Synthesis and Insecticidal Activity of Derivants of 3,13-Ester Mesaconitine

[Objective] The experiment aimed to explore the relations between structure of aconitine, aconitine derivatives and insecticide. [Method] The aconitine derivatives such as 3-acetyl mesaconitine, 3-propionyl mesaconitine, 3-acetyl-13-Benzoyl-mesaconitine and 3-propionyl-13-Benzoyl-mesaconitine were synthesized by mesaconitine with acetic anhydride or propionic anhydride and their insecticidal activities were also determined. [Result] When the concentration was 500 mg/L, the insecticidal activities of mesaconitine against Nilaparvata legen and Aphis were 50% and 30% respectively while the insecticidal activities of 3-acetyl mesaconitine, 3-propionyl mesaconitine, 3-acetyl-13-Benzoyl-mesaconitine and 3-propionyl-13-Benzoyl-mesaconitine against Aphis medicagini were 40%, 30%, 30% and 20% respectively at 500 mg/L. [Conclusion] After hydroxyl esterification, the insecticidal activity of mesaconitine was declined and the existence of hydroxyl at 3rd position in mesaconitine played very important influences on insecticidal activity.

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.

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.

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.

Application of Oats to Control the Weeds in the Seedling Period of Alfalfa Sowed in Spring

[Objective]The aim was to solve the weeds existed in seedling period of alfalfa in Chongqing.[Method]The naked oat and common oat were selected.Through the analysis of determiners such as plant number,plant height,yield and weeds ratio,the oat type and sowing method were screened through all the accompany sowing crops to better control weeds in the seedling period of alfalfa.[Result]Compared with common oat,the tested naked oat as the companion crop of alfalfa seeding can prevent weeds more effectively;the proper companion-seeding disposal of alfalfa sowed in spring in Chongqing area was 15 kg/hm2 of alfalfa and 120 kg/hm2 of naked oat,sowing in strip in 40 cm row spacing;the proper mowing period of oat was during the time from May 11th to May 21st;the proper mowing period of alfalfa was ten days later.[Conclusion]Spring sowing oats with alfalfa can control seedling weeds effectively.

Study on Spatial Distribution Pattern of Thrip (Odentot hrips lati) on Alfalfa (Medicago sativa)

[Objective] The aim of this study was to reveal and analyze the spatial distribution pattern of thrip（Odentot hrips lati） on alfalfa（Medicago sativa）.[Method] Within alfalfa plantation areas,experimental plots with the sizes of 10×10,10×20,10×30,20×30,20×40,20×80,30×30,30×40,30×50,40×40,40×50,40×80 and 50×50 m2 respectively were set up in the randomly selected sampling sites of 1 m ×2 m in size.During bud-emerging period and florescence period of alfalfa in the experimental plots,the number of thrips was counted and their distribution pattern was measured using XU Ru-mei＇s method.[Result] During bud-emerging period of alfalfa,the spatial distribution of thrips assumed an elliptic shape sized of 40×30-40×40 cm2 at horizontal level and that presented an ascending-falling trend of tress density down from the top at vertical level.During the florescence period of alfalfa,it showed a homogeneous distribution or randomly distributed pattern at horizontal level and a falling trend of tress density from the top to the apex to the root at vertical level.[Conclusion] Our results provide theoretical basis for field selection of insect-resistant alfalfa varieties.

SELECTION OF METHIONINE-RESISTANT VARIANT OF MEDICAGO SATIVA

A variant cell line of Medicago saliva L. resistant to methionine (100 mmol/L) was selected .from calli treated with 1.5 mmol/L sodium azide. Plants were regenerated from this selected cell line. After growing for 6 months on the medium devoid of selection pressure, the variant cell line still showed high level of resistance, which was 7.2-fold higher than that of the wild type. This indicated the stability of the resistance expression. The variant cell line also expressed a 3.3-fold higher cross-resistance to ethionine, than that of the wild type. The contents of methionine, lysine, threonine and isoleucine in the variant calli were respectively 2.0, 1.43, 1.71 and 1.57 times higher than those in the wild type. However, in the variant regenerated plantlets they respectively were 2.0, 1.33, 1.22 and 1.22 times higher than those in the wild type regenerants. Four new protein bands, viz. 65 kD, 23 kD, 20 kD and 18 kD, appeared in the SDS-PAGE pattern of soluble proteins in the variant calli. In the peroxidase isoenzyme electrophoresis pattern, two new bands were also observed in the variant calli.

