The Identification Of MiRNA And Related Genes Of Regulatng Fall Dormancy On The Light Signal Transduction,Glycometabolism In Alfalfa

Fall dormancy is an adaptive growth characteristic of alfalfa,with the changes of the shorter day-length and lower temperature.This growth characteristic was significantly positive correlation with the cold resistance of plant and production performance.In recent years,research has shown that fall dormancy and cold resistance are two relatively independent characters,and the photoperiod is the key factor which effects fall dormancy.In order to identify target genes and related enzymes which were involved in regulation of fall dormancy in alfalfa,we carried out these following experiments.Based on alfalfa transcriptome sequencing,we selected key genes(difference multiples,the Fold Change2 or higher),such as blue light receptors(Cryptochrome2B)CRY2B,far red light,and red light receptors(Phytochrome A,PHYA;Phytochrome B,PHYB)on light signal transduction pathways as the research objects.We applied high-throughput Small RNA(sRNA)sequencing to identify some miRNAs that were responsive to fall dormancy in standard variety(Maverick and CUF101)of alfalfa:(1)We identified 20 known miRNAs and eight new miRNA candidates that were responsive to fall dormancy,and identified using fluorescence PCR verification and Northern hybrid.(2)On the basis of the transcriptome and sRNA sequencing,we select PHYA,PHYB,CRY2 B and FHY1(PHYA downstream regulation factor)on the light signal transduction pathways.We measured m RNA expressions of PHYA,PHYB,CRY2 B and FHY1 under the conditions of t different months and PHYA,PHYB silence transgenic plants.(3)We constructed the overexpression vector of CRY2 B and obtained the transformed plants of Accaribou.(4)On the sugar metabolism,we measured alpha amylase,beta amylase enzyme activity and theirs m RNA expression.(5)Finally,we measured IAA,GA3 and ZR,ABA content of two different fall dormancy alfalfa;Experiments included five parts as follows:?.Identification of different dormant Medicago sativa L.micro RNAs in response to fall dormancyFour sRNA libraries were generated and sequenced from alfalfa leaves in two typical varieties at distinct seasons.Through integrative analysis,we identified 51 novel miRNA candidates of 206 families.Additionally,we identified 28 miRNAs associated with fall dormancy,including 20 known miRNAs and eight novel miRNAs.Both high-throughput sequencing and RT-q PCR confirmed that eight known miRNA members were up-regulated and six known miRNA members were down-regulated in response to fall dormancy.Among the 51 novel miRNA candidates,five miRNAs were up-regulated and three miRNAs were down-regulated in response to fall dormancy,and five of them were confirmed by Northern blot analysis.Our data provide a useful resource for investigating miRNA-mediated regulatory mechanisms of fall dormancy in alfalfa,and these findings are important for our understanding of the roles played by miRNAs in the response of plants to abiotic stress in general and fall dormancy in alfalfa.Based on the transcriptome sequencing results,we chose PHYA,PHYB,CRY2 B and FHY1 as the key genes on the light signal transduction pathways in the experiment.?.Expression levels of genes derived from light signal transduction pathwaysThe expression levels of m RNAs of PHYA,PHYB,CRY2 B and FHY1 genes from the light signal transduction pathways were performed,accompanying with m RNA expression levels in transgenic plants.At transcription level,CRY2 B plays vital roles in regulating fall dormancy(FD)in alfalfa,among the light signal transduction pathway.Prior to transcription,PHYA and PHYB function as light signal transduction factors to participate in the circadian cycle via regulating the downstream regulatory elements.Depending on this,we assume that PHYA and PHYB regulate FD prior to transcription,or combine with related miRNAs,while CRY2 B regulates FD at transcription level.PHYA,PHYB and CRY2 B are indispensably all involved in regulating FD in light signal transduction pathways.?.Enzyme activity and mRNA expression levels of alpha amylase and beta amylase in alfalfa with different FD classesFD has a positive correlation with winter hardiness,while the decline in fructose and starch related to winter hardiness.Research on Euphorbia esula showed an increase of 16,000-fold in beta amylase between different growth stages of bud dormancy.In this experiment,Enzyme activities and m RNA expression levels of Maverick(FDC1),Vernal(FDC2),UC-1887(FDC10)and UC-1465(FDC11)were examined.The results showed that there were consistence expression levels of beta amylase in Maverick and Vernal,at different growing stages.There was an increased trending of beta amylase in Maverick and Vernal,but their expression levels were not so high as in Euphorbia esula.?.Construction of overexpression vector and transformation of CRY2BIn this section,p CAMBIA3301(contains Ca M 35 S promoter and Nos poly A terminator)was used to construct the overexpression vector,by adding Bsp HI and Pml I recognition sites of Ncol I isocaudomer.The transformed plants of Accaribou were obtained by utilizing Agrobacterium-mediated transformation.This laid a foundation for further study of CRY2 B in regulating FD in alfalfa.?.Determination of contents of endogenous hormones IAA,GA3,ZR and ABAThe endogenous hormone levels of IAA,GA3,ZR and ABA of Maverick(FDC1),Vernal(FDC2),UC-1887(FDC10)and UC-1465(FDC11)were analysed.The results inferred that there was a negative correlation between ABA and IAA,GA3,and ZR.There were different roles of these four hormones even at different growing stages.At the beginning stage of alfalfa,IAA,GA3 and ZR promote growth.However,IAA,GA3 and ZR were inhibited under the induction of high temperature and FD.ABA begins to take advantage in this situation.ABA increased significantly in September.Similar changes in both PHYB and ABA were confirmed.The results indicated that IAA,GA3,ZR and ABA work together to regulate the growth and FD in alfalfa.The regulation mechanism of alfalfa fall-dormancy is a complex network,PHYA,PHYB were involved in the regulation of alfalfa fall-dormancy before transcription,or its corresponding micro RNAs combination made them suppressive(in 28 key micro RNAs,these highly conserved micro RNAs,such as mi R156,mi R164,mi R172 were involved in the process of growth,dormancy,and flowering),but CRY2 B was involved in the regulation of alfalfa fall-dormancy on transcription level.PHYA,PHYB and CRY2 B participated the regulation of alfalfa fall-dormancy;the beta amylase m RNA expression of alfalfa standard varieties Maverick(FDC1)and CUF101(FDC2)in different months were compared with theirs enzyme activity,alpha amylase were not obvious;Under high temperature and fall-dormancy induce,ABA content increased in September,however,compared with July,ABA content increased more in July,the synthesis of ABA was similar with PHYB,the experiment also proved the PHYB regulated by fall-dormancy endogenous hormone indirectly.