Cloning And Functional Analysis Of MiR396a Precursor Sequence Of Chrysanthemum Indicum

With the growing needs of people for a better life,the landscape gardening industry has ushered in more opportunities and challenges in the cause of improving the human living environment and building a beautiful China,and at the same time,the development of garden plants has also ushered in a broader prospect.Chrysanthemum morifolium is a traditional famous flower in China,with a wide variety of species and great industrial value,but its quality upgrade and application are limited by abiotic stresses.However,due to the complex genomes of commercial varieties of Chrysanthemum and the small number of resistance genes,molecular breeding of Chrysanthemum resistance has encountered a bottleneck.Chrysanthemum indicum,a diploid species of the genus Chrysanthemum,has strong resistance and is an important resource for resistance gene mining and genetic improvement of ornamental Chrysanthemums in the genus Chrysanthemum.miRNA is a class of endogenous non-coding small molecule RNA that plays an important function in the regulation of plant resistance gene expression,but its function in response to abiotic stress in Chrysanthemum indicum has not been reported.However,its function in response to abiotic stresses in Chrysanthemum indicum has not been reported.However,its function in response to abiotic stresses has not been reported yet.In order to further verify the function of cin-miR396a,the overexpression vector of cin-MIR396a was constructed and successfully transformed into Chrysanthemum indicum,and the function of the transgenic Chrysanthemum indicum was verified using fluorescence.Functional verification was carried out,and the relationship between the action of cin-miR396a and the target genes was also verified using fluorescence quantitative PCR and transient transformation techniques,and the results were as follows:1.Real-time quantitative PCR results showed that cin-miR396a was affected by salt,drought and heat stresses.The miR396 precursor sequence of 203 bp in length and the upstream promoter region of 2546 bp in length were searched and cloned from C.indicum by PCR,and the miR396 precursor sequence was named as cin-MIR396a.By bioinformatics analysis,it was found that the cin-miR396a precursor sequence could form a stable stem and and the mature miR396a base sequences are consistent with the sequences of most other miR396 family members.The promoter region contains abscisic acid response element,anaerobically induced response element,etc.and MYB binding site,MYC binding site,etc.2.The cin-MIR396a overexpression vector was successfully constructed,and the C.indicum was transformed using agrobacterium-mediated leaf disc method to obtain cin-MIR396a overexpression transgenic strains,and five positive strains were obtained by rooting screening and post-transcriptional level detection.The results of real-time quantitative PCR showed that the expression of cin-MIR396a was significantly higher in the overexpression positive strains compared with the control strains,indicating that the cin-MIR396a gene had been integrated into the genome of C.indicum.3.Combined with the degradome data of the group,the expression level of miR396a target gene in C.indicum transgenic to cin-MIR396a gene was detected,and the target gene overexpression vector containing GFP was constructed.The relationship between cin-miR396a and target genes was demonstrated by three methods:degradome sequencing,fluorescence quantification and transient transformation of tobacco,and the results showed that cin-miR396a regulates the expression of CiGRF1 and CiGRF5 in C.indicum.4.The seedlings of C.indicum overexpressing cin-MIR396a were treated with salt,drought and heat stresses,and the fluorescence quantification of cin-miR396a and its target genes after the stresses was performed.The relative leaf water content decreased,the relative leaf conductivity became larger,the leaf MDA content increased,the leaf free proline content decreased,and the total chlorophyll content did not differ significantly from that of WT after stress in transgenic C.indicum with cin-MIR396a gene compared with the control WT.Na+content in leaves and roots of transgenic C.indicum increased significantly after salt stress,K+content did not change much compared with WT,and Na+/K+ ratio increased after stress in transgenic plants.The expression of CiGRFl and CiGRF5,the target genes of cin-miR396a,was changed after salt,drought and high temperature stresses,and C.indicum may respond to abiotic stresses through the miR396-GRF pathway.