Identification Of Differentially Expressed MiRNA Of Elongated Phase And Cell Wall Thickening And The Wall Melting Phase In Ramie

As China’s important ramie textile material, the ramie fiber with moisture absorption heatfaster, breathe freely, not personal, antibacterial, tinggua aesthetic advantages and so on, notonly have“The king of natural fibers”, but also is the best quality, the intensity of hemp thebiggest fiber. But most species of ramie fibre fineness is thick, rigidity, spinning withproducing more than 60 is difficult, and the current domestic ramie varieties in a singlecharacter only stay improvement, not yet in a single fibre fineness and fiber to achieve theoptimal output of varieties. To solve these problems that must seek the molecular mechanismof ramie fibre development from the fundamental solution to influence the bottleneck problemof the fiber count.MiRNA is a kind of exist widely in eukaryotic cells of the kind of endogenous the codingRNA molecules, about the size of 22 nt or so, in plant growth and development plays animportant role in the process. At present miRNA regulation in arabidopsis, cotton fiberdevelopment on study more, with cotton fiber miRNA development of the research has alsomade a lot of progress, not only an important development of cotton fiber regulation functionmiRNA, about the development of cotton fiber related miRNA also has been declared a patent.As you can see, is the fiber plant miRNA growth and differentiation of fine regulaton.In this study, plant miRNA probes ( chip containing probe 1959 ) is used to tetectedmiRNA expression in elongating period of fiber and cell wall thickening of the end walldissolving period of limonene One ramie two developmental stages of , Screening 7miRNAs，which differentially expressed in multiples of 2, miR1450 miR156h , miR172b1 ,miR159a , miR172a , miR172b2 , miR482, deteted the four developmental stages of the ramiefiber and flowers , leaves , roots , stems and other organs in the expression studiesbysemi-quantitative RT-PCR . And real-time quantitative PCR is used to deteced the expressionof differences of miR482、miR172a and miR172b2 in the four developmental stages of the ramie fiber for further verification . The results are as follows :(1) using CTAB method, we successfully extracted total RNA from the ramie fiber atdifferent developmental stages and flowers, leaves, roots and underground stems and othertissues , and 51 miRNAs wered found differentially expressed form elongating period of fiberto cell wall thickening of the end wall dissolving period in microarray experiments, 9 ofthemdifferences in multiples of more than 2 , the highest differences in multiples is 6.857 .These miRNAs belonging to 11miRNA families .(2) Semi-quantitative experiments , in addition to the outside of 172b2 , the remaining sixmiRNAs expressen in various tissue, majority of the highest expression leavel in all tissues ismeristem period of fiber( miR156h , miR172b1 , miR159a , miR172a , miR172b2 ) , miR1450expressed the highes in fiber maturity period of fiber, and miR482 is the highest expressionlevel in roots . The differential expression trend in Semi-quantitative RT-PCR experiment ofelongating period of fiber and cell wall thickening of the end wall dissolving period is same tomicroarray experiments, except for miR172b1 and miR172b2. difference in multiples ofmiR1450 is 2.669 , slightly higher than the microarray experiments, differences in multiples of1.990 , differences in multiples of the remaining miRNA is less than the microarrayexperiments’s. The smallest differences in multiples is miR159a and miR172a, the differencein multiples are only 1.048 and 1.229, while the difference multiples in the microarrayexperiments are of 2.109 and 2.216 .(3) From the result of Real-time quantitative PCR, we can see differentially expressedquantities of miR482 , miR172a , miR172b2 form elongating period of fiber to cell wallthickening of the end wall dissolving period are 6.98,11.00 and 96.00 times , in microarrayexperiments , differences in multiples are 1.841, 2.216 and 3.492 , the difference multiples areobviously higher than the results of microarray experiment .(4) From the result of Quantitative real-time PCR experiment, we found that in meristemperiod of fiber and maturity period of fiber, miR172b2 expressed both in the 50bp and 100bp ,unlike the single expression of other miRNAs .In this study, we stretched and identified differentially expressed miRNAs fromelongating period of fiber to cell wall thickening of the end wall dissolving period to find theregulatory role of miRNAs from the fiber development. This study is the foundation of thebasic preparatory works of the research of the function these genes and the regulationmechanism of target genes, also prepare for further study of fiber development mechanismfunction.