Effects Of CiKN6 On Leaf Shape And Photosynthesis Of Citrus And Creation Of Transgenic Materials

The diversity of plant leaves has always been the focus of developmental biology research.Leaf shape is closely related to photosynthetic efficiency and transpiration efficiency of plants and can affect yield.In almost all the citrus trees with single leaf character,the unique character of three compound leaves can be used as a marker character for crossbreeding.If the key gene regulating the development of citrus leaf shape can be found out,the gene could be used as a molecular marker for the selection of citrus hybrid progeny,which would facilitate the early selection of breeding.In this study,Citrus limon and Citrus trifoliata were used as research materials.There are 9 KNOTTED1-like homeobox(KNOX)family genes involved in leaf shape development were screened out by RNA-seq analysis,It was found that Ci KN6 was closely related to leaf shape development,so Ci KN6 was selected for further study.and the mechanism of Ci KN6 involved in citrus leaf shape development was also discussed.The main results are as follows:1.Transcriptome sequencing(RNA-seq)was used to analyze key genes in leaf shape development.The leaf development of Citrus limon and Citrus trifoliata was divided into four stages: the initial stage of lobed leaf formation(S1);fully formed stage of lobed leaves(S2);middle leaf development(S3)and mature leaves(S4).Transcriptomic sequencing was performed on Citrus limon and Citrus trifoliata leaves at S1 stage;Citrus limon leaves at S1 stage and Citrus trifoliata leaves at S2 stage.The differentially expressed genes(DEGs)of the two samples were compared,and 604 candidate genes related to lobed leaf formation were screened out,including 9 KNOX homologous family genes.Compared the expression patterns of Ci KN6 in citrus with different leaf shapes,it was found that the expression level of Ci KN6 in Citrus trifoliata was higher than that in Citrus limon at four leaf development stages,especially at lobed leaf formation stage.When Ci KN6 was transformed into Arabidopsis,the overexpressed Arabidopsis produces leaf splitting,suggesting that the expression of Ci KN6 could affect the leaf shape development of plants.When the 35S:Ci KN6 overexpression vector was stably transformed into Citrus limon,the leaf edge of the transgenic Citrus limon appeared serration,and the anatomical structure of the Citrus limon leaf became more similar to that of Citrus trifoliata.2.Bioinformatics analysis of 35S:Ci KN6 Citrus limon transcriptome showed that DEGs was enriched in cytoskeleton construction,stress response,hormone metabolism,cell cycle and other biological processes.The results showed that the auxin content of 35S:Ci KN6 was significantly higher than that of the wild-type.q RT-PCR analysis of auxin synthesis and auxin transport genes in the margins and middle of wild-type and 35S:Ci KN6 Citrus limon leaves.The results showed significant differences in the expression of these genes in the margins and middle of the leaves.The distribution of auxin in the leaves of 35S:Ci KN6 Citrus limon was visualized by DR5.Compared with the wild-type Citrus limon,the auxin signal in the leaves of 35S:Ci KN6 Citrus limon was significantly distributed at the leaf margin and leaf fold.These results suggest that the development of 35S:Ci KN6 Citrus limon leaf shape is regulated by auxin.In conclusion,the overexpression of Ci KN6 in Arabidopsis and Citrus limon can make the leaves appeared the phenotype of deep splitting or serration,and Ci KN6 has different expression patterns in Citrus limon and Citrus trifoliata,suggesting that Ci KN6 is involved in the regulation process of citrus leaf shape development.Through a series of experiments,including transcriptome sequencing analysis,hormone determination,auxin related gene q RT-PCR and auxin localization of Ci KN6 overexpressed Citrus limon,we can conclude that Ci KN6 is involved in auxin mediated development pathway of citrus leaf shape.The specific role of Ci KN6 in this regulatory network needs to be further explored.In addition,the photosynthetic properties of 35S:Ci KN6 lemon leaves were investigated,and it was found that the expression of Ci KN6 affected the photosynthetic rate of the plant.