Function Analysis Of Transcription Factor GhMYB201

In China,cotton is an important economic crop.Cotton fiber is the most used natural fiber in the textile industry and has great application value.The fiber cell is extremely elongated single cell.There are four independent but partially overlapping periods of cotton fiber growth: initiation,elongation,secondary cell wall synthesis,and dehydration.Cotton fiber has a longer cell elongation period and secondary wall synthesis period,which is an ideal material for studying cell elongation and secondary wall synthesis.At the same time,the elongation period of the fibroblasts and the secondary wall synthesis period determine the yield and quality of mature fibers.Studying the development and regulation of cotton fibers is an important part of analyzing the mechanism of fiber production and quality formation.Transcription factors are the major regulatory proteins of eukaryotic gene transcription.They can bind to promoters,enhancers,or other regulatory regions of genes and regulate the transcription level of related genes through direct or indirect interaction with RNA polymerase II.In view of the important function of transcription factors in gene regulation,transcription factors have always been the focus of molecular biology research.The analysis of the function and mechanism of transcription factors in the development of cotton fibers is a basic content,and it is also the important basis and target for molecular regulation and improvement of traits.Based on previous reports and previous VIGS experiments,it was found that the transcription factor GhMYB201 may be involved in the regulation of cotton fiber elongation mechanism.Therefore,the goal of this study is to use the transcriptome data from different tissues of cotton to identify specific or dominantly expressed transcription factor genes during cotton fiber development,and to identify the role of transcription factor GhMYB201 in cotton fiber development through bioinformatics analysis,expression analysis and VIGS analysis.And lay the foundation for further analysis of gene regulation network and its transgene regulation during cotton fiber development in the final.The main results are as follows: 1.Functional positioning of GhMYB201According to the previous transcriptome and VIGS data,a transcription factor gene GhMYB201 that was highly expressed during cotton fiber development was obtained.To verify whether it is a transcription factor,mature tobacco leaves were transiently expressed by a specific vector constructed,and fluorescence observation was performed two days later.According to the subcellular localization map at 800 times magnification of the confocal microscope,it was found that the GhMYB201 gene can be observed in the nucleus from the subcellular localization map,and it is initially determined that the GhMYB201 gene should be a transcription factor.At the same time,the GhMYB201 homologous gene sequence alignment analysis revealed that it contains two MYB-type structural regions in common R2-MYB and R3-MYB and three functional structural segments of M1,M2 and M3.2.Bioinformatics analysis of GhMYB201 in upland cottonPhylogenetic analysis of GhMYB201-related genes revealed that GhMYB201 has 3 MYB-like homologous genes,namely MYB192,MYB201,and MYB105.Through q-PCR analysis of different homologous genes of GhMYB201 gene,it was found that the D subfamily expression level of GhMYB201 gene was the highest.Among them,the fiber is highly expressed in the fast elongation phase of the fiber,and the expression is low in other tissues and organs.At different developmental stages of ovule fibers,the expression level of Gh MYB201 gradually increased with the elongation of the fibers,reached a peak at about 10 DPA(Day Post Anthesis),and then gradually decreased.The results indicate that GhMYB201 plays an important role in the development and elongation of cotton fibers.Gh MYB201 is expressed in all tissues of cotton,with the highest fibers,followed by leaves and sepals.3.Cloning and expression analysis of GhMYB201According to the five functional segments contained in GhMYB201,the sequence containing different segments was constructed on the pGBKT7 BD vector,and the yeast two-hybrid self-activation test was performed to verify the main functional regions that function.The previous yeast two-hybrid experiments showed that R2R3 + M2,M1 + M2 + M3,and Full length had self-activation.Combined analysis showed that the M2 region may be the main region for GhMYB201 gene function.The M2 region and M2 region knock-out sequences were individually ligated to the pGBKT7 BD vector,and a yeast two-hybrid self-activation test was performed to verify the interaction region.The results showed that there was self-activation in the M2 region of the vector,but there was no self-activation in the vector without the M2 region and without the M2 region sequence,which proved that the main region in which GhMYB201 functions is the M2 region.4.Inhibition of GhMYB201 gene expression inhibits transgenic fiber elongationGhMYB201 gene is highly expressed during the rapid elongation of fibroblasts,which may be closely related to fiber elongation.A VIGS vector containing GhMYB201 and YFP was constructed to inject cotton,and the GhMYB201 gene was silenced by VIGS technology.It was found that the transcription factor gene GhMYB201,which is specifically expressed during the elongation of cotton fibers,was silenced.The corresponding GhMYB201 gene expression in cotton fibers was lower,and the corresponding cotton fiber length was shorter.It indicated that GhMYB201 gene could promote the elongation of cotton fiber,and further verified the genetic transformation of cotton by constructing different vectors.5.Changing the expression level of GhMYB201 affects the growth and development of cotton plantsThe phenotypic analysis of the knockout material found that the knockout material grew more slowly than the wild type,and the plants were shorter and the plants were shorter,flowers,petals,and sepals are smaller,stamens are reduced,stigmas are significantly higher than anthers,and the leaves are dark green in color,and fruit cannot stabilize after flowering.At the same time,the epidermis of leaves,petioles,and primary tender leaves is shorter.From the knockout materials,it was seen that the epidermal hair of the plants that had knocked out the GhMYB201 gene became shorter,and it is speculated that it may also have the same effect on cotton fibers.It is currently waiting for the boll to burst for further verification.6.GhMYB201 can affect cotton fiber elongation through response to hormone ABAABA is a natural plant hormone that affects all stages of plant development.The purpose of this experiment is to investigate whether ABA has an effect on the expression of GhMYB201 gene through ovule culture,which ultimately affects the elongation and development of cotton fibers.Through the treatment of ABA stress at different concentrations and analysis of expression levels,ABA with different concentration gradients will affect the expression level of GhMYB201 gene.Within a certain range of concentrations,an increase in ABA concentration will lead to an increase in the expression level of cotton fiber GhMYB201 and the Cotton fiber length will increase accordingly。When the ABA concentration reaches a certain value,the length of cotton fibers will decrease with the increase of ABA concentration,and excessive ABA will even inhibit the development of cotton fibers.This indicates that changes in ABA concentration will affect the expression level of Gh MYB201,and thus affect the development of cotton fibers.