The Regulatory Function Of MiR159 And MiR319 During Pollen Development In Brassica Campestris

miR159 and miR319 are highly conserved miRNAs that regulate target genes and play an important role in plant growth and development.The production patterns,secondary structures,and mature sequences of miR159 and miR319 are highly similar,and all come from the same ancestor.The two are classified into the same family and have similar effects in plant growth and development.However,due to differences in target genes and spatiotemporal expression,they have functional differentiation.In the pre-laboratory study,high-throughput sequencing analysis revealed that miR159 and miR319 were significantly different in‘Aijiaohuang' genic male sterility AB line(ajhGMS ‘Bcajh97-01A/B'),suggesting that they may be involved in pollen development.After the whole genome triplication event,the number of members of the coding gene and miRNA family increased,and their expression patterns,biological functions and regulatory pathways changed.In this paper,we conducted bioinformatics analysis of miR159 and miR319 family members of Brassica campestris L.subsp.chinensis(syn.B.rapa subsp.chinensis),and studied their evolution and expression patterns.Subsequently,overexpression vectors of miR159 and miR319 were constructed to observe the phenotypic differences between different transgenic plants in B.campestris,and the biological functions of different members of miR159 and miR319 were compared.Combined with 5' RACE,tobacco co-expression and qRT-PCR experiments,the regulatory pathways of miR159 and miR319 in B.campestris were studied.The main results and conclusions obtained are as follows:(1)After searching by BLASTN,seven precursor genes of miR159 and miR319 were identified in B.campestris,respectively,in which miR159 a and miR159 b have the same precursor gene.The precursor genes of miR159 and miR319 had high retention rates in B.campestris L.subsp.chinensis.Except for the precursor genes of miR159 c and miR319 b,there were three copies of other precursor genes,and the sequence similarity of the three copies was higher.Through sequence alignment analysis,it was found that the mature and pre-gene sequences of miR159 and miR319 members are highly conserved in B.campestris L.subsp.chinensis and Arabidopsis thaliana,,and their mature sequences were among 21 bases and 17 were identical.The secondary structures of miR159 and miR319 members were analyzed and found to be highly similar to miR159 and miR319 in A.thaliana,forming a "bi-duplex" type of stem-loop structure.(2)The phylogenetic tree of the precursor genes of miR159 and miR319 in B.campestris L.subsp.chinensis and A.thaliana was constructed.The results showed that there were differences in the evolutionaryrelationship between miR159 and miR319 members in A.thaliana.The three precursor genes of miR319 a only Bra-MIR319a2 was closely related to the evolution of the precursor gene of miR319 a in Arabidopsis,the other two copies are one with Bra-MIR319 b and have a long relationship with the miR319 a and miR319 b precursor genes in Arabidopsis.In B.campestris,the precursor genes of miR319 c and miR159 c are far from the evolutionary relationship between miR159 a and miR319 a precursor genes.It was found by promoter GUS staining that the miR159 and miR319 members of B.campestris L.subsp.chinensis had different spatiotemporal expression patterns.The spatiotemporal expression of the three precursor gene promoters of miR159 a had differences in mature pollen and sepal.Three copies of miR319 a only the Bra-MIR319a2 promoter was expressed in the late anthers and open flower petals,the three precursor genes of miR319 c were only expressed in anthers by the Bra-MIR319c3 promoter,the spatiotemporal expression pattern of Bra-MIR319c2 and Bra-MIR159 c promoters was restricted to the detachment at the late flower development stage,other precursor genes did not detect the spatiotemporal expression pattern of the promoter.Different spatiotemporal expression patterns indicate that they have functional differentiation during the growth and development of B.campestris L.subsp.chinensis.(3)By constructing the MIR159 a and MIR159 c overexpression vectors in B.campestris,genetically transforming the B.campestris L.subsp.chinensis and observing the phenotype of the transgenic plants,it was found that overexpression of MIR159 a and MIR159 c in B.campestris caused pollen abortion,and pollen abortion was caused by degradation of pollen grain inclusions,causing the inner wall of the pollen to disappear and the pollen to shrink.Overexpressed MIR159 a and MIR159 c plants of B.campestris L.subsp.chinensis,the pollen appeared to have a different period of abortion,overexpression of MIR159 a produced a higher rate of pollen abortion than overexpressing MIR159 c plants,and overexpression of MIR159 a in B.campestris also caused abnormal pollen tube germination,indicating that there were different regulatory patterns in miR159 a and miR159 c of B.campestris L.subsp.chinensis.(4)A.thaliana was expressed heterologously by MIR319 a,MIR319b and MIR319 c of B.campestris L.subsp.chinensis.The phenotypes of transgenic plants were observed.It was found that MIR319 a,MIR319b and MIR319 c of B.campestris could affect the division and differentiation of leaf and petal cells,leading to leaf and petal wavy.The heterologous expression of MIR319 a in B.campestris L.subsp.Chinensis also caused pollen abortion,which was similar to the reason that overexpressed MIR159 in B.campestris L.subsp.chinensis to produce pollen abortion,that could cause the degradation of pollen grain content and affect the construction of pollen inner wall,indicating that miR159 and miR319 a had the same target gene.The MIR319 c overexpression vector was transformed into the B.campestris L.subsp.chinensis and the transgenic phenotype was observed.It was found that overexpression of miR319 c in B.campestris L.subsp.chinensis could also affect cell division and produce larger floral organs.Excessive expression of miR319 c in B.campestris L.subsp.chinensis could also cause degradation of pollen contents,leading to pollenabortion.(5)By 5' RACE and tobacco in vitro co-expression experiments,it was found that the common target gene BcMYB101-1 exists in miR159 and miR319 in B.campestris L.subsp.chinensis.In addition,miR159 and miR319a inhibited the expression of BcMYB101-2,but miR319 c could not act on BcMYB101-2.Differences in the temporal and spatial expression patterns of miR159 and miR319 in B.campestris and BcMYB101-1 and BcMYB101-2 resulted in differentiation of the regulatory patterns of BcMYB101-1 and BcMYB101-2 by miR159 and miR319.Compared with the common target genes of miR159 and miR319 in A.thaliana,the regulatory pathways of miR159 and miR319 were altered after the triploidization of B.campestris.