Effect of Mixed Silage of Alhagi sparsifolia and Medicago sativa at Initial Flowering Stage on Degradation

[Objective] The influence of mixed silage of A.sparsifolia and M.sativa at initial flowering stage on degradation was studied to provide theoretical basis for production practice.[Method] Using A.sparsifolia and M.sativa at initial flowering stage as materials,the changes of in vitro degradation rate,gas production,pH value,ammoniacal nitrogen（NH3-N）and volatile fatty acid（VFA）content in degradation solution were analyzed by means of artificial rumen technique,and the effect of mixed silage of A.sparsifolia and M.sativa at initial flowering stage on degradation was studied in order to find out suitable mixed silage ratio of A.sparsifolia and M.sativa.[Result] Compared with single M.sativa,NH3-N content in degradation solution was very significantly decreased in the mixed silage of A.sparsifolia and M.sativa,and there was a reduction in gas production,while the content of acetic acid,propionic acid and butyric acid increased significantly;for the treatment of 70% A.sparsifolia and 30% M.sativa,NH3-N content（22.93 mmol/L）and gas production（40.50 ml）was lowest,while degradation rate（32.27%）,acetic acid（5.48 mmol/L）,propionic acid（2.08 mmol/L）,butyric acid（2.52 mmol/L）and their total content（10.08 mmol/L）were highest,so its degradation effect was the best.[Conclusion] The mixed silage of A.sparsifolia and M.sativa at initial flowering stage could improve the utilization efficiency of protein and carbohydrate in M.sativa and the treatment of 70% A.sparsifolia and 30% M.sativa had the best degradation effect.

Disruption of nifA Gene Influences Multiple Cellular Processes in Sinorhizobium meliloti

Sinorhizobium meliloti nifA is important in fixing nitrogen during symbiosis. A nifA null mutant induces small white invalid nodules in the roots of host plant. The additional phenotypic alterations associated with the disruption of the nifA gene are reported in this study. Under a free-living state, S. meliloti nifA mutant reduces its ability to swarm on a half-solid plate. Interestingly, the AHL （Acylhomoserine lactones） contents in the nifA mutant are lower than that of the wild type during the lag phase, whereas it is reversed in the logarithmic and stationary phases. Quantitative spectrophotometric assays reveal that the total amount of extracellular proteins of the nifA mutant are lower than that of the wild type. In addition, the mutant abolishes its nodulation competitive ability during symbiosis. These findings indicate that NifA plays a regulatory role in multiple cellular processes in S. meliloti.

Growth Performance of Alfalfa(Medicago sativa)under Different Nitrogen Application Levels

Nitrogen addition is rather important to the growth of alfalfa. In this study, the effects of different nitrogen application levels on various growth characteristics of alfalfa were investigated. The results showed that nitrogen application exhibited no significant effects on plant height and stem diameter but posed significant effects on branch number of alfalfa. Branch number of alfalfa in each cutting raised with the increasing application level of nitrogen, with significant differences among different treatments （P〈0.05）; individual aboveground biomass increased with the increasing application level of nitrogen, but the increasing trend gradually tended to be steady from the first to the fourth cutting. Nitrogen application affected significantly （P〈0.05） individual aboveground biomass of alfalfa in the first, second and fourth cutting, but exhibited no significant effects on individual aboveground biomass of alfalfa in the third cutting. With the increase of nitrogen application level, total surface area, total volume, collar diameter and crossing number of alfalfa roots increased gradually, but total length, average diameter and furcating number of alfalfa roots increased first and then declined. Total length, average diameter and furcating number of alfalfa roots reached the maximum in N60 treatment.

