Cloning And Analysis Of Photoperiod Insensitive Gane In Oats

Oats are important food and forage crops. The common cultivated oat species inthe world is Avena sativa L., which is hexaploid (2n=6x=42) species and has sevenchromosome number. Oat is also a great nutritional valued crop. It is cultivated widelythroughout the world, distributing in more than40countries on five continents mainlyin the northern hemispheric temperate regions. In China, Oat cultivation dated backmore than2100years, slightly earlier than the rest of the world. China's oat is mainlydistributed in north, northwest and southwest. Environmental conditions andadaptability are very important factors for oat growth. Photoperiod, being one of themost important factors, not only affects growth and development but also productsmaking it an important consideration for breeding superior varieties. Oat is generallylong-day crop, meaning that seedlings have to go through long-day to reach floweringand fruiting. Therefore, photoperiod make oat particularly restricted in geographicalarea and sowing time. Different breeding practices have shown that flowering andfruiting of some of the oat varieties depend on short day, so these are photoperiodinsensitive in the day-length. Therefore, breeding for photoperiod insensitive oatstrains which mature earlier that enabled effective use of the frost-free growth periodfrom single-cropping to doube-cropping a year and hence increasing yield per unitarea, becomes very important.A total of11oat varieties including Chinese and Canadian varieties were usedfor observing growth in this study. These oat varieties were planted in the screenhouse at the same time and were assayed for flowering-time in the normal conditions.The results show that there are early flowering varieties:60-62days, includingBaiyan-8, Baiyan-9, Baiyan-10varieties; middle flowering:66-68days, including theAC-Baton, VOA-8, AC-Loffa varieties and late flowering:73-76days, includingBaiyan-1, Baiyan-2, AC-Ernie, AC-Navan and AC-Gehe varieties. The latest flowering varieties is Baiyan-1(76days). From the above results we selectedBaiyan-10as early flowering, VOA-8as middle flowering, AC-Navan and Baiyan-1as late flowering. These were treated as samples and planted in the incubator which isdivided into two groups i.e. long-day:14hours light,24oC; short day:10hours light,24oC. The results were: in the long day light treatment, all4varieties enteredflowering after72days while for87days of treatment in the short day light, only onevariety (VOA-8) entered flowering (at the68th day). In long-day conditions, Baiyan-1variety was the latest flowering while VOA-8can enter flowering in both the twolight treatment conditions. Therefore, VOA-8may be having photoperiod insensitivegenes in its genome.The two varieties (Baiyan-1and VOA-8) were then selected for further assays.cDNA-AFLP and64MseI/EcoRI (8×8) combinations of primer pairs to assess theirgene expression differences. We discovered that there were1987Transcript-DerivedFragments (TDF). Among them323fragments were differentially expressed,accounting for about16%, indicating that the genotype differences of the two speciesis relatively large. Sequence analysis of108differentially expressed TDF indicatedthat28of the TDF have high homology with already functionally known genes. Thesegenes are involved in photosynthesis and energy (7,6.48%), signal transduction(5,4.63%), metabolism (4,3.70%), transcription (3,2.78%), translation (3,2.78%),secondary metabolism (2,1.85%), transposable elements (2,1.85%), defense response(1,0.93%) and transport (1,0.93%). It was found that TDF140-one of the VOA-8fragment from short day has high homology with wheat photoperiod gene (Ppd-D1).The Ppd-D1a wheat mutant is a photoperiod-insensitive, so that the VOA-8genomewith Ppd gene may be related to photoperiod-insensitive. Also TDF144exist in theVOA-8and has higher homology to the gene that regulates maize flowering-time(Rap2.7). This gene may be associated with oat flowering-time regulation.Real-time qRT-PCR analysis of the resulted unknown candidate gene indicatedthat TDF140in short day treatment expressed3-7times higher in the varieties VOA-8than Baiyan-1. Thus, experimental results show that the VOA-8variety has photoperiod related gene (OPpd). The OPpd gene expression results combined withthe VOA-8phenotype (long and short day flowering), we can say that OPpd caninduce VOA-8variety in shortday to normal flowering. This can lead us to speculatethat the OPpd is photoperiod insensitive gene. We named this oat photoperiodinsensitive gene as OPpdi. At the same time we used Real-time qRT-PCR to analyzeTDF144but the resulted sequence was not long enough find a matching location todesign high specificity primers in the sequence, so the outcome is not satisfactory.This result can not be used to prove this gene's regulatory role of oats at floweringstage. In addition, we used Real-time qRT-PCR to analyze the three signaltransduction homologs TDF108, TDF124, TDF171and two transcription factorhomologous genes TDF116, TDF268. The results showed that photoperiod affectsthese5genes' expression, i.e. long-day conditions promote their expression levels.The expression patterns of five genes compare to Baiyan-1and the VOA-8aresimilarly very sensitive to the Day length; also the expression level of everyindividual gene in every oat variety was the same in different varieties for each geneexpression. These results indicate that these genes are not oats photoperiod insensitiverelated genes.The RACE technique was used for amplification of unknown gene OPpdi. Weused3'RACE and5'RACE to obtain a high specificity fragment. The joinedconsensus3' and5' terminal sequence results into2669bp full-length gene sequencesof already known TDF140sequence. ORF analysis of the1983bp complete openreading frame suggested that it encodes660amino acids. These results proved thatthis fragment has a complete coding region of a gene, and therefore gene cloning andfunctional analysis can be carried out. Using the BLAST homology analysis revealedthat OPpdi gene protein is homology to PRR proteins of wheat, barley and otherplants. PRR is the protein that regulates photoperiod in plants while in wheat andbarley this protein is resulted from translation of photoperiod coding gene. The Ppdmutant is a photoperiod insensitive gene which allows early flowering in wheat andbarley in the short and long day conditions. Using. NCBI (http://www.ncbi.nlm.nih. gov/Structure/cdd/wrpsb.cgi), conserved domains of this gene was predicted. Theanalysis of the results shows that the conserved domains of the superfamily of OPpdicontain signal receiver protein (REC), response regulator (Response_reg) conserveddomain of the CCT superfamily conserved domain. Among them, response regulator(Response_reg) protein is encoded by wheat and barley photoperiod genes (The Ppd).The multiple amino acids comparison of the PRR gene from different plants (barley,wheat and goat grass, short grass, sorghum, rice) using DNAMAN, indicate that thisgene has high homology to other PRR related genes with an Identity index of70.41%.Comparison of the protein from conserved domain with that of multiple sequencesgave more certain that OPpdi gene and PRR genes identity is even higher. Thecombination of the phylogenetic tree analysis showed that this gene's protein withthose of wheat and barley PRR protein was very close. Therefore, we cloned oatphotoperiod gene OPpdi which has the same function and role with wheat and barleyphotoperiod genes (Ppd). Throughout this research, we can fundamentally affirm thatthe OPpdi gene is photoperiod insensitive genes of oats.Therefore, in this study we have been able to show that the oat VOA-8is aphotoperiod-insensitive variety. We have successfully used the information from geneexpression level to carry out the cloning of oats photoperiod insensitive gene. Wehave improved important information concerning our previous awareness of thephotoperiod insensitive genes. This will play important role in the future use ofphotoperiod insensitive oat variety for genetic improvement of oat and fordouble-year production of oat.