Research Of MicroRNA Of Lforal Development Between Gossypium Harknessii Cytoplasmic Male Sterile Line And Its.Maintainer Line

Comparing with inbred cotton, hybrid F1cotton shows obvious superiority on growth potential,resistance, yield and quality. The r Hybrid F1is grown in more than90%of the whole area for cotton inYangtze River Basin. The application of cytoplasmic male sterile lines can not only provide an effectiveway for the high-volume commercial production of hybrid cotton seed, but also have significantadvantages in utilizing heterosis and hybridization. The infertility mechanism of sterile lines having notyet been studied thoroughly, boththe choice of restorer linesand the improvment of CMS materials arelimited, to a certain extent, the application of the three-line breeding is blocked. Based on the study ofthe differences of mitochondrial DNA, protein and miRNA sequencing on Gossypium harknessiicytoplasmic male sterile (CMS) together with its maintainer line which has the different cytoplasmic butsame nuclear background by our research group, seven miRNAs and their target genes which related toanther development and energy metabolism were analyzed by RT-PCR on the CMS line and maintainerline in order to get the relation of miRNA and cytoplasmic male sterile.The main results and conclusions are as follows:1. From free-hand section and paraffin section of different developmental stages (length1-5mm) ofmicrosporgenesis for ZB and ZBA, the abortive stage of ZBA was preliminary confirmed before thestage of meiotic pachytene,namedthe stage of2-3.1mm.2. Seven miRNAs(miR156、miR160、miR167、miR168、miR319、miR482, including the newlyidentified miR-gh-3), which may be associated with fertility according to the relevant studies andhave statistical differences between ZB and ZBA, were analyzed byRT-PCR. The expression levelsof them were gradually increased with the development of pollen in ZB and ZBA;Each miRNA havedifferent degree of variation between the CMS line and its maintainer line,especially in3mm-stage,the expression of miR156in ZBA is significantly lower than ZB, for the other six miRNA, arehigheror signally higher than ZB.3.22of SPLs gene family in cotton which were widely recognized as the target genes of miR156wereanalyzed by RT-PCR, the expression levels of SPL1、SPL6、SPL8、SPL15、SPL18and SPL21whichreversed the trend of miR156,gradually decreasing with the development of pollen in ZB and ZBA.Only8SPLs’ expression was not markedly different between ZB and ZBA. Previous researchesreported that miR156affects male gametogenesis by regulating the expression of the SPL genes.4.16target genes of the seven miRNAs which relate to anther development and energy metabolismwere analyzed by RT-PCR,4genes are not memorably different between ZB and ZBA, the otherssuch as gultathione transferase, ARF8, tublin binding cofactor C,NADH dehydrogenase subunit andso on,were dramaticallydifferent between ZB and ZBA.5. The sequences of TBCC and ARF8were acquired using rapid amplification of cDNA ends (RACE)approach, and both of them were especially observably different between ZB and ZBA. The full length of TBCC and ARF8sequence were2044bp and3274bp. The ORF of TBCC gene encoded570amino acid residues, and the conserved domain of TBCC and CARP were observed in the predictedprotein sequence (KC488331). In the same way,the ORF of ARF8gene encoded823amino acidresidues, and the conserved domain of B3DNA binding,Aux/IAA-ARF dimerization and AuxinResponse were included in the predicted protein sequence.