conditions with wild type as a control.Samples were analyzed for bioactive compounds, degradation fractions, truly digestible nutrients, energetic values and in vitro ammonia productions in ruminant systems. Furthermore, relationships between physiochemical, metabolic and fermentation characteristics and molecular spectral parameters were determined using vibrational molecular spectroscopy. Results showed that the HB12i had higher lignin, while TT8i had higher phenolics. Both silenced genotypes had higher rumen slowly degraded carbohydrate fractions and truly digestible neutral detergent fiber, but lower rumen degradable protein fractions. Moreover, the HB12i had lower truly digestible crude protein, energetic values and ammonia production compared with other silenced genotypes. In addition, in relation to the nutritive values of alfalfa, structural carbohydrate parameters were negatively correlated, whereas alpha/beta ratio in protein structure was positively correlated. Furthermore, good predictions were obtained for degradation of protein and carbohydrate fractions and energy values from molecular spectral parameters. In conclusion, silencing of the TT8 and HB12 genes decreased protein availability and increased fiber availability. Silencing of the HB12 gene also increased lignin and decreased energy and rumen ammonia production. Moreover, nutritional alterations were closely correlated with molecular spectral parameters. Therefore, gene modification through silencing the TT8 and HB12 genes in alfalfa influenced physiochemical, metabolic and fermentation characteristics.

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.

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。

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。

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.

Callus Induction of Alfalfa and Evaluation of Callus Tolerance to Acid and Aluminum

Medicago is an important grass in the legume family,which is suitable to be grown in neutral or alkalescent soils,and hence,can be widely spread in south of China with low pH value.It is one of major objectives to cultivate acid-aluminum tolerant species.In the research,6 high-quality species with high-yielding potential were taken as materials to make evaluation on acid-aluminum tolerance by cell-culture technique,and the results showed that tolerance capacity from high to low was GT13R＞Muge 701=Muge 702＞Acrora＞AC-3＞Sheshou No.2=Medicago sativa.

紫花苜蓿耐盐性相关NAC转录因子的挖掘及表达分析

NAC(NAM,ATAF1/2,CUC2)是植物特有的一类转录因子家族,可调控植物响应盐胁迫。本研究基于盐胁迫下紫花苜蓿(Medicago sativa L.)耐盐品种GIB和盐敏感品种LS根和叶的转录组数据,通过生物信息学方法筛选出17个差异表达的MsNAC转录因子家族成员,并对其序列特征、系统进化、顺式作用元件及盐胁迫下的表达模式等进行分析。结果显示,17个MsNACs均具有NAC典型的NAM结构域,且蛋白结构相似。系统进化分析将17个MsNACs分成8个亚族,各亚组成员具有高度相似的保守基序。顺式作用元件分析发现,有15个MsNACs具有ABA响应顺式元件ABRE。转录组数据表达模式分析与RT-qPCR验证结果显示MsNAC1,MsNAC2,MsNAC35,MsJUB1和MsNAC40基因在盐处理下GIB叶中上调表达,在LS叶中下调或者未发生变化,推测这些基因参与调控紫花苜蓿的耐盐反应。本研究为进一步研究紫花苜蓿NAC转录因子响应盐胁迫功能提供参考依据。

补播苜蓿对退化草地植物群落结构和生产力的影响途径

近年来,紫花苜蓿(Medicago sativa L.)和黄花苜蓿(Medicago falcata L.)被广泛选作补播材料,用于修复退化草地。尽管研究表明二者的植物学特征存在差异,但鲜有人关注它们是否会通过不同的方式影响退化草地的生产力。为了探究二者对退化草地的修复效果是否存在差异,并确定影响补播地生产力的限制因素,本研究选取黄花苜蓿和紫花苜蓿作为补播种对退化草甸草原进行了补播,并在隔年对草地植被、土壤理化性质和微生物量进行监测。结果表明,两种苜蓿都可以显著增加退化草地生产力并改善土壤健康状况。然而,紫花苜蓿通过增加土壤铵态氮改变植被群落结构,其补播样地生产力也由土壤速效养分影响;黄花苜蓿通过增加土壤速效磷含量改变植被群落结构,其补播样地生产力由植被群落结构和土壤微生物量共同影响。初步结果表明,黄花苜蓿更适宜在养分贫瘠的退化草地进行补播。此外在选择紫花苜蓿补播时,可以适当施肥以提高补播成功率。

外源lea3基因转化紫花苜蓿的研究

将来源于大麦的lea3基因通过基因枪法导入紫花苜蓿栽培品种“中苜一号”的胚性愈伤组织中,经过膦丝菌素类除草剂(PPT)的4次筛选获得抗性愈伤组织,诱导分化后共获得21株抗性再生植株。经叶片涂抹除草剂试验和lea3基因的PCR分子检测证明,lea3基因已整合到紫花苜蓿的基因组中。与对照植株相比,获得的转基因植株具有显著增强的耐盐能力,表明遗传转化lea3基因可用于苜蓿抗旱耐盐新品种的选育。