The Mechanism Of MiRNA In Mepiquat Chloride Inhibiting Internode Elongation Of Cotton

Cotton（Gossypium hirsutum L.）is one of the most important economic crops,witch is an important source of edible oil and natural fibers.China is an important cotton producer,with a cotton planting area of3,392,000 hectares（50.88 million mu）.As a major agricultural product related to national livelihoods,increasing cotton production is an important issue in agricultural production.Mepiquat chloride（MC）chemical control technology is currently the most widely used chemical topping method in the process of cotton planting.It can effectively inhibit the excessive vegetative growth of cotton and break the top advantage,thereby shortening the internodes,reducing plant height,and compact planting.MC treatment can effectively regulate the energy transfer from vegetative growth to reproductive growth of cotton seedlings,promote the development of cotton fiber and seed,and improve economic benefits.As a class of important post-transcriptional gene expression regulators,micro RNAs（miRNAs）play important regulatory roles in plant growth and development.However,the regulatory mechanism of miRNAs in MC-induced cotton growth inhibition is still unclear.At present,the research on cotton miRNAs mainly focuses on the regulation mechanism of fiber initiation and elongation,anther development,male sterility,somatic embryogenesis,stress response and disease resistance,ect.Although there have been reported miRNAs involved in plant height control crops,but so far,plant height related miRNAs studies rarely reported.In order to explore the role of miRNA in MC regulation of plant high,we used Upland cotton cotton（Gossypium hirsutum cv.CCRI49）as experimental material,and collected the second internode（from bottom to top）at 0h,48 h,72h and 96 h after 80 mg/L MC spraying at the third leaf stage of cotton seedlings to construct and sequence small RNA（sRNA）library.The main results of miRNA in response to MC in this study are as follows:（1）By observing the plant morphology after MC treatment,it was found that the treatment significantly inhibited the length of each internode,among which the new internode was the most significantly inhibited,so we extracted Total RNA from neonatal internode samples at 4 stages to construct4 sRNA libraries.High-throughput sequencing showed that 84.18% of the sequences were successfully matched to the cotton（G.hirsutum TM-1）genome,clean reads ranged from 23,159,571 to 25,918,608,unique reads ranged from 11,681,583 to 14,262,096.In addition,t RNAs,rr RNAs,snor RNAs and sn RNAs accounted for 0.00%-2.35% of the total reads.According to the statistics of the sequence length of 18-30 nt in the four libraries,the 24 nt sRNA was the largest one in all the four stages（about 50%）,followed by the21 nt sequence（about 17%）,which was consistent with the previous reports on cotton and other plants.Sequencing data and data statistics reflect that high-quality SRNA library has been obtained in this study,which can be used for subsequent analysis.（2）Unique reads screened from four cotton sRNA libraries were matched with all known plant mature miRNA sequences from miRBase 22.1.In this study,we identified 508 known miRNAs belonging to 197 miRNA families,of which 21 belonged to highly conserved miRNA families.Currently,a total of 378 mature miRNAs from 217 families of cotton have been registered in miRBase（22.1）.A total of 309 cotton miRNAs（81.7%）were identified,belonging to 177 families,indicating that the sequencing coverage of these 4 sRNA libraries was good.（3）A total of 8 novel miRNAs were identified in this study,including 3 new members of the known miRNA family.The length of these novel miRNAs and miRNA* ranged from 20 nt to 25 nt,and the abundance of most novel miRNAs was relatively low compared to conserved miRNAs.（4）A total of 104 differentially expressed genes including 98 known miRNAs and 6 novel miRNAs were identified by comparing the miRNAs expression levels between the control group（0h）and the MC treatment group（48h,72 h and 96h）.Sixty-three DEG miRNAs（61%）were highly conserved miRNAs,and 20 were cottonspecific miRNAs,which have been identified only in cotton.The differentially expressed miRNAs at 48,72 and96 h after MC treatment were 60（24 up-regulated and 36 down-regulated）,65（34 up-regulated and 31 downregulated）and 77（29 up-regulated and 48 down-regulated）,respectively.With the extension of treatment time,the number of differentially expressed miRNAs gradually increased.Among these differentially expressed miRNAs,14 miRNAs were continuously inhibited by MC,12 known miRNAs were continuously induced by MC,and only ghr-miRN4 expression was continuously increased in novel miRNAs.Most DEG miRNAs were altered in response to MC at early or late post treatment time.These data suggest that DEG miRNAs play an important role in MC induced inhibition of internode elongation.（5）Nine differentially expressed miRNAs were randomly selected for qRT-PCR verification,and the results showed that the trend obtained by qRT-PCR was basically consistent with the variation of sRNA-seq multiple.Therefore,miRNA expression profile data obtained by deep sequencing is reliable and can be used for further analysis.（6）In this experiment,a total of 1639 target genes of 71 known miRNAs and 162 target genes of 4 novel miRNAs were predicted.Many targets of the miRNA were transcription factors,including GRF,TCP,MYB,SPL,ARF,NAC and SCL,etc.In addition to the previously identified conserved targets,many new targets have also been identified for known miRNAs.（7）Ten known miRNA target genes were verified by RLM-5’ RACE,and the specific splicing sites between the complementary sequences of miRNA and its target genes were found.These include four highly conserved miRNA family members miR160,miR164,miR319,and miR393;Six non-conserved family miRNAs were first verified in cotton: miR403,miR535,miR7505,miR858,miR8634,and miR8746,of which miR8746,miR7505,and miR8634 are cottons-specific miRNAs.