Effects Of MiRNA160 On Leaf Water Loss And Fruit Development In Tomato By SIARF10

Auxin response factor (ARFs) is a kind of transcription factor, which participated in many physiological and biochemical processes of plants by regulating the expression of downstream genes. In addition, the transcription level of ARF is regulated by miRNA, and it is not clear whether the complex regulation proceess is involved in the growth and development of tomato. Based on previous research and research group, SImiRNA160 could regulate the target genes SIARF10, SIARF16 and SIARF17, SImiRNA167 could control the SIARF6 and SIARF8. In order to further clarify the regulation of miRNA160 and miRNA167 on the target gene ARFs are involved in tomato growth and development process. In this study, we used the 35S:mSIARF10-6 (resistant ro miR160 version) transgenic materials, it was found that SIARF10 regulated by miR160 affected tomato leaf water loss rate and clearly ARF10 by acting directly affect stomatal development and the aquaporin expression to accelerate leaf hydraulic conductace. Secondly, using dgt and gib-3 mutants, we study the ARFs/miRNAs in tomato fruit developmental effects of early fruit development. It was confirmed that ARFs regulated by miR160 and miR167 regulate cell division and expansion process of the pericarp mediated by IAA and GA. Secondly, using In addition, the transgenic plants and T1 generations of miR160 overexpression were obtained. It was found that overexpression of miR160 could have an important role on the tomato leaf development. This study laid a foundation for further study on the biological function of ARF/miRNA and the mechanism of auxin on the mechanism of tomato. The main results are as follows:1. Using mSIARF10 overexpression transgenic tomato (resistant miR160 version), through phenotypic analysis it was found that the leaves were significantly narrower (length/width), stomatal size became larger and stomatal density became smaller. Usually narrow blade has a smaller water loss rate, however, compared with WT,35S:mSIARF10-6 showed a greater rate of leaf water loss. During the dehydration assay,35S:mSIARF10-6 accumulated a higher ABA content, and the sensitivity to ABA was significantly higher than that of the WT. But further analysis revealed that 35S:mSIARF10-6 actual leaf water loss was not consistent with the calculated water loss relying on stoma. This demonstrated that there are other ways of 35S:mSIARF10-6 leaf water loss. Further use of the aquaporins (AQPs) inhibitor HgCl2 treatment confirmed that 35S:mSIARF10-6 has a higher AQP activity, with a higher hydraulic conductivity. These results show that the increase of 35S:mSIARF10-6 leaf water loss is the result of the interaction of stomatal and AQPs.2.Screening by RNA sequencing revealed that five AQP family genes, seven ABA biosynthesis/signal genes and three stomatal development realted genes were significantly altered in 35S:mSIARF10-6 plants, which SIABI5 with ABA dependent transcription factor activity. Promoter analysis of the up-regulated expression of AQP genes shhowed that upregulation of the promoter of AQPs contained ABRE or AuxRE promoter elements. Selecting upregulated remarkable three genes (SITIPl-1-like, SIPIP2;4 and SINIP-type like) promoter assay found that SIPIP2;4 and SINIP-type like promoter region containing AuxRE in 35S:mSIARF10-6 enhanced the GUS activity. EMSA and yeast one-hybrid assay confirmed that SIARF10 could be combined with the AuxRE prmoter element in AQP gene promoter. The transcription factor SlABI5-like which expression was siginificantly increased in 35S:mSIARF10-6. It could also be combined with the ABRE promoter element in the promoter of AQP gene by yeast one-hybrid assay. Therefore, the expression of AQPs in 35S:mSIARF10-6 may be the result of upregulation expression of ARF10 and ABI5. However, through SIARFIO silence and SIABI5 overexpression transient expression assay, the downregulation of SIARFIO can reduce the tomato leaf water loss, while overexpression of S1ABI5 before dehydration treatment 6h significantly decreased the rate of water loss. So the high water loss of 35S:mSIARF10-6 leaf is not resulted from the effect of SlARB15, but the SIARF10 directly action. This study shows that despict the SIARF10 increased ABA biosynthesis and signal response through the regulation of stomatal opening to reduce moisture loss, but SIARF10 by affecting stomatal development and aquaporins activity to accelerate water loss. The regulation of miR160 on SIARF10 has important role on maintaining the balanced of water in leaves.3. Application exogenous IAA on tomato GA-deficient (gib-3) and exogenous GA on IAA signaling pathway blocked (dgt). Morphological results showed that IAA resulted in the pericarp thickening and cell layer number increasing, while GA did not increase the number of cell layer number. RT-PCR study found that the expression levels of SIARF6, SIARF8, SIARF10 and SIARF16 were significantly downregulated in gib-3 with IAA treatment, while the expression levels of miR160 and miR167 were significantly increased. The expression levels of SIARF6, SIARF8, SIARF10 and SIARF16 were upregulated in dgt with GA application, while the expression levels of miRNAs were significantly reduced in the dgt mutant. These results imply that the expression levels of SIARF6, SIARF8, SIARFIO and SIARF16 were negatively correlated with the number of cell layers in the pericarp during early stage of tomato fruit set development. Further use of 35S:mSIARF10-6 transgenic tomato fruit pericarp was analyzed. Results revealed that mSIARF10 overexpression resulted in fewer cell layers during fruit set. Together, it suggests that GA- and IAA-mediated miRNAs and their target ARFs may be involved in the proliferation of pericarp in the early stage of tomato fruit development.4. miRNA160 overexpression vector was successfully constructed by using Gateway technique. The tomato miRNA160 mature is connected with a strong promoter of pB7WG2D,1 vector. Transgenic tomato plants were successfully obtained by optimizing the genetic transformation system of tomato. And through the PCR test, GFP fluorescence detection method successfully verified the T2 generation of transgenic tomato plants. The functional identification of T1 transgenic plants showed that over expression of miR160 has an effect on the leaf width, size and less leaf lobes; compound leaves growth promisingly vegetative organs increased growth and development of tomato leaf. It suggests the regulation of miR160 on the molecular mechanism of tomato leaves development.