| The fall dormancy(FD)of alfalfa(Medicago sativa L.)refers to a state of reduced or ceased plant growth with the decline of photoperiod and temperature in the autumn.According to FD classification with 9 grades,alfalfa varieties were divided into three groups: dormant type(fall dormancy classes 1-3,FDC1-3),semi-fall dormant type(FDC4-6),and non-dormant type(FDC7-9).Fall dormancy is negatively associated with alfalfa yield to some extent,however,that is positive related to winter survival.It is the first index to select suitable alfalfa varieties as we grow alfalfa.The objiective of this study was to select suitable alfalfa varieties and explore the relationship among production performance,quality,biologic traits of different FD alfalfa varieties,and we carried out a preliminary functional analysis with associated fall dormancy genes by using the proteomics and molecular biotechnology.Experiment 1 Evaluation of production performance and quality of alfalfa varieties with different fall dormancy classesTo explore the effects of alfalfa varieties with different FDC on production performance and quality,17 commercial alfalfa varieties with FDC ranging from 3 to 8 and 3 self-cultured strains by Henan Agricultural University(H-1,H-2,H-3)were studied in Xinxiang,Henan province,China,in 2016-2017,using a randomized block design with 4 repetitions.The results showed:(1)H-3 had the highest dry matter yield(with 14.29 t/hm2),and was significantly higher than those of Ju'neng 7(FD4),WL363(FD5)and WL366(FD5)(P<0.05),however,there were no significant differences among other varieties(P>0.05);In general,annual DM yield of non-dormant alfalfa varieties was higher than that of dormant varieties,and that of semi-fall dorman alfalfa varieties between dormant and non-fall dormant alfalfa varieties.According to annual DM yield,H-3 and Eurek G+ varieties were suitable in Henan province.(2)The crude proteinn(CP)contents of WL-366(FD5)and RFV were the highest,but those of Zhongmu 3(FD3)were opposite.According to the decision coefficient of each items,the top three varieties with suitable varieties are: H-3,Eurek G+ and Bluemoon,respectively.(3)There was a highly significant positive correlation among the decision coefficient index,fall dormancy class,total dry matter yield and plant height.As a consequence,semi-dormant and non-dormant alfalfa varieties were suitable in Henan province.Experiment 2 Ms EGs gene cloning,protein prokaryotic expression and purificationAccording to sequence results of apical buds iTRAQ of two standard alfalfa varieties Maverick(FD1)and CUF101(FD9)in autumn,we selected endo-(1,3;1,4)-?-D-glucanase(glucan hydrolase family 17 [GH17])and studied its relationship with fall dormancy.The results indicate:(1)According to sequence information of Medicago truncatula,we successfully choned endo-(1,3;1,4)-?-D-glucanase of Medicago sativa,including Ms EGs c DNA of 731 bp coding sequence,which encoded a protein of 243 amino acid residues with a molecular mass of 25.6KD and a p I of5.54.(2)Prokaryotic expression vector of Ms EGs was built,and polyclonal antibodies was produced by using soluble proteins.Those had satisfactory biological value and specificity.Experiment 3 Preliminary study of Ms EGs on the regulation of fall dormancyJust as mentioned above in experiment 2,the functions of Ms EGs and its relationship with alfalfa fall dormancy were studied by using the molecular biotechnology.The result showed that:(1)20 OE-transgenic plants of WL-903 variety were obtained by agrobacterium-mediated transformation,and those grew slowly and stunted compared with wild;and their abundance of m RNA and protein were increased by using q RT-PCR and westenblot technology examined.(2)The protein expression locus of Ms EGs was in the cell wall by immunohistochemistry.In conclusion,it has not been reported of Ms EGs expression in cell wall in alfalfa.our results demonstrate that(1,3;1,4)-?-D-glucan is not unique to the monocotyledon but is exist in alfalfa cell wal.Meanwhile,it may participate in circadian rhythm of alfalfa fall dormancy. |
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