Study On Callus Differentiation And MicroRNA396 Function Of Different Types Of Eucalyptus Urophylla × Eucalyptus Grandis

Micro RNAs(miRNAs)in plants are a kind of non coding small RNA molecules with regulatory function.They play an important regulatory role in plant morphological construction,growth and development,plant hormone response,biological and abiotic stress and so on.However,there are few studies on the role of miRNA396 in plant callus differentiation and adventitious bud growth.In order to preliminarily explore the differentiation of different types of callus of Eucalyptus urophylla × Eucalyptus grandis and the biological function of miRNA396 in the callus formation and differentiation of E.urophylla × E.grandis,the stem explants of DH32-29 E.urophylla × E.grandis aseptic seedlings were cultured under different conditions in this paper.After differentiating different forms of callus,their physiological and biochemical differences and the content of endogenous hormones were measured.At the same time,transcriptome sequencing was performed on the calli of different forms of E.urophylla× E.grandis,and the relative expression of GRFs and CKXs genes in the four calli were compared and analyzed.In addition,the CRISPR/Cas9 editing vector of miRNA396 was constructed to study the transformation of E.urophylla × E.grandis callus,so as to preliminarily determine the differentiation differences of different types of E.urophylla× E.grandis callus and the effect of miRNA396 on the formation and differentiation of E.urophylla × E.grandis callus.The main research contents and results are as follows.1.The stem explants of E.urophylla × E.grandis were dark cultured on the callus induction medium,and the yellow callus was differentiated after two weeks.A part of the callus continued to be dark cultured,and the callus differentiated into white after one week.Some calli were transferred to bud differentiation medium for dark culture for one week,and the calli differentiated into green.The remaining yellow calli were taken out and cultured under light.After one week,the calli differentiated into red.The determination and analysis of physiological and biochemical properties of different forms of E.urophylla × E.grandis callus showed that compared with green callus,the differences of protein content and CAT activity of other three color calli reached highly significant level,the differences of SOD activity reached significant level,the differences of POD activity reached highly significant level in red calli,but there was no significant difference in white and yellow calli,The difference of PPO activity reached highly significant level in white and yellow callus,but not in red callus.The enzyme activity of white,green and yellow calli is enhanced,which can better eliminate the reactive oxygen species produced by the external environment in plants,and can make stress response to the adverse environment in time,while the adaptability of red calli to the outside world is relatively weak.And the enrichment degree of endogenous auxin and cytokinin in different calli is quite different.There is a two-way link between enzyme system and hormone signal pathway,their interaction affects the biomass accumulation and physiological metabolism of E.urophylla × E.grandis.2.Transcriptome analysis of different forms of E.urophylla × E.grandis callus showed that compared with green callus,there were 2203 differentially expressed genes in red callus,including 764 up-regulated genes and 1439 down regulated genes;There were 2485 differentially expressed genes in white callus,of which 1493 were upregulated and 992 were down regulated;There were 2078 differentially expressed genes in yellow callus,including 977 up-regulated genes and 1101 down regulated genes.The results of heat map of differentially expressed genes showed that the four calli had significant clustering differences.The target gene GRFs of miRNA396 and cytokinin dehydrogenase related gene CKXs were screened from the differentially expressed genes,and enriched them with GO and KEGG.It is speculated that miRNA396 mainly affects the formation and differentiation of callus of E.urophylla × E.grandis by controlling the formation of cell wall and cytokinin signal transduction and response.3.RNA from four different types of calli of E.urophylla × E.grandis was extracted and reverse transcribed into c DNA.Fluorescence quantitative PCR was performed on the target gene GRFs of miRNA396 and cytokinin dehydrogenase related gene CKXs.It was found that the expression of other GRFs in red E.urophylla ×E.grandis calli was down-regulated compared with green calli,except that the expression of GRF5 was up-regulated,while the expression of GRFs in white and yellow E.urophylla × E.grandis calli was up-regulated compared with green calli,At the same time,the expression of CKXs in red callus was up-regulated,while the expression of CKXs in white and yellow callus was up-regulated,and the difference reached a significant or very significant level.It was preliminarily determined that miRNA396 could affect the formation and differentiation of callus and the expression of CKXs by regulating the level of endogenous hormones in E.urophylla × E.grandis.4.Two g RNAs were designed according to the sequences of miRNA396 A and miRNA396 B of E.urophylla × E.grandis.GRNA of miRNA was amplified with p CBC plasmid as template.The amplified band size was in line with expectations.The successfully digested plasmid p HEE401E-35S-RUBY was recombined with g RNA of miRNA to form a new vector plasmid p HEE401E-35S-RUBY-gmiRNA396.After transformed the new vector into E.coli competent cells,the PCR results of bacterial solution and plasmid were in line with the expectation,and the base sequencing results were in line with the expected sequence,indicating that the construction of p HEE401E-35S-RUBY-gmiRNA396 vector was successful.Agrobacterium tumefaciens competent cells were transformed with the recombinant vector,and the PCR results of bacterial solution were in line with the expectation,indicating that the engineering strain was successfully constructed.The engineering bacteria transformed the callus of E.urophylla × E.grandis,and the transformants were screened for RUBY gene red phenotype and molecular identification.The expression of RUBY gene was the highest in the red positive transformation,,indicating that the callus transformation of E.urophylla × E.grandis was successful.In the existing sample testing,the successful editing of miRNA396 in E.urophylla × E.grandis has not been found,and the sampling amount needs to be further increased for identification.In conclusion,miRNA396 mainly affects the formation and differentiation of E.urophylla × E.grandis callus by regulating hormone signal transduction,hormone response and cell wall lignin biosynthesis..This study provides a scientific basis for the establishment of efficient regeneration and genetic transformation system of E.urophylla×E.grandis.