The Screenging And Identity Of Phytochrome Interacting Factors Of Soybean

Soybean is one of the most important economic crops in the world, which posesas the main source of plant proteins (67%) and oils (28%) for human being. Moreover,soybean and Bradyrhizobium japonicum form the root nodule together, which canconvert ammonia to semicarbazone compounds for plants absorbing. Based on theseadvantages, soybean is considered to be an environment friendly crop that applies tothe agricultural model of crop rotation or intercropping. So soybean has a veryimportant significance to protect our country’s oil security strategy and the sustainabledevelopment of agriculture.There are various environmental factors that can influence growth anddevelopment of soybean as well as affecting the quality of soybean. Among thesefactors, light plays a extrusive role not only in providing the sources of energy thatused in photosynthesis, but also as a signal regulator in many ways of the plant lifecycle. Light can play its role through adjusting the early seedling photomorphogenesisand photoperiodic response. Growth period is an important index of photoperiodicresponse, which closely related with the yield and quality of soybean. The length ofgrowth period determines the planting latitude and planting season.Soybean is a thermophilic sensitive short day crop. Heilongjiang province is themajor soybean producing areas. In recent years, due to the impact of high yield cropssuch as rice and maize, the main planting area has been made by I and II accumulatedtemperature zone north gradually transferred to the III、IV and V zone which islocated in high latitudes, the climate is cold, frost free period is short, and varietybreeding did not keep up. So urgently needed this area is the super early maturingcultivar insensitive to photoperiod response. Therefore, the molecular mechanism ofsoybean genes involved in the regulation of light signaling pathways and photoperiodresponse can provide theoretical basis for the molecular breeding and directionaltransformation suitable varieties.In order to change the perception in the growth environment of light signals andtimely response, soybean evolved a series of light receptors, regulating the soybeanconsistent with its growth environment. As a type of significant photoreceptor,phytochromes are responsible for absorbing red and far-red light and through a series of interacting proteins playing an important role in photomorphogenesis andphotoperiod reaction process.Our previous studies showed that in soybean E4is between the linkage group ISSR maker satt496and satt354, encodes GmPhyA2. The analysis of E4near isogeniclines, e4is due to the insertion of a LTR type retrotransposons in the first exton leadedto mutation. We also found four other E4gene mutant type (e4)(kes, oto, tus4andkam), sequence analysis revealed that these four e4encoding GmPhyA2lack of Cterminal domain. Based on these results, futher studies found that diverse geneticmechanisms underline photoperiod insensitivity in soybean. At least three multi-locusgenotypes consisting of various allelic combination at E1, E3and E4conferredper-flowering photoperiod insensitivity, the phyA genes E3and E4are majorcontrollers underlying not only pre-flowering but also post-flowering photoperiodresponses. Using Arabidopsis genetic transformation technology that overexpressede4gene, we identified C terminal domain of GmPhyA2domain on early seedlingphotomorphogenesis. Then GmPIF3gene was isolated from Harosoy, we verified theinteraction between GmPhyA2and GmPIF3by yeast two hybrid system andbiomolecular fluorence complementation and characterized the function of GmPIF3gene.1.Genetic variation in four maturity genes led to different responses tophotoperiod during post-flowering vegetative and reproductive photoperiod responsesThe molecular bases of four maturity genes (E1through E4) have beendetermined. To determine the alleic constitutions at the four loci, we genotyped the4genes in each of the53photopreriod-insensitive accessions by using previouslyallele-specific DNA makers. We classified the53photoperiod-insensitive accessionsinto6genotypic groups, the difference in flowering datas between ND and LDconditions was small (that is,5days or less). However, these accessions differedmarkedly in their post-flowering vegetative and reproductive growth characteristics,such as reproductive period and stem growth after flowering. The phytochromeA-regulated of the determinate growth habit gene Dt1, an ortholog of ArabidopsisTERMINAL FLOWER1, was involved in the persistence of the vegetative acticity atthe stem apical meristem of flower-induced plants under long-day conditions. ThephyA genes E3and E4are major controllers underlying not only pre-flowering butalso post-flowering photoperiod reponses.2. Functional analysis of e4gene To characterize functions of e4gene, the vectors containing each e4gene drivenby35S promoter were genetically transformed into Arabidoposis. Under far-red lightcondition, transgenic plants and Arabidopsis phyA mutants showed similar length ofhypocols, which were longer than these of wide types. But under red light condition,all of the plants showed analogous length of hypocotyls. The datas indicatd that PASand HKRD domain might affect the growth of hypocotyl.3. Verification of the interaction between GmPhyA2and GmPIF3Within the publicly available soybean GeneBank, there are several highly similarsequences in BLAST search to Arabidopsis PIFs. Program MEGA4.0was used toconstruct the phylogenetic tree for these sequences and the data showed that threecandidate GmPIF3and the AtPIF3were included and aligned. These GmPIF3bothhad the bHLH domain and APB domain that could interact with phycochromes.Designing the specific primers to get their full length cDNAs, they were named asGmPIF3-a（Glyma10g28290.1）、GmPIF3-b（Glyma19g41261.2）and GmPIF3-c（Glyma20g22291.1）. Using yeast two hybrid and biomolecular fluorencecomplementation methods, we verificated the interaction between GmPhyA2andthree GmPIF3, the results indicated that GmPhyA2could only interact with GmPIF3that encoded by GmPIF3-b, but could not interact with the other two GmPIF3.4. Screening more proteins interact with GmPhyA2in yeast cDNA libraryThe yeast two hybrid cDNA libary was screened with the bait ofpGBKT7-GmPhyA2. The results showed that32clones could activate the reportgenes. Then PCR analysis and sequencing were done for these positive clones.7clones of them had right open reading frams and3of them had homologoussequences with the genes in photosystem. But the results need to be confirmed.5. Overexpression of GmPIF3in soybeanTo futher characterize the functions of GmPIF3-b, the construct containing targetgene was transformed into soybean system mediated by agrobacterium tumefaciens.In transgenic plants, GmPIF3-b gene was over expressed. Under low R:FRincandescent light, the length of hypocotyls of transgenic plants were longer thanthese of control plants DongNong50, this indicated that GmPIF3down regulated thegrowth of hypocotyl. Flowering time in transgenic lines was longer than Dongnong50,which may be related to GmPIF3-b, but the results need further validation of thestatistical work in more detail.