Phenotypic Analysis Of Overexpression Of BdAGL6 Transgenic Arabidopsisand Overexpression Of BdFUL Transgenic Rice

Brachypodium distachyon,the new model plant of grasses,its genome is relatively small,which is merely 272 Mbp.Moreover it has collinearity with rice,maize and wheat,the genetic relationship between wheat and Brachypodium distachyon is quite close,they both belong to Pooideae.Meanwhile Brachypodium distachyon is easy to grow,its growth period only takes 3 months,it also has the characteristics including self-pollination,easily-breeding and structural similarity with rice and wheat.This report focuses on AGL6（AGAMOUS LIKE 6）,FUL1（FRUITFULL1）and FUL2（FRUITFULL2）,which among contain MADS-box family conserved domains,they are so imperative in regard to the regulation of floral development in plants.After three genes have been analysed,biological function of Brachypodium distachyon was preliminary revealed,in order to provide a huge number of gene resources into our grass grain crop genetic breeding.Our laboratory used Brachypodium distachyon cDNA as template andcloned the whole sequence of BdAGL6,BdFUL1 and BdFUL2 genes.BdAGL6 and BdFUL1 analysis showed that gene alternative splicing existed here,but there is no alternative splicing in BdFUL2 gene.BdAGL6 transcription formed 3 transcripts: they were respectively BdAGL6-1（7 amino acids more than BdAGL6-3）,BdAGL6-2（5 amino acids more than BdAGL6-3）and BdAGL6-3,followed by constructing BDO1,BDO2 and BDO3 overexpression vector,which were transformed successively into Arabidopsis thaliana;BdFUL1 gene transcription produced 2 transcripts,they were respectively BdFUL1-1 and BdFUL1-2,then constructing the BdF1O1,BdF2O2 and BdF2 O overexpression vectors so as to process rice transformation.Based on the previous work,by using hygromycin（50mg/ml）for resistance screening of transgenic Arabidopsis thaliana seeds,PCR detection and phenotypic analysis,the biological function of BdAGL6 gene was preliminary revealed,then we compared the differences about product function of three transcripts;Simultaneously preliminary analysis observation has proceeded on transgenic positive plants of overexpression BdFUL1 and BdFUL2 gene on the basis of genetic transformation in rice.The main results of this report are presented as follows:1.The T₀ generation（Agrobacterium infection Arabidopsis thaliana contemporary harvested seeds）transgenicarabidopsis seeds is used for hygromycin resistance screening,6 strains were obtained with BDO1 T1 generation seeds,9 strains were obtained with BDO2 T1 generation seeds,12 strains were obtained with BDO3 the T1 generation seeds;further,T1 generation transgenic seeds were screened and identified,finally obtained 43 BDO1 strains,84 BDO2 strains and 82 BDO3 strains T2 generation transgenic positive plants respectively.2.Based on enough amount of transgenic Arabidopsis thaliana plants,this research recorded and statistically analyzed the flowering time and the number of branches,meanwhile plants leaf and Arabidopsis thaliana flowers were observed and photographed,The expression of 3 transcripts were analyzed so as to compare the similarities and differences between the function of the three transcripts products.Phenotypic analysis showed that there were some phenotypes among these strains: flowering time change,plant variation,leaf type specific and abnormal flower development.The results of qRT-PCR showed that there was no significant disparity in the expression level of the three transcripts.3.Among the BdFUL1 overexpression lines,the transgenic rice with 2 F1O1 and 2 F1O2 are identified,their expression level were analyzed as well,simultaneously the phenotypes were preliminarily observed.In transgenic rice plants,the level of expression of FUL1-1 and FUL1-2 was significantly higher than in wild type rice plants,but the expression of FUL1-2 was stronger.Preliminary observation and analysis of F1O2 transgenic rice plants showed that the phenomenon from plant containing short,small,early-flowering and low-fertility,however the disparities of F1O1 transgenic lines and wild type was not significant.Therefore it infered that BdFUL1-1 probably has no this function,and BdFUL1-2 is likely to be the main reason result in decrease of fertility and early flowering in rice.Of course,we need more enough evidence to prove it.4.Among the BdFUL2 overexpression lines,10 strains of F2 O transgenic rice plants has been identified and acquired.Initial observation and analysis showed that in F2 O rice plants,rice plants of BdFUL2 high expression level performed a dwarf and early flowering phenomenon.Because the number of transgenic plants is limited,which need to obtain much more transgenic plants,in order to count the flowering time,so that it can provide sufficient evidence as to the phenomenon of early flowering.In conclusion,this paper mainly studied the biological function of BdAGL6、BdFUL1 and BdFUL2 genes in Brachypodium distachyon by genetic methods and molecular biology.The analysis results presented that the BdAGL6 gene has three functional differences transcripts,but the specific molecular mechanism need more evidence;BdFUL1 and BdFUL2 gene has a regulatory role in the control of floral organs and seeds development,we need to further study in flowering time.These results laid a foundation for revealing the molecular mechanism of flower development in Brachypodium Distachyon,it had certain theoretical significance.