Leaf Developmental Regulation Of MiRNA319 Gene In Poplar

The leaves are the main organs of plant photosynthesis and transpiration.They can not only convert light energy into chemical energy,but also reduce the temperature of plants by gas exchange,and provide a stable growth environment for the normal growth and development of plants.miRNA319 a and target genes are key regulators of plant development in many species,such as leaf morphology,petal and pollen grain fullness,and plant cold tolerance.In order to study the effect of miRNA319 a on the leaf development of poplar,agrobacterium-mediated transformation of miRNA319 a gene was established using 84 K poplar leaves as material.A total of 14 transgenic plants were obtained.By identifying the expression of miRNA319 a in transgenic plants,investigating the leaf traits,seedling growth traits,leaf lignin content,chlorophyll content and photosynthetic rate of transgenic plants;analyzing transgenic data to construct a regulatory network of miRNA319 a during leaf development,the main results are as follows.(1)In order to identify the overexpression of miRNA319 a in poplar,the miRNA319 a overexpressing plants and wild-type leaves were used as materials in May,and total RNA was extracted from leaves by standard CTAB method.miRNA319 a overexpression was identified by qRT-PCR,and the expression levels of the genetically modified plants were higher than those of the wild type,and the transgenic lines with the highest expression of miRNA319a-1,miRNA319a-2,miRNA319a-7,miRNA319a-8 and miRNA319a-13 were found,among them,the expression level of miRNA319a-7 was almost five times higher than that of wild type.(2)On the phenotype,miRNA319 a overexpressing plants showed obvious zigzag and leaf curvature from the seedling stage to the stocking stages.The plant height and ground diameter of 14 overexpressing plants in seedling stage were lower than wild type,and the ground diameter of 14 overexpressing plants in stocking stages was larger than that in wild type;the plant height of overexpressing plants in stocking stages was still smaller than wild type.It indicated that the plant height of miRNA319 a overexpressing plants grew slowly,while the lateral thickening growth was significantly better than wild type.(3)To identify the effects of miRNA319 a overexpression on chlorophyll content and photosynthetic rate of poplar leaves,the study compared the content of chlorophyll and the photosynthesis of 14 overexpressing lines and 3 wild type,respectively,and found that both the content of chlorophyll and the photosynthetic rate of miRNA319 a overexpresses poplar were smaller than the wild type.(4)In order to study the effect of miRNA319 a overexpression in poplar on vascular cells and vascular bundles and sheath cells in the main veins of the leaves,we made slices of miRNA319 a overexpressing plants and wild type of main vein sections on the sixth section.We found that the number of cell layers in the vascular bundle and vascular bundle sheath in the main vein on the same number of nodes increased with respect to the wild type,and was more easily stained red by the pyrogallol staining solution during the same staining time.This result indicated that the cells of miRNA319 overexpressing the main veins of the plants were pre-lignified;it was also found that the vascular cells of the transgenic plants were stained dark red,while the cells of the vascular bundles were stained reddish,indicating that the composition of vascular bundle cells and vascular bundle sheath cells of lignin are different.(5)To analyze the differential genes in the leaves of miRNA319 a overexpressing plants,the transcriptome data analysis was performed.The RNA-seq data was aligned with the P.simonii genome using HiSAT2,and then the FPKM value of the annotated gene was calculated using Cufflink.41123 differentially expressed genes were screened,among which 1198 genes were significant differential genes.Through analysis of GO enrichment and protein domain,it was found that overexpression of miRNA319 in poplar mainly affects photosynthesis and some protein of photosystem II.(6)In order to establish a regulatory network that affects the development of poplar leaves by miRNA319 a,all differential genes were further screened.The top-down GGM regulatory network was used to discover that miRNA319 a directly caused changes in poplar phenotypes through MEE35/TCP4,TCP2 and TCP2-1.These three genes are approximately complementary to miRNA319 a in the sequence and belong to differentially expressed genes that are significantly down-regulated in response to overexpression of miRNA319 a.(7)In order to clarify the function of miRNA319 a target gene,93 target genes were screened and phylogenetic trees were constructed with TCPs genes related to leaf growth and development in rice and Arabidopsis.A rootless phylogenetic tree of poplar,rice and Arabidopsis was made using MEGA 7.0.42 TCPs were clustered in the first clade along with AtTCP3,AtTCP4,AtTCP10 and OsaPCF1,22 in the second clade along with AtTCP2,and 29 in the third clade along with AtTCP24 and OsTCP8.For the three targets genes of miRNA319 a,MEE35/TCP4 presented in the first clade while TCP2 and TCP2-1 appeared in the second clade.