Screening And Identification Of Genes And Proteins Regulationg Alfalfa Fall Dormancy

Alfalfa(Medicago sativa L.)is a perennial and high-quality forage crop cultivated worldwide,including in China,and plays an important role in improving the quality of milk,meat and eggs.Fall Dormancy(FD)is defined as the reduction in growth during the autumn,FD is one of the most important factors influencing adaptation and production performance,as well as in choosing cultivable alfalfa varieties for specific regions.However,its molecular mechanism remain almost unknown,at present,a key gene and protein involved in the regulation of alfalfa fall dormancy have not been identified,so the purpose of this study is to screen and determine the genes and proteins regulating fall dormancy.Firstly,important genes regulating fall dormancy were selected by comparison of transcriptomes of different fall-dormant alfalfa(Maverick and CUF101)leaves at FD and non FD,and q RT-PCR and correlation analysis showed that the m RNA content of Monogalactosyldiacylglycerol synthase 3 and IAA-amino acid hydrolase ILR1-like 1 was significantly correlated with the photoperiod,and the m RNA content of CyclinD5-2 was negatively correlated with photoperiod;and the m RNA content of Ribulose bisphosphate carboxylase/oxygenase(rubisco)activase and Monocopper oxidase-like protein SKU5 was significantly correlated with temperature,the m RNA content of Abscisic acid receptor PYL8 was negatively correlated with temperature.In addition,ten differential expressed unannoted genes in RNA-SEQ were chosen by foldchange value and open reading frame(ORF)length and their m RNA and DNA were obtained by RACE method,at last m RNA and DNA of only three unannoted genes(comp71142,comp 32802 and comp 59277)were successfully amplified,cloned and sequenced.Secondly,i TRAQ-based proteome was taken to compare the terminal buds of the Maverick(FD variety)and CUF101(non-FD variety)in autumn.A total of 3,872 protein species were firstly annotated,90 differential abundance protein species(DAPS)(difference >1.2-fold and p-value <0.05)were identified between Maverick terminal buds and CUF101 terminal buds.A functional analysis suggested that DAPS were mainly involved in amino acid and protein synthesis and metabolism,auxin polar transport,water-soluble vitamin metabolic process,etc.,and the phenylpropanoid biosynthesis,circadian rhythm-plant,flavonoid biosynthesis,etc.pathways by GO and KEGG analysis.m RNA abundance change sixteen proteins in DAPS were understood from non-FD to FD by q RT-PCR,and it is proved that the decrease of L-asparaginase,thiazole biosynthetic enzyme,aldo/keto reductase family oxidoreductase and CAD-like protein induce the fall dormancy of Maverick,the increase of chalcone and stilbene synthase family protein promotes the growth of CUF101 in autumn,and the increase of glycoside hydrolase family 1 protein enhances the resistance of CUF101 to autumn and promotes its growth.In addition,the specific high expression of RBP47 C protein in Maverick terminal bud was determined,it may also be involved in the fall dormancy of alfalfa.A novel protein was identified by the combination analysis of leaf transcriptome and terminal bud proteome.From the results of leaf transcription and terminal bud proteome,three known genes(RBP47C,SPL1 and L-asparaginase)and three unknown genes(71142,32802 and MSTPR)were selected and further verified and studied their function and mechanism in regulating alfalfa fall dormancy.At present,compared with the negative transgenic plants and wild plants,only the phenotype of WL903(nondormant alfalfa)of SPL1 over-expressed and Reindeer(fall dormancy alfalfa)of 32802 over-expressed change significantly,growth of the WL903 of SPL1 overexpressed becomes slow and its stem becomes thin and the Reindeer(fall dormancy alfalfa)of 32802 over-expressed grows slowly and its stem becomes thicken,so increase of SPL1 and reduction of 32802 induce alfalfa fall dormancy.At the same time,targeted genes of SPL1 was screened by pull-down technique.In addition,RBP47 C,SPL1and L-asparaginase,71142,32802 and MSTPR were expressed using prokaryotic expression system,purified by affinity chromatography and their polyclonal antibodies were prepared by the mice immunized.