Cloning Of Medicago Sativa Phychrome B Cdnaand Construction Of Its RNA Interference Expression Vector

Alfalfa(Medicago sativa)has been gradually concerned as an important forage legume,the characteristics of fall dormancy is related with cold hardiness and production performance,it is the first index of choosing suitable variety in different areas.Fall dormancy of alfalfa exists a Photoperiodic Effect,meanwhile,Phytochrome is the receptor of Photoperiodic Effect.Phytochrome B is also the important photoreceptor in plants.So,we presumed that there has certain relationship between the Phytochrome B and alfalfa's dormancy,based on it,hopes to reveal the the molecular mechanism of alfalfa's fall dormancy.This study used the method of RNA interference in plants to inhibit the expression of the phyB by constructing the vector of RNA interference,and made an analysis on phenotype and the characteristic of light-regulation about transgenic plant, the result was to study the function of phyB and the molecular'regulational mechanism of alfalfa's fall dormancy.Main results are as follows:Trial I:The total RNA of Vernal was extracted successfully,the primers were designed.The partial cDNA of phyB(333bp) was amplified from total RNA by PCR,after purification and recovery,the target fragment was cloned into pGEM?-T Easy vector.The results proved that the similarity degree reached mor than 96% between DNA sequences of insert fragment and the phyB'sequences of WL-525HQ,Vernal, Medicago truncatula in Genbank by detection of PCR product,double enzyme digestion analysis using ClaI/XbaI and KpnI/XhoI, analysis of sequencing results.Successive test can be studied.In order to obtain purification and recovery's product,recombinant plasmid contains target fragments were double enzyme digested,it provided the material for the construction of RNAi vector.Trial II:"pHANNIBAL/positive"vector was obtained by linking between positive fragment and the Intermediate vector digested with ClaI/XbaI;After it was digested with KpnI/XhoI,purification and recovery and linked to reverse fragment,"pHANNIBAL/positive/reverse"was obtained.It is proved that the positive and the reverse fragments had been inserted the Intermediate vector wholely and correctly.The ihpRNA structure was obtained after the "pHANNIBAL/positive/reverse"vector was digested with NotI,linked it to the pART27 vector digested with NotI,the RNA interference vector was obtained.After that transferred to the Agrobacterium EHA105 by using the method of frozen thawed,material preparation for deuteric transformation was achieved and lay a foundation for further studying about the function of phyB of Medicago sativa.