Study on the Correlation Between Leaf Area and Drought Resistance in Different Alfalfa Varieties

[Objective] This study was to reveal the correlation between leaf area and drought resistance in different varieties of alfalfa.[Method] Using various alfalfa varieties as experimental materials,the drought resistance of leaves of drought-stressed alfalfa plants was assessed by measuring the content of free proline for analyzing its correlation with leaf area.[Result] Under drought condition,the drought resistance of alfalfa is directly related to leaf area in a positive correlation.[Conclusion] Leaf area could be used as an institutional assistant index to reflect the resistance of different alfalfa varieties.

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.

Bioinformatics Analysis of Zinc Transporter from Baoding Alfalfa

[Objective] This study aimed to perform the bioinformatics analysis of Zinc transporter （ZnT） from Baoding Alfalfa. [Method] Based on the amino acid sequence, the physical and chemical properties, hydrophilicity/hydrophobicity, secondary structure of ZnT from Baoding alfalfa were predicted by a series of bioinformatics software. And the transmembrane domains were predicted by using different online tools. [Result] ZnT is a hydrophobic protein containing 408 amino acids with the theoretical pl of 5.94, and it has 7 potential transmembrane hydrophobic regions. In the sec- ondary structure, co-helix （Hh） accounted for 48.04%, extended strand （Ee） for 9.56%, random coil （Cc） for 42.40%, which was accored with the characteristic of transmembrane protein. [Conclusion] mZnT is a member of CDF family, responsible for transporting Zn^2＋ out of the cell membrane to reduce the concentration and toxicity of Zn^2＋.

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.

Research Progress on Alfalfa(Medicago sativa L.) Adaptability under Drought Stress

Alfalfa （Medicago sativa L） is one of the most important leguminous forage in China, and distributed most widely in the world. Because of Its high nutritional value, large biomass and strong reproducibility and improved soil green ma- nure plants, it is a kind of green manure plant capable of improving soil and known as ＂the king of forage＂. Alfalfa has strong adaptability under drought stress, and could adapt drought conditions through its physiological metabolism, structure devel- opment and morphology construction. Drought resistance in alfalfa is always a hot research subject of forage, and the research has been gradually developed from the morphological level to more-deeply physiological, biochemical and molecular biology fields, and has made many valuable research achievements.

Phosphorus Solubilizing Capability,IAA Secretion and Characteristics of Phosphorus-solubilizing Bacteria in Rhizosphere of Alfalfa in Guizhou Province

[Objective] The study aimed to isolate and screen efficient phosphorus-sol- ubilizing strains from the root soil of alfalfa in Guizhou, and investigate its growth promoting mechanism. [Method] Phosphorus solubilizing bacteria were isolated from rhizosphere of alfalfa with inorganic and organic phosphorous medium and the strains with higher capacity of dissolving phosphorus were screened through dissolv- ing phosphate zone and further researched. Meanwhile the phosphorus solubilizing capability of strains cultured in liquid medium was investigated by molybdenum blue colorimetric method. [Result] The capacity of dissolving calcium phosphate of 11 se- lected strains differed significantly and tended to be volatile from 150.40 to 268.20 μg/ml; phosphate solubilization capacity, IAA secretion and the pH of culture media did not show any significant correlation; all strains could produce IAA, and the se- cretion was from 12.09 to 22.16 μg/ml; the selected strains all could produce alka- line matter, and most of colonies were pale or milky white, irregular, opaque, jagged, flat and non-pigmented; utilization of carbon sources by different strains varied signifi- cantly. [Conclusion] The study laid the foundation for relieving phosphorus in unpro- ductive land, saving phosphate resources, reducing environmental pollution and pro- viding fertilizer in alfalfa production.

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.