| Soybean(Glycine Max(L.)Merr.)is one of the most important food and oil crops originated in China.Thus,its high-yield breeding is always the aim in the breeding process.The architecture of the plants is an important factor affecting crop yield.To explore the key regulators which related to the plant architecture and their molecular mechanism are very important.The leaf shape is one of the important part in the plant architecture,also act as the primary source of photoassimilate and the foundation of the "source,sink and flow".As the foundation of the "sink",flower is the direct organ for seeds obtain,and has a close relationship with yield breeding.In addition,the structure and fertility characteristics of floral organs are all the important characters for utilization of crop heterosis.Thus,the studies of the morphogenesis and the functions of both leaf and flower are very important.To date,there were many advances for the leaf shape and lots factors have been identified.For flower development,there also has the "ABC" model and the extended "ABCDE"model which can describe this process brilliantly.However,the researchers found that there were phenomena cannot be explained still,and the connections between the leaf and flower development are unclear.In our study,we identified a new mutant display chicken toe-like leaf and petalody flower named ctp in soybean.Compared with its wild-type,the mutant has impaired leaf and flower development and sterility in both male and female.Inheritance analysis showed that the locus charged for the phenotypes was a single Mendelian gene.Map-based cloning was used for isolated the gene which function was studied in this dissertation.The main results are as follows:1)Phenotypic analysis showed that there is not significant difference of mature plant height and the leaflet length between ctp mutant and its wild type.In addition,the stem diameter of the mutant is smaller,while its branch number is increased.However,the leaf width,leaf shape index,leaf area of ctp mutant decrease dramatically.The phenotype variation of mutant appears at an early development stage which can be identified through gross observation at the fifth day after seeding.Scanning electron microscope(SEM)analysis showed that the mutant epidermal cells were uniformly larger but lost the natural regularity compared with the wild-type in both the adaxial and adaxial leaf sides.Paraffin section analysis indicated that the mutant leaflet were significantly thicker than the wild type,while the arrangement of the palisade/spongy tissue cells also lost their natural regularity and arranged sparsely.In addition to the chicken toe-like leaf,the mutant floral organs also have dramatic petalody variation.In detail,the petal numbers are increased,and the morphological characters and position of the standard,wing,and keel petals all become weakened and disordered.In the third whorl of the flower,the stamens number was decreased and showing obvious petalody,and the anther was impaired severely,even with no anther.Taken together,these severe defects in leaf and flower morphogenesis of the ctp mutant suggested that this mutant is a new mutant with abnormal leaf and flower and sterility simultaneously.2)On the basis of the preliminary mapping,to isolate the gene,map-based cloning approach was performed using the F2 and the derived segregated populations F2:3.2200 recessive plants were used and the locus was finally mapped to 37 kb region of the A1 linkage group,and there are three ORFs(Open Reading Frame)predicted.Then these three predicted genes were sequenced.Among them,only one gene differed in sequence between the wild-type and the mutant,and the mutant harbored a 33 bp deletion in its first exon but with no frameshift resulting in 11 amino acids deletion of the CTP protein.Bioinformatics analysis suggested that the CTP gene coded a protein without known functions.To validation of CTP gene function,both forward and reverse genetic means were used.First,co-segregation analyses were performed between different background materials and F10 residual heterozygous population by an InDel marker designed based on the deletion of the gene.Then the Tobacco Rattle Virus induced gene silencing(TRV-VIGS)was also used for validation the homologous gene of CTP in Nicotiana.3)In this study,the dramatically alternative splicing(AS)pattern were also found,and T-A cloning and monoclonal sequencing experiments were performed to test the transcripts.At the premise of the same numbers of the clones,there are ten AS patterns in the wild-type,and only six were obtained in the mutant,and the transcripts are not all the same between wild-and mutant types.However,there are two patterns which with the highest abundance were presented in both wild-and mutant types.Such results showed that the AS maybe involved in the gene function.4)The real-time quantitative PCR detection showed that CTP could universally expressed in various tissues during development stages,including SAM(shoot apical meristem),axillary bud,young root,stem leaf,flower and young pod.Because of the impaired phenotypes of the mutant were displayed obviously in leaf and flower,so the relative expression levels of the gene in the wild and the mutant type were compared.The relative expression level of the gene were decreased in both leaf and flower tissues of the ctp mutant,and more significantly in the flower.This result indicated that the dramatically expression variation of the CTP gene maybe a main reason caused the seriously impaired of leaf and flower phenotypes.5)Phylogenetic analysis showed that the CTP proteins are conserved in moss,ferns,gymnosperms and angiosperms which belonged to higher plants and the similarity in dicots achieved 60%.Compared with the land plants,typical feature of algae is lacking of root,stem and leaf tissues and the predominant expression level of CTP is in root and leaf.The results also showed that the first conserved proline amino acids of CTP which was deleted in ctp is absent in gymnosperms whereas presents in the angiosperms.Comparing with the angiosperms flowers,the gymnosperms have no fruit structure,ovule exposed and no ovary.It indicates that the conserved proline may play a key role in the angiosperms floral organ development.To sum up,the CTP act as one of the key regulator involved in lateral organ development may take an important role during the emergence and evolution of the leaf and flower in the angiosperms.6)Bioinformatics analysis didn't obtain the localization signal and the conserved motif of the CTP protein.To determine the subcellular localization of CTP,two means in Arabidopsis protoplasts and Nicotiana benthamiana leaf mesophyll cell were both used.The results showed that the CTP protein localized in nucleus and cell membrane.These results suggested that the CTP protein may act as a normal transcriptional factor.To validate it,the dual luciferase reporter(DLR)assay system was used to test the transcriptional activity.The results showed that CTP has medium transcriptional activation ability,and the activation activity of ctp protein was reduced to some extend compared with CTP.7)Based on the dramatic mutant phenotypes of ctp leaf and flower,the relative expression levels of many key genes involved in leaf and flower development were compared between the wild and mutant type.The auxin-related genes,KNOX genes,WOX genes,YAB genes,KAN1 and AS1/2 were tested in both leaf and flower tissues.Compared with the wild-type,the expression of the YUC2 and TAA1 which involved in auxin biosynthesis were both down-regulated.The ABCB1 and PIN1 which act as the transporter and the ARF6(AUXIN RESPONSE FACTOR 6)of the auxin were up-regulated when the ABP1(AUXIN BINDING PROTEIN 1)was down-regulated.These results suggested that the auxin signaling pathway was influenced by the ctp mutation.Furthermore,the expression levels of YAB4 were down-regulated significantly in ctp leaf,and the KNOX genes were significantly up-regulated.It suggested that the CTP was upstream of these genes.Based on the results of this study and combined with the previous studies,the CTP regulation of the WOX1 and KNOX were via the YAB.The "ABC" model was expanded into "ABCDE" in floral organs development,qRT-PCR results showed that compared with the wild-type,all of the A/B/C/D/E-class genes relative expression were down-regulated in the ctp mutant,especially the B-class gene PI which was involved in the petals and stamens development simultaneously.These results indicated that CTP is required for normal expression of the A/B/C/D/E-class genes and may act as a transcript factor upstream of them.At the same time,compared with the wild-type,the relative expression of the STM,AS1/2 and YAB were all up-regulated in ctp mutant flower.It indicated that the CTP has a negative regulation of these genes in floral organs development. |
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