| In the cultivation process of facility tomato of winter and spring,malformed fruit is easy to produce,which seriously affects the quality and economic benefits of tomato.Therefore,the prevention and control of malformed fruit is an important issue that needs to be solved urgently.The study showed that the locule number of tomato was positively correlated with the occurrence of malformed fruit,that is the more locule number,the more likely it was to produce malformed fruit.Genetic studies have found two important locus that control the locule number of tomato: the fas(fasciated)locus and the lc(locule number)locus.Among them,the action value of the fas locus is as high as 37%.Different researchers had come to different conclusions about the function of these two genes,some people think that YABBY2 b control the tomato ovary locule formation,while others think CLV3 control tomato ovary locule formation,is not yet out,more failed to clear the mechanism of action of fas locus control tomato locule number,thus further parse gene of fas loci controlling tomato locule number,that is of great significance for developed the key technology of prevention of malformed fruit.In this study,mutants of the candidate genes at the fas locus-SlCLV3 and SlYABBY2 b,were constructed to analyze the changes in the number of flower organs and the number of fruit locules in homozygous mutants,and to verify the candidate gene at the fas locus regulating the locule formation of tomato ovary.Again through the analysis of fas locus candidate gene of mutant transcriptome data and expression levels of differentially expressed genes in genetic background materials at different fas locus,screen the downstream candidate genes of the fas locus candidate genes,and through the expression pattern analysis and evolutionary tree analysis preliminary discussion the important role of downstream candidate genes in the process of tomato flower development,and build the transgenic plants of downstream candidate gene,in order to further verify the gene function and the preliminary discussion the action mechanism of SlCLV3 gene through downstream candidate gene regulation of tomato locule number provides the basis material.The main results are as follows:1.Using CRISPR/Cas9 technique,the homozygous mutants of tomato fas locus candidate genes-SlCLV3 and SlYABBY2 b were successfully obtained.The number changes of flower organs and fruit locule of clv3 and yabby2 b homozygous mutants were analyzed,and it was found that the number of sepals,petals,stamens and carpels of clv3 mutants was significantly increased compared with that of wild type,fruit locule number and fruit weight were also significantly increased.However,there was no significant change in the number of flower organs and fruit locule in yabby2 b mutants,indicating that SlCLV3 gene is the major gene that regulates the number of flower organs and fruit locule,and it is a candidate gene of fas locus that regulates the formation of ovary locule in tomato.It was also found that clv3 mutants had many inflorescence branches,stamen abortion and other phenotypes.In conclusion,SlCLV3 gene is a candidate gene of fas locus that regulates locule number in tomato,and also plays an important role in the development of inflorescence branches and stamens.In addition,it was confirmed that SlYABBY2 b gene did not regulate the locule number of tomato,but played an important role in regulating plant height.2.Two candidate genes as downstream of SlCLV3 gene: SlGIF1 and SlLET6 were selected by analyzing the transcriptomics of flower buds at the early carpel formation of tomato clv3 mutants and gene expression levels in different fas locus genetic background materials.Expression level of SlGIF1 and SlLET6 genes in flower differentiation of flower buds increased significantly after SlCLV3 gene mutation,and expression in fas locus mutant background material was also significantly higher than that of fas loci of wild type background material,the results suggest that SlCLV3 gene may regulate expression of SlGIF1 and SlLET6.3.Through analysis of tomato SlGIF1 and SlLET6 gene expression pattern,the results showed that SlGIF1 gene expresses in leaf,shoot apical meristem,the flower/inflorescence meristem,young flower bud,open flower,fruit and roots,and highly expresses in the shoot apical meristem,flower/inflorescence meristem,young flower bud.SlLET6 also expressed in leaves,shoot apical meristem,flower/inflorescence meristem,young flower buds,open flowers,fruits and roots,and highly expressed in shoot apical meristem,flower/inflorescence meristem and young flower buds.These results suggest that SlGIF1 and SlLET6 genes may play important roles in flower development.4.tomato SlGIF1 gene encodes a calcium response trans-activator,which contains the SSXT domain and is a member of the SSXT proteins family.SlLET6 encodes 1 Knotted-like domain protein LET6,which contains the KNOX domain and is a member of the KNOX protein family.Phylogenetic tree analysis showed that there were SSXT family members in Arabidopsis,tomato,rice,wheat and apple,and tomato SlGIF1 was highly homologous to Arabidopsis At AN3.KNOX family members were found in Arabidopsis,tomato,soybean and apple.The functions of members of this gene family are mainly related to the regulation of meristematic tissue and the development of leaf and flower.Tomato SlLET6 is highly homologous to Arabidopsis At STM.5.CRISPR/Cas9 vectors and overexpression vectors of tomato SlGIF1 and SlLET6 genes were successfully constructed.gif1 mutant plants have been successfully constructed,including 3 homozygous mutant plants,4 chimeras/chimeras plants,7 plants of SlLET6 gene overexpressed have been successfully constructed with SlLET6 gene expression level increased by 2 to 7 times.The successful construction of tomato transgenic plants of SlGIF1 and SlLET6 genes provided basic materials for further verification of the functions of SlGIF1 and SlLET6 genes and preliminary discussion on the mechanism of SlCLV3 gene regulating the locule number of tomato through SlGIF1 and SlLET6 genes. |